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Sample records for albumin bsa protein

  1. Bovine serum albumin (BSA) can replace patient serum as a protein source in an in vitro fertilization (IVF) program.

    PubMed

    Benadiva, C A; Kuczynski-Brown, B; Maguire, T G; Mastoianni, L; Flickinger, G L

    1989-06-01

    Alternate protein sources have been suggested to replace the commonly used cord or patient serum for in vitro fertilization (IVF) procedures. During an 11-month period 127 patients treated for in vitro fertilization had either their serum (N = 71) or bovine serum albumin (BSA; N = 56) used as the protein source in the insemination and growth media. Ham's F-10 + 0.5% BSA was used for sperm swim-up and insemination media and 1% BSA was used for the growth media. Patient's serum was added to Ham's F-10 culture media at concentrations of 7.5 and 15% for insemination and growth, respectively. Embryo transfer was performed with Ham's F-10 containing 90% maternal serum in both groups. Fertilization rate of 259 oocytes inseminated in medium containing patient's serum did not differ when compared with 200 oocytes inseminated in medium containing BSA. Likewise, rates of abnormal fertilization, cleavage, and pregnancy were similar in both groups. In a second experiment, 148 normally fertilized oocytes were transferred after 24 hr in culture to growth media containing two different concentrations of BSA (0.5 or 1%). Cleavage rates for the two groups were similar and the percentage of embryos developed to greater than or equal to 4 cells did not differ significantly. We conclude that a single concentration of BSA can safely be used to supplement culture media in human IVF with several practical and economical benefits.

  2. Spectroscopic Study on the Interaction between Naphthalimide-Polyamine Conjugates and Bovine Serum Albumin (BSA).

    PubMed

    Tian, Zhi-Yong; Song, Li-Na; Zhao, Yuan; Zang, Feng-Lei; Zhao, Zhong-Hua; Chen, Nan-Hao; Xu, Xue-Jun; Wang, Chao-Jie

    2015-01-01

    The effect of a naphthalimide pharmacophore coupled with diverse substituents on the interaction between naphthalimide-polyamine conjugates 1-4 and bovine serum albumin (BSA) was studied by UV absorption, fluorescence and circular dichroism (CD) spectroscopy under physiological conditions (pH = 7.4). The observed spectral quenching of BSA by the compounds indicated that they could bind to BSA. Furthermore, caloric fluorescent tests revealed that the quenching mechanisms of compounds 1-3 were basically static type, but that of compound 4 was closer to a classical type. The Ksv values at room temperature for compound-BSA complexes-1-BSA, 2-BSA, 3-BSA and 4-BSA were 1.438 × 10⁴, 3.190 × 10⁴, 5.700 × 10⁴ and 4.745 × 10⁵, respectively, compared with the value of MINS, 2.863 × 10⁴ at Ex = 280 nm. The obtained quenching constant, binding constant and thermodynamic parameter suggested that the binding between compounds 1-4 with BSA protein, significantly affected by the substituted groups on the naphthalene backbone, was formed by hydrogen bonds, and other principle forces mainly consisting of charged and hydrophobic interactions. Based on results from the analysis of synchronous three-dimensional fluorescence and CD spectra, we can conclude that the interaction between compounds 1-4 and BSA protein has little impact on the BSA conformation. Calculated results obtained from in silico molecular simulation showed that compound 1 did not prefer either enzymatic drug sites I or II over the other. However, DSII in BSA was more beneficial than DSI for the binding between compounds 2-4 and BSA protein. The binding between compounds 1-3 and BSA was hydrophobic in nature, compared with the electrostatic interaction between compound 4 and BSA. PMID:26378511

  3. Spectroscopic Study on the Interaction between Naphthalimide-Polyamine Conjugates and Bovine Serum Albumin (BSA).

    PubMed

    Tian, Zhi-Yong; Song, Li-Na; Zhao, Yuan; Zang, Feng-Lei; Zhao, Zhong-Hua; Chen, Nan-Hao; Xu, Xue-Jun; Wang, Chao-Jie

    2015-09-11

    The effect of a naphthalimide pharmacophore coupled with diverse substituents on the interaction between naphthalimide-polyamine conjugates 1-4 and bovine serum albumin (BSA) was studied by UV absorption, fluorescence and circular dichroism (CD) spectroscopy under physiological conditions (pH = 7.4). The observed spectral quenching of BSA by the compounds indicated that they could bind to BSA. Furthermore, caloric fluorescent tests revealed that the quenching mechanisms of compounds 1-3 were basically static type, but that of compound 4 was closer to a classical type. The Ksv values at room temperature for compound-BSA complexes-1-BSA, 2-BSA, 3-BSA and 4-BSA were 1.438 × 10⁴, 3.190 × 10⁴, 5.700 × 10⁴ and 4.745 × 10⁵, respectively, compared with the value of MINS, 2.863 × 10⁴ at Ex = 280 nm. The obtained quenching constant, binding constant and thermodynamic parameter suggested that the binding between compounds 1-4 with BSA protein, significantly affected by the substituted groups on the naphthalene backbone, was formed by hydrogen bonds, and other principle forces mainly consisting of charged and hydrophobic interactions. Based on results from the analysis of synchronous three-dimensional fluorescence and CD spectra, we can conclude that the interaction between compounds 1-4 and BSA protein has little impact on the BSA conformation. Calculated results obtained from in silico molecular simulation showed that compound 1 did not prefer either enzymatic drug sites I or II over the other. However, DSII in BSA was more beneficial than DSI for the binding between compounds 2-4 and BSA protein. The binding between compounds 1-3 and BSA was hydrophobic in nature, compared with the electrostatic interaction between compound 4 and BSA.

  4. Effect of bovine serum albumin (BSA) on enzymatic cellulose hydrolysis.

    PubMed

    Wang, Hui; Mochidzuki, Kazuhiro; Kobayashi, Shinichi; Hiraide, Hatsue; Wang, Xiaofen; Cui, Zongjun

    2013-06-01

    Bovine serum albumin (BSA) was added to filter paper during the hydrolysis of cellulase. Adding BSA before the addition of the cellulase enhances enzyme activity in the solution, thereby increasing the conversion rate of cellulose. After 48 h of BSA treatment, the BSA adsorption quantities are 3.3, 4.6, 7.8, 17.2, and 28.3 mg/g substrate, each with different initial BSA concentration treatments at 50 °C; in addition, more cellulase was adsorbed onto the filter paper at 50 °C compared with 35 °C. After 48 h of hydrolysis, the free-enzyme activity could not be measured without the BSA treatment, whereas the remaining activity of the filter paper activity was approximately 41 % when treated with 1.0 mg/mL BSA. Even after 96 h of hydrolysis, 25 % still remained. Meanwhile, after 48 h of incubation without substrate, the remaining enzyme activities were increased 20.7 % (from 43.7 to 52.7 %) and 94.8 % (from 23.3 to 45.5 %) at 35 and 50 °C, respectively. Moreover, the effect of the BSA was more obvious at 35 °C compared with 50 °C. When using 15 filter paper cellulase units per gram substrate cellulase loading at 50 °C, the cellulose conversion was increased from 75 % (without BSA treatment) to ≥90 % when using BSA dosages between 0.1 and 1.5 mg/mL. Overall, these results suggest that there are promising strategies for BSA treatment in the reduction of enzyme requirements during the hydrolysis of cellulose.

  5. Spectroscopic study on the interaction between mononaphthalimide spermidine (MINS) and bovine serum albumin (BSA).

    PubMed

    Tian, Zhiyong; Zang, Fenglei; Luo, Wen; Zhao, Zhonghua; Wang, Yueqiao; Xu, Xuejun; Wang, Chaojie

    2015-01-01

    The interaction mononaphthalimide spermidine (MINS, 1) and bovine serum albumin (BSA) was studied by UV/vis absorption, fluorescence and circular dichroism spectra (CD) under physiological conditions (pH=7.4). The observed spectral quenching of BSA by compound 1 indicated compound 1 could bind to BSA. Further fluorescent tests revealed that the quenching mechanism of BSA by compound 1 was overall static. Meanwhile, the obtained binding constant and thermodynamic parameters on compound-BSA interaction showed that the type of interaction force of compound 1 and BSA was mainly hydrophobic. The analysis of synchronous, three-dimensional fluorescence and CD showed that compound 1 had weak influence on the conformational changes in BSA. Molecular docking simulation was performed and docking model in silico suggested that the configuration of compound 1 was localized in enzymatic drug site II in BSA. Furthermore, naphthalimide moiety of compound 1 greatly contributed to the hydrophobic interaction between compound 1 and BSA protein, as confirmed by experimental data.

  6. Characterization of Silver/Bovine Serum Albumin (Ag/BSA) nanoparticles structure: morphological, compositional, and interaction studies.

    PubMed

    Gebregeorgis, A; Bhan, C; Wilson, O; Raghavan, D

    2013-01-01

    The primary objective of this study was to elucidate the structure of protein conjugated silver nanoparticles prepared by chemical reduction of AgNO(3) and bovine serum albumin (BSA) mixture. The role of BSA in the formation of Ag/BSA nanoparticles was established by UV-Vis Spectroscopy. The association of silver with BSA in Ag/BSA nanoparticles was studied by the decrease in the intensity of absorbance peak at 278 nm in UV-Vis spectra and shift in cathodic peak potential in cyclic voltammogram. The molar ratio of silver to BSA in the Ag/BSA nanoparticles is 27:1, as ascertained by thermogravimetric analysis and atomic absorption spectrometry. Based on atomic force microscopy, dynamic light scattering and transmission electron microscopy (TEM) measurements, the average particle size of nanoparticles was found to be range of 11-15 nm. TEM image showed that the nanoparticle has two distinct phases and selected area electron diffraction pattern of nanoparticles indicated that the silver phase in Ag/BSA is fcc. X-ray photo electron spectroscopy measurements of freshly prepared and argon sputtered nanoparticles provided evidence that the outer and inner region of nanoparticles are mainly composed of BSA and silver respectively. The structural and compositional findings of nanoparticles could have a strong bearing on the bioavailability and antimicrobial activity of nanoparticles.

  7. Albumin (BSA) Adsorption over Graphene in Aqueous Environment: Influence of Orientation, Adsorption Protocol, and Solvent Treatment.

    PubMed

    Vilhena, J G; Rubio-Pereda, Pamela; Vellosillo, Perceval; Serena, P A; Pérez, Rubén

    2016-02-23

    We report 150 ns explicit solvent MD simulations of the adsorption on graphene of albumin (BSA) in two orientations and using two different adsorption protocols, i.e., free and forced adsorption. Our results show that free adsorption occurs with little structural rearrangements. Even taking adsorption to an extreme, by forcing it with a 5 nN downward force applied during the initial 20 ns, we show that along a particular orientation BSA is able to preserve the structural properties of the majority of its binding sites. Furthermore, in all the cases considered in this work, the ibuprofen binding site has shown a strong resilience to structural changes. Finally, we compare these results with implicit solvent simulations and find that the latter predicts an extreme protein unfolding upon adsorption. The origin of this discrepancy is attributed to a poor description of the water entropic forces at interfaces in the implicit solvent methods.

  8. Albumin (BSA) Adsorption over Graphene in Aqueous Environment: Influence of Orientation, Adsorption Protocol, and Solvent Treatment.

    PubMed

    Vilhena, J G; Rubio-Pereda, Pamela; Vellosillo, Perceval; Serena, P A; Pérez, Rubén

    2016-02-23

    We report 150 ns explicit solvent MD simulations of the adsorption on graphene of albumin (BSA) in two orientations and using two different adsorption protocols, i.e., free and forced adsorption. Our results show that free adsorption occurs with little structural rearrangements. Even taking adsorption to an extreme, by forcing it with a 5 nN downward force applied during the initial 20 ns, we show that along a particular orientation BSA is able to preserve the structural properties of the majority of its binding sites. Furthermore, in all the cases considered in this work, the ibuprofen binding site has shown a strong resilience to structural changes. Finally, we compare these results with implicit solvent simulations and find that the latter predicts an extreme protein unfolding upon adsorption. The origin of this discrepancy is attributed to a poor description of the water entropic forces at interfaces in the implicit solvent methods. PMID:26799950

  9. Protein Crystal Serum Albumin

    NASA Technical Reports Server (NTRS)

    1998-01-01

    As the most abundant protein in the circulatory system albumin contributes 80% to colloid osmotic blood pressure. Albumin is also chiefly responsible for the maintenance of blood pH. It is located in every tissue and bodily secretion, with extracellular protein comprising 60% of total albumin. Perhaps the most outstanding property of albumin is its ability to bind reversibly to an incredible variety of ligands. It is widely accepted in the pharmaceutical industry that the overall distribution, metabolism, and efficiency of many drugs are rendered ineffective because of their unusually high affinity for this abundant protein. An understanding of the chemistry of the various classes of pharmaceutical interactions with albumin can suggest new approaches to drug therapy and design. Principal Investigator: Dan Carter/New Century Pharmaceuticals

  10. Comprehensive spectroscopic probing the interaction and conformation impairment of bovine serum albumin (BSA) by herbicide butachlor.

    PubMed

    Liu, Xiaoyi; Ling, Zhaoxing; Zhou, Xing; Ahmad, Farooq; Zhou, Ying

    2016-09-01

    Butachlor is an effective herbicide to deal with undesired weeds selectively and is used at high levels in Asian countries. However, its interaction and impairment effect on BSA was still not clear. In this study, we investigated the interaction between butachlor and bovine serum albumin (BSA) by multi-spectroscopic methods including UV absorption, circular dichroism (CD) spectra, Fourier transform infrared (FTIR) spectra and fluorescence spectra under physiological conditions (pH=7.4). The results revealed that there was a static quenching of BSA induced by butachlor stemmed from the formation of complex. Based on thermodynamic data, the interaction of butachlor with BSA was due to happen, and van der Waals force as well as hydrogen bond were the major forces contributed to the interaction. The binding constant Kb and number of binding site of butachlor with BSA were 5.158×10(5) and 1.372 at 303K, respectively. The distance r between donor (BSA) and acceptor (butachlor) was 0.113nm, obtained according to the Förster theory. The results revealed that butachlor induced conformational changes in BSA but the secondary structure of BSA was still retained. In addition, the microenvironment around chromophore residues of BSA, for example, tryptophan, changed as well, resulting from the formation of more hydrogen bonds. PMID:27419617

  11. Comprehensive spectroscopic probing the interaction and conformation impairment of bovine serum albumin (BSA) by herbicide butachlor.

    PubMed

    Liu, Xiaoyi; Ling, Zhaoxing; Zhou, Xing; Ahmad, Farooq; Zhou, Ying

    2016-09-01

    Butachlor is an effective herbicide to deal with undesired weeds selectively and is used at high levels in Asian countries. However, its interaction and impairment effect on BSA was still not clear. In this study, we investigated the interaction between butachlor and bovine serum albumin (BSA) by multi-spectroscopic methods including UV absorption, circular dichroism (CD) spectra, Fourier transform infrared (FTIR) spectra and fluorescence spectra under physiological conditions (pH=7.4). The results revealed that there was a static quenching of BSA induced by butachlor stemmed from the formation of complex. Based on thermodynamic data, the interaction of butachlor with BSA was due to happen, and van der Waals force as well as hydrogen bond were the major forces contributed to the interaction. The binding constant Kb and number of binding site of butachlor with BSA were 5.158×10(5) and 1.372 at 303K, respectively. The distance r between donor (BSA) and acceptor (butachlor) was 0.113nm, obtained according to the Förster theory. The results revealed that butachlor induced conformational changes in BSA but the secondary structure of BSA was still retained. In addition, the microenvironment around chromophore residues of BSA, for example, tryptophan, changed as well, resulting from the formation of more hydrogen bonds.

  12. Residual bovine serum albumin (BSA) quantitation in vaccines using automated Capillary Western technology.

    PubMed

    Loughney, John W; Lancaster, Catherine; Ha, Sha; Rustandi, Richard R

    2014-09-15

    Bovine serum albumin (BSA) is a major component of fetal bovine serum (FBS), which is commonly used as a culture medium during vaccine production. Because BSA can cause allergic reactions in humans the World Health Organization (WHO) has set a guidance of 50 ng or less residual BSA per vaccine dose. Vaccine manufacturers are expected to develop sensitive assays to detect residual BSA. Generally, sandwich enzyme-linked immunosorbent assays (ELISA) are used in the industry to detect these low levels of BSA. We report the development of a new improved method for residual BSA detection using the SimpleWestern technology to analyze residual BSA in an attenuated virus vaccine. The method is based on automated Capillary Western and has linearity of two logs, >80% spike recovery (accuracy), intermediate precision of CV <15%, and LOQ of 5.2 ng/ml. The final method was applied to analyze BSA in four lots of bulk vaccine products and was used to monitor BSA clearance during vaccine process purification.

  13. Glycation does not modify bovine serum albumin (BSA)-induced reduction of rat aortic relaxation: The response to glycated and nonglycated BSA is lost in metabolic syndrome

    PubMed Central

    Rubio-Ruiz, Maria Esther; Díaz-Díaz, Eulises; Cárdenas-León, Mario; Argüelles-Medina, Rabindranath; Sánchez-Canales, Patricia; Larrea-Gallo, Fernando; Soria-Castro, Elizabeth; Guarner-Lans, Verónica

    2008-01-01

    The effects of nonglycated bovine serum albumin (BSA) and advanced glycosylation end products of BSA (AGE-BSA) on vascular responses of control and metabolic syndrome (MS) rats characterized by hypertriglyceridemia, hypertension, hyperinsulinemia, and insulin resistance were studied. Albumin and in vitro prepared AGE-BSA have vascular effects; however, recent studies indicate that some effects of in vitro prepared AGEs are due to the conditions in which they were generated. We produced AGEs by incubating glucose with BSA for 60 days under sterile conditions in darkness and at 37°C. To develop MS rats, male Wistar animals were given 30% sucrose in drinking water since weanling. Six month old animals were used. Blood pressure, insulin, triglycerides, and serum albumin were increased in MS rats. Contraction of aortic rings elicited with norepinephrine was stronger. There were no effects of nonglycated BSA or AGE-BSA on contractions in control or MS rats; however, both groups responded to L-NAME, an inhibitor of nitric oxide synthesis. Arterial relaxation induced using acetylcholine was smaller in MS rats. Nonglycated BSA and AGE-BSA significantly diminished relaxation in a 35% in the control group but the decrease was similar when using nonglycated BSA and AGE-BSA. This decrease was not present in the MS rats and was not due to increased RAGEs or altered biochemical characteristics of BSA. In conclusion, both BSA and AGE-BSA inhibit vascular relaxation in control artic rings. In MS rats the effect is lost possibly due to alterations in endothelial cells that are a consequence of the illness. PMID:18458031

  14. SANS study of interaction of silica nanoparticles with BSA protein and their resultant structure

    NASA Astrophysics Data System (ADS)

    Yadav, Indresh; Aswal, V. K.; Kohlbrecher, J.

    2014-04-01

    Small angle neutron scattering (SANS) has been carried out to study the interaction of anionic silica nanoparticles (88 Å) with globular protein Bovine Serum Albumin (BSA) (M.W. 66.4 kD) in aqueous solution. The measurements have been carried out on fixed concentration (1 wt %) of Ludox silica nanoparticles with varying concentration of BSA (0-5 wt %) at pH7. Results show that silica nanoparticles and BSA coexist as individual entities at low concentration of BSA where electrostatic repulsive interactions between them prevent their aggregation. However, as the concentration of BSA increases (≥ 0.5 wt %), it induces the attractive depletion interaction among nanoparticles leading to finally their aggregation at higher BSA concentration (2 wt %). The aggregates are found to be governed by the diffusion limited aggregation (DLA) morphology of fractal nature having fractal dimension about 2.4.

  15. SANS study of interaction of silica nanoparticles with BSA protein and their resultant structure

    SciTech Connect

    Yadav, Indresh Aswal, V. K.; Kohlbrecher, J.

    2014-04-24

    Small angle neutron scattering (SANS) has been carried out to study the interaction of anionic silica nanoparticles (88 Å) with globular protein Bovine Serum Albumin (BSA) (M.W. 66.4 kD) in aqueous solution. The measurements have been carried out on fixed concentration (1 wt %) of Ludox silica nanoparticles with varying concentration of BSA (0–5 wt %) at pH7. Results show that silica nanoparticles and BSA coexist as individual entities at low concentration of BSA where electrostatic repulsive interactions between them prevent their aggregation. However, as the concentration of BSA increases (≥ 0.5 wt %), it induces the attractive depletion interaction among nanoparticles leading to finally their aggregation at higher BSA concentration (2 wt %). The aggregates are found to be governed by the diffusion limited aggregation (DLA) morphology of fractal nature having fractal dimension about 2.4.

  16. Binding of an Oligomeric Ellagitannin Series to Bovine Serum Albumin (BSA): Analysis by Isothermal Titration Calorimetry (ITC).

    PubMed

    Karonen, Maarit; Oraviita, Marianne; Mueller-Harvey, Irene; Salminen, Juha-Pekka; Green, Rebecca J

    2015-12-16

    A unique series of oligomeric ellagitannins was used to study their interactions with bovine serum albumin (BSA) by isothermal titration calorimetry. Oligomeric ellagitannins, ranging from monomer to heptamer and a mixture of octamer-undecamers, were isolated as individual pure compounds. This series allowed studying the effects of oligomer size and other structural features. The monomeric to trimeric ellagitannins deviated most from the overall trends. The interactions of ellagitannin oligomers from tetramers to octa-undecamers with BSA revealed strong similarities. In contrast to the equilibrium binding constant, enthalpy showed an increasing trend from the dimer to larger oligomers. It is likely that first the macrocyclic part of the ellagitannin binds to the defined binding sites on the protein surface and then the "flexible tail" of the ellagitannin coats the protein surface. The results highlight the importance of molecular flexibility to maximize binding between the ellagitannin and protein surfaces.

  17. Binding of an Oligomeric Ellagitannin Series to Bovine Serum Albumin (BSA): Analysis by Isothermal Titration Calorimetry (ITC).

    PubMed

    Karonen, Maarit; Oraviita, Marianne; Mueller-Harvey, Irene; Salminen, Juha-Pekka; Green, Rebecca J

    2015-12-16

    A unique series of oligomeric ellagitannins was used to study their interactions with bovine serum albumin (BSA) by isothermal titration calorimetry. Oligomeric ellagitannins, ranging from monomer to heptamer and a mixture of octamer-undecamers, were isolated as individual pure compounds. This series allowed studying the effects of oligomer size and other structural features. The monomeric to trimeric ellagitannins deviated most from the overall trends. The interactions of ellagitannin oligomers from tetramers to octa-undecamers with BSA revealed strong similarities. In contrast to the equilibrium binding constant, enthalpy showed an increasing trend from the dimer to larger oligomers. It is likely that first the macrocyclic part of the ellagitannin binds to the defined binding sites on the protein surface and then the "flexible tail" of the ellagitannin coats the protein surface. The results highlight the importance of molecular flexibility to maximize binding between the ellagitannin and protein surfaces. PMID:26608224

  18. Hepatoma-Targeted Radionuclide Immune Albumin Nanospheres: (131)I-antiAFPMcAb-GCV-BSA-NPs.

    PubMed

    Lin, Mei; Huang, Junxing; Zhang, Dongsheng; Jiang, Xingmao; Zhang, Jia; Yu, Hong; Xiao, Yanhong; Shi, Yujuan; Guo, Ting

    2016-01-01

    An effective strategy has been developed for synthesis of radionuclide immune albumin nanospheres ((131)I-antiAFPMcAb-GCV-BSA-NPs). In vitro as well as in vivo targeting of (131)I-antiAFPMcAb-GCV-BSA-NPs to AFP-positive hepatoma was examined. In cultured HepG2 cells, the uptake and retention rates of (131)I-antiAFPMcAb-GCV-BSA-NPs were remarkably higher than those of (131)I alone. As well, the uptake rate and retention ratios of (131)I-antiAFPMcAb-GCV-BSA-NPs in AFP-positive HepG2 cells were also significantly higher than those in AFP-negative HEK293 cells. Compared to (131)I alone, (131)I-antiAFPMcAb-GCV-BSA-NPs were much more easily taken in and retained by hepatoma tissue, with a much higher T/NT. Due to good drug-loading, high encapsulation ratio, and highly selective affinity for AFP-positive tumors, the (131)I-antiAFPMcAb-GCV-BSA-NPs are promising for further effective radiation-gene therapy of hepatoma.

  19. Hepatoma-Targeted Radionuclide Immune Albumin Nanospheres: 131I-antiAFPMcAb-GCV-BSA-NPs

    PubMed Central

    Lin, Mei; Huang, Junxing; Zhang, Dongsheng; Jiang, Xingmao; Zhang, Jia; Yu, Hong; Xiao, Yanhong; Shi, Yujuan; Guo, Ting

    2016-01-01

    An effective strategy has been developed for synthesis of radionuclide immune albumin nanospheres (131I-antiAFPMcAb-GCV-BSA-NPs). In vitro as well as in vivo targeting of 131I-antiAFPMcAb-GCV-BSA-NPs to AFP-positive hepatoma was examined. In cultured HepG2 cells, the uptake and retention rates of 131I-antiAFPMcAb-GCV-BSA-NPs were remarkably higher than those of 131I alone. As well, the uptake rate and retention ratios of 131I-antiAFPMcAb-GCV-BSA-NPs in AFP-positive HepG2 cells were also significantly higher than those in AFP-negative HEK293 cells. Compared to 131I alone, 131I-antiAFPMcAb-GCV-BSA-NPs were much more easily taken in and retained by hepatoma tissue, with a much higher T/NT. Due to good drug-loading, high encapsulation ratio, and highly selective affinity for AFP-positive tumors, the 131I-antiAFPMcAb-GCV-BSA-NPs are promising for further effective radiation-gene therapy of hepatoma. PMID:26981334

  20. Spectrometry researches on interaction and sonodynamic damage of riboflavin (RF) to bovine serum albumin (BSA)

    NASA Astrophysics Data System (ADS)

    Wang, Zhiqiu; Li, Jushi; Wang, Jun; Zou, Mingming; Wang, Siyu; Li, Ying; Kong, Yumei; Xia, Lixin

    2012-02-01

    In this paper, the riboflavin (RF) was used to study the interaction and sonodynamic damage to bovine serum albumin (BSA) by fluorescence and UV-vis spectroscopy. The results showed that the RF could efficiently bind to BSA in aqueous solution. Under ultrasonic irradiation, the RF could obviously damage the BSA. In addition, synchronous fluorescence spectroscopy revealed that the RF showed more accessible to tryptophan (Trp) residues than to tyrosine (Tyr) residues. Also, it damaged Trp residues more seriously than Tyr residues under ultrasonic irradiation. At last, the generation of reactive oxygen species (ROS) in sonodynamic process was estimated by the method of Oxidation-Extraction Spectrometry (OES). And then, several radical scavengers were used to determine the kind of ROS. It was found that at least the singlet oxygen ( 1O 2) and hydroxyl radicals ( rad OH) were generated.

  1. Preparation of Bovine Serum Albumin (BSA) nanoparticles by desolvation using a membrane contactor: a new tool for large scale production.

    PubMed

    Yedomon, B; Fessi, H; Charcosset, C

    2013-11-01

    Albumin nanoparticles are attractive drug delivery systems as they can be prepared under soft conditions and incorporate several kinds of molecules. The aim of this study was to upscale the desolvation process for preparing Bovine Serum Albumin (BSA) nanoparticles using a membrane contactor. At a first step, the BSA nanoparticles were prepared at small scale using a syringe pump. BSA nanoparticles of 139 nm in size, with a polydispersity index of 0.046, were obtained at the optimal conditions: pH 8.2, 100 mg mL(-1) BSA albumin solution (2 mL), and 1 mL min(-1) flow rate of ethanol addition (8 mL). The upscaling with a membrane contactor was achieved by permeating ethanol through the pores of a Shirasu Porous Glass (SPG Technology Co., Japan) membrane and circulating the aqueous phase tangentially to the membrane surface. By increasing the pressure of the ethanol from 1 to 2.7 bars, a progressive decrease in nanoparticle size was obtained with a high nanoparticles yield (around 94-96%). In addition, the flow rate of the circulating phase did not affect the BSA nanoparticle characteristics. At the optimal conditions (pH 8.2, 100 mg mL(-1) BSA albumin solution, pressure of ethanol 2.7 bars, flow rate of the circulating phase 30.7 mL s(-1)), the BSA nanoparticles showed similar characteristics to those obtained with the syringe pump. Large batches of BSA nanoparticles were prepared up to 10 g BSA. The BSA nanoparticles were stable at least during 2 months at 4 °C, and their characteristics were reproducible. It was then concluded that the membrane contactor technique could be a suitable method for the preparation of albumin nanoparticles at large scale with properties similar to that obtained at small scale.

  2. Spectroscopic and molecular docking studies of binding interaction of gefitinib, lapatinib and sunitinib with bovine serum albumin (BSA).

    PubMed

    Shen, Guo-Feng; Liu, Ting-Ting; Wang, Qi; Jiang, Min; Shi, Jie-Hua

    2015-12-01

    The binding interactions of three kinds of tyrosine kinase inhibitors (TKIs), such as gefitinib, lapatinib and sunitinib, with bovine serum albumin (BSA) were studied using ultraviolet spectrophotometry, fluorescence spectroscopy, circular dichroism (CD), Fourier transform infrared spectroscopy (FT-IR) and molecular docking methods. The experimental results showed that the intrinsic fluorescence quenching of BSA induced by the three TKIs resulted from the formation of stable TKIs-BSA complexes through the binding interaction of TKIs with BSA. The stoichiometry of three stable TKIs-BSA complexes was 1:1 and the binding constants (Kb) of the three TKIs-BSA complexes were in the order of 10(4)M(-1) at 310 K, indicating that there was a strong binding interaction of the three TKIs with BSA. Based on the analysis of the signs and magnitudes of the free energy change (ΔG(0)), enthalpic change (ΔH(0)) and entropic change (ΔS(0)) in the binding process, it can be deduced that the binding process of the three TKIs with BSA was spontaneous and enthalpy-driven process, and the main interaction forces between the three TKIs and BSA were van der Waals force and hydrogen bonding interaction. Moreover, from the results of CD, FT-IR and molecular docking, it can be concluded that there was a significant difference between the three TKIs in the binding site on BSA, lapatinib was located on site II (m) of BSA while gefitinib and sunitinib were bound on site I of BSA, and there were some changes in the BSA conformation when binding three TKIs to BSA but BSA still retains its secondary structure α-helicity.

  3. Spectroscopic and molecular docking studies of binding interaction of gefitinib, lapatinib and sunitinib with bovine serum albumin (BSA).

    PubMed

    Shen, Guo-Feng; Liu, Ting-Ting; Wang, Qi; Jiang, Min; Shi, Jie-Hua

    2015-12-01

    The binding interactions of three kinds of tyrosine kinase inhibitors (TKIs), such as gefitinib, lapatinib and sunitinib, with bovine serum albumin (BSA) were studied using ultraviolet spectrophotometry, fluorescence spectroscopy, circular dichroism (CD), Fourier transform infrared spectroscopy (FT-IR) and molecular docking methods. The experimental results showed that the intrinsic fluorescence quenching of BSA induced by the three TKIs resulted from the formation of stable TKIs-BSA complexes through the binding interaction of TKIs with BSA. The stoichiometry of three stable TKIs-BSA complexes was 1:1 and the binding constants (Kb) of the three TKIs-BSA complexes were in the order of 10(4)M(-1) at 310 K, indicating that there was a strong binding interaction of the three TKIs with BSA. Based on the analysis of the signs and magnitudes of the free energy change (ΔG(0)), enthalpic change (ΔH(0)) and entropic change (ΔS(0)) in the binding process, it can be deduced that the binding process of the three TKIs with BSA was spontaneous and enthalpy-driven process, and the main interaction forces between the three TKIs and BSA were van der Waals force and hydrogen bonding interaction. Moreover, from the results of CD, FT-IR and molecular docking, it can be concluded that there was a significant difference between the three TKIs in the binding site on BSA, lapatinib was located on site II (m) of BSA while gefitinib and sunitinib were bound on site I of BSA, and there were some changes in the BSA conformation when binding three TKIs to BSA but BSA still retains its secondary structure α-helicity. PMID:26555641

  4. Ascorbic acid and BSA protein in solution and films: interaction and surface morphological structure.

    PubMed

    Maciel, Rafael R G; de Almeida, Adriele A; Godinho, Odin G C; Gorza, Filipe D S; Pedro, Graciela C; Trescher, Tarquin F; Silva, Josmary R; de Souza, Nara C

    2013-01-01

    This paper reports on the study of the interactions between ascorbic acid (AA) and bovine serum albumin (BSA) in aqueous solution as well as in films (BSA/AA films) prepared by the layer-by-layer technique. Regarding to solution studies, a hyperchromism (in the range of ultraviolet) was found as a function of AA concentration, which suggested the formation of aggregates from AA and BSA. Binding constant, K, determined for aggregates from BSA and AA was found to be about 10(2) M(-1), which indicated low affinity of AA with BSA. For the BSA/AA films, it was also noted that the AA adsorption process and surface morphological structures depended on AA concentration. By changing the contact time between the AA and BSA, a hypochromism was revealed, which was associated to decrease of accessibility of solvent to tryptophan due to formation of aggregates. Furthermore, different morphological structures of aggregates were observed, which were attributed to the diffusion-limited aggregation. Since most of studies of interactions of drugs and proteins are performed in solution, the analysis of these processes by using films can be very valuable because this kind of system is able to employ several techniques of investigation in solid state.

  5. Ascorbic Acid and BSA Protein in Solution and Films: Interaction and Surface Morphological Structure

    PubMed Central

    Maciel, Rafael R. G.; de Almeida, Adriele A.; Godinho, Odin G. C.; Gorza, Filipe D. S.; Pedro, Graciela C.; Trescher, Tarquin F.; Silva, Josmary R.; de Souza, Nara C.

    2013-01-01

    This paper reports on the study of the interactions between ascorbic acid (AA) and bovine serum albumin (BSA) in aqueous solution as well as in films (BSA/AA films) prepared by the layer-by-layer technique. Regarding to solution studies, a hyperchromism (in the range of ultraviolet) was found as a function of AA concentration, which suggested the formation of aggregates from AA and BSA. Binding constant, K, determined for aggregates from BSA and AA was found to be about 102 M−1, which indicated low affinity of AA with BSA. For the BSA/AA films, it was also noted that the AA adsorption process and surface morphological structures depended on AA concentration. By changing the contact time between the AA and BSA, a hypochromism was revealed, which was associated to decrease of accessibility of solvent to tryptophan due to formation of aggregates. Furthermore, different morphological structures of aggregates were observed, which were attributed to the diffusion-limited aggregation. Since most of studies of interactions of drugs and proteins are performed in solution, the analysis of these processes by using films can be very valuable because this kind of system is able to employ several techniques of investigation in solid state. PMID:23984366

  6. Ascorbic acid and BSA protein in solution and films: interaction and surface morphological structure.

    PubMed

    Maciel, Rafael R G; de Almeida, Adriele A; Godinho, Odin G C; Gorza, Filipe D S; Pedro, Graciela C; Trescher, Tarquin F; Silva, Josmary R; de Souza, Nara C

    2013-01-01

    This paper reports on the study of the interactions between ascorbic acid (AA) and bovine serum albumin (BSA) in aqueous solution as well as in films (BSA/AA films) prepared by the layer-by-layer technique. Regarding to solution studies, a hyperchromism (in the range of ultraviolet) was found as a function of AA concentration, which suggested the formation of aggregates from AA and BSA. Binding constant, K, determined for aggregates from BSA and AA was found to be about 10(2) M(-1), which indicated low affinity of AA with BSA. For the BSA/AA films, it was also noted that the AA adsorption process and surface morphological structures depended on AA concentration. By changing the contact time between the AA and BSA, a hypochromism was revealed, which was associated to decrease of accessibility of solvent to tryptophan due to formation of aggregates. Furthermore, different morphological structures of aggregates were observed, which were attributed to the diffusion-limited aggregation. Since most of studies of interactions of drugs and proteins are performed in solution, the analysis of these processes by using films can be very valuable because this kind of system is able to employ several techniques of investigation in solid state. PMID:23984366

  7. Probing into the binding interaction between medroxyprogesterone acetate and bovine serum albumin (BSA): spectroscopic and molecular docking methods.

    PubMed

    Fang, Fang; Pan, Dong-Qi; Qiu, Min-Jie; Liu, Ting-Ting; Jiang, Min; Wang, Qi; Shi, Jie-Hua

    2016-09-01

    To further understand the mechanism of action and pharmacokinetics of medroxyprogesterone acetate (MPA), the binding interaction of MPA with bovine serum albumin (BSA) under simulated physiological conditions (pH 7.4) was studied using fluorescence emission spectroscopy, synchronous fluorescence spectroscopy, circular dichroism and molecular docking methods. The experimental results reveal that the fluorescence of BSA quenches due to the formation of MPA-BSA complex. The number of binding sites (n) and the binding constant for MPA-BSA complex are ~1 and 4.6 × 10(3)  M(-1) at 310 K, respectively. However, it can be concluded that the binding process of MPA with BSA is spontaneous and the main interaction forces between MPA and BSA are van der Waals force and hydrogen bonding interaction due to the negative values of ΔG(0) , ΔH(0) and ΔS(0) in the binding process of MPA with BSA. MPA prefers binding on the hydrophobic cavity in subdomain IIIA (site II'') of BSA resulting in a slight change in the conformation of BSA, but BSA retaining the α-helix structure. Copyright © 2016 John Wiley & Sons, Ltd.

  8. Evaluation of BSA protein release from hollow hydroxyapatite microspheres into PEG hydrogel.

    PubMed

    Fu, Hailuo; Rahaman, Mohamed N; Brown, Roger F; Day, Delbert E

    2013-05-01

    Implants that simultaneously function as an osteoconductive matrix and as a device for local drug or growth factor delivery could provide an attractive system for bone regeneration. In our previous work, we prepared hollow hydroxyapatite (abbreviated HA) microspheres with a high surface area and mesoporous shell wall and studied the release of a model protein, bovine serum albumin (BSA), from the microspheres into phosphate-buffered saline (PBS). The present work is an extension of our previous work to study the release of BSA from similar HA microspheres into a biocompatible hydrogel, poly(ethylene glycol) (PEG). BSA-loaded HA microspheres were placed in a PEG solution which was rapidly gelled using ultraviolet radiation. The BSA release rate into the PEG hydrogel, measured using a spectrophotometric method, was slower than into PBS, and it was dependent on the initial BSA loading and on the microstructure of the microsphere shell wall. A total of 35-40% of the BSA initially loaded into the microspheres was released into PEG over ~14 days. The results indicate that these hollow HA microspheres have promising potential as an osteoconductive device for local drug or growth factor delivery in bone regeneration and in the treatment of bone diseases.

  9. Characterizing the binding interaction between antimalarial artemether (AMT) and bovine serum albumin (BSA): Spectroscopic and molecular docking methods.

    PubMed

    Shi, Jie-Hua; Pan, Dong-Qi; Wang, Xiou-Xiou; Liu, Ting-Ting; Jiang, Min; Wang, Qi

    2016-09-01

    Artemether (AMT), a peroxide sesquiterpenoides, has been widely used as an antimalarial for the treatment of multiple drug-resistant strains of plasmodium falciparum malaria. In this work, the binding interaction of AMT with bovine serum albumin (BSA) under the imitated physiological conditions (pH7.4) was investigated by UV spectroscopy, fluorescence emission spectroscopy, synchronous fluorescence spectroscopy, Fourier transform infrared spectroscopy (FT-IR), circular dichroism (CD), three-dimensional fluorescence spectroscopy and molecular docking methods. The experimental results indicated that there was a change in UV absorption of BSA along with a slight red shift of absorption wavelength, indicating that the interaction of AMT with BSA occurred. The intrinsic fluorescence of BSA was quenched by AMT due to the formation of AMT-BSA complex. The number of binding sites (n) and binding constant of AMT-BSA complex were about 1 and 2.63×10(3)M(-1) at 298K, respectively, suggesting that there was stronger binding interaction of AMT with BSA. Based on the analysis of the signs and magnitudes of the free energy change (ΔG(0)), enthalpic change (ΔH(0)) and entropic change (ΔS(0)) in the binding process, it can be concluded that the binding of AMT with BSA was enthalpy-driven process due to |ΔH°|>|TΔS°|. The results of experiment and molecular docking confirmed the main interaction forces between AMT and BSA were van der Waals force. And, there was a slight change in the BSA conformation after binding AMT but BSA still retains its secondary structure α-helicity. However, it had been confirmed that AMT binds on the interface between sub-domain IIA and IIB of BSA.

  10. Characterizing the binding interaction between antimalarial artemether (AMT) and bovine serum albumin (BSA): Spectroscopic and molecular docking methods.

    PubMed

    Shi, Jie-Hua; Pan, Dong-Qi; Wang, Xiou-Xiou; Liu, Ting-Ting; Jiang, Min; Wang, Qi

    2016-09-01

    Artemether (AMT), a peroxide sesquiterpenoides, has been widely used as an antimalarial for the treatment of multiple drug-resistant strains of plasmodium falciparum malaria. In this work, the binding interaction of AMT with bovine serum albumin (BSA) under the imitated physiological conditions (pH7.4) was investigated by UV spectroscopy, fluorescence emission spectroscopy, synchronous fluorescence spectroscopy, Fourier transform infrared spectroscopy (FT-IR), circular dichroism (CD), three-dimensional fluorescence spectroscopy and molecular docking methods. The experimental results indicated that there was a change in UV absorption of BSA along with a slight red shift of absorption wavelength, indicating that the interaction of AMT with BSA occurred. The intrinsic fluorescence of BSA was quenched by AMT due to the formation of AMT-BSA complex. The number of binding sites (n) and binding constant of AMT-BSA complex were about 1 and 2.63×10(3)M(-1) at 298K, respectively, suggesting that there was stronger binding interaction of AMT with BSA. Based on the analysis of the signs and magnitudes of the free energy change (ΔG(0)), enthalpic change (ΔH(0)) and entropic change (ΔS(0)) in the binding process, it can be concluded that the binding of AMT with BSA was enthalpy-driven process due to |ΔH°|>|TΔS°|. The results of experiment and molecular docking confirmed the main interaction forces between AMT and BSA were van der Waals force. And, there was a slight change in the BSA conformation after binding AMT but BSA still retains its secondary structure α-helicity. However, it had been confirmed that AMT binds on the interface between sub-domain IIA and IIB of BSA. PMID:27327124

  11. CdSe-ZnS quantum dots as temperature sensors during thermal coagulation of bovine serum albumin (BSA) solder

    NASA Astrophysics Data System (ADS)

    Jaschinski, Evelin; Wehner, Martin

    2012-06-01

    Laser tissue soldering (LTS) has variously interesting applications such as wound closure, anastomosis of blood vessels, and sealing corneal wounds. Since tissue properties such as optical absorption or thermal conductivity may differ, temperature control is essential to obtain full coagulation and to minimize thermal side effects. In this article, a non-invasive technique is proposed for temperature sensing by using CdSe-ZnS quantum dots (QDs) dissolved in protein solder, namely bovine serum albumin (BSA). The temperature measurement is conducted by monitoring the change in the photoluminescence spectra of the QDs. It is shown that the peak emission wavelength of about 653 nm of CdSe-ZnS QDs shifts linearly in a temperature range from 30 °C to 70 °C, with a coefficient of 0.153 nm °C-1 with increasing temperature. The wavelength shift can be determined by applying a small spectrometer with a CCD-array detector. The uncertainty associated with this method is estimated to be less than 6 °C in temperature. As the temperature increases, the measured signal strength initially remains constant and then falls off abruptly when exceeding 55 °C. The signal drop correlates with a phase change from a clear, low-scattering protein solution to strong-scattering solid material.

  12. Interaction of different polyphenols with bovine serum albumin (BSA) and human salivary alpha-amylase (HSA) by fluorescence quenching.

    PubMed

    Soares, Susana; Mateus, Nuno; Freitas, Victor de

    2007-08-01

    Phenolic compounds are responsible for major organoleptic characteristics of plant-derived food and beverages; these substances have received much attention, given that the major function of these compounds is their antioxidant ability. In the context of this study, our major aim was study the binding of several phenolic compounds such as (+)-catechin, (-)-epicatechin, (-)-epicatechin gallate, malvidin-3-glucoside, tannic acid, procyanidin B4, procyanidin B2 gallate, and procyanidin oligomers to different proteins (bovine serum albumin and human alpha-amylase) by fluorescence quenching of protein intrinsic fluorescence. From the spectra obtained, the Stern-Volmer, the apparent static, and the bimolecular quenching constants were calculated. The structure of polyphenols revealed to significantly affect the binding/quenching process; in general, the binding affinity increased with the molecular weight of polyphenol compounds and in the presence of galloyl groups. For catechin monomer and procyanidin dimer B4, the K(SV) was 14,100 and 13,800 M(-1), respectively, and for galloyl derivatives, the K(SV) was 19,500 and 21,900 M(-1), respectively. Tannic acid was shown to be the major quenching molecule for both proteins. However, comparing different proteins, the same polyphenol showed different quenching effects, which are suggested to be related to the three-dimensional structure of the proteins studied. For (+)-catechin and BSA, the K(SV) was 8700 M(-1), and with alpha-amylase, it was 14,100 M(-1); for tannic acid, the K(SV) was 10,0548 and 11,0674 M(-1), respectively. From the results obtained, besides the main binding analysis performed, we conclude that this technique is more sensitive than thought because we can detect several interactions that have not been proven by other methods, namely, nephelometry. Overall, fluorescence quenching has proven to be a very sensitive technique with many potentialities to analyze the interaction between polyphenols and proteins.

  13. Preparation and properties of BSA-loaded microspheres based on multi-(amino acid) copolymer for protein delivery.

    PubMed

    Chen, Xingtao; Lv, Guoyu; Zhang, Jue; Tang, Songchao; Yan, Yonggang; Wu, Zhaoying; Su, Jiacan; Wei, Jie

    2014-01-01

    A multi-(amino acid) copolymer (MAC) based on ω-aminocaproic acid, γ-aminobutyric acid, L-alanine, L-lysine, L-glutamate, and hydroxyproline was synthetized, and MAC microspheres encapsulating bovine serum albumin (BSA) were prepared by a double-emulsion solvent extraction method. The experimental results show that various preparation parameters including surfactant ratio of Tween 80 to Span 80, surfactant concentration, benzyl alcohol in the external water phase, and polymer concentration had obvious effects on the particle size, morphology, and encapsulation efficiency of the BSA-loaded microspheres. The sizes of BSA-loaded microspheres ranged from 60.2 μm to 79.7 μm, showing different degrees of porous structure. The encapsulation efficiency of BSA-loaded microspheres also ranged from 38.8% to 50.8%. BSA release from microspheres showed the classic biphasic profile, which was governed by diffusion and polymer erosion. The initial burst release of BSA from microspheres at the first week followed by constant slow release for the next 7 weeks were observed. BSA-loaded microspheres could degrade gradually in phosphate buffered saline buffer with pH value maintained at around 7.1 during 8 weeks incubation, suggesting that microsphere degradation did not cause a dramatic pH drop in phosphate buffered saline buffer because no acidic degradation products were released from the microspheres. Therefore, the MAC microspheres might have great potential as carriers for protein delivery.

  14. Interaction of bovine serum albumin protein with self assembled monolayer of mercaptoundecanoic acid

    NASA Astrophysics Data System (ADS)

    Poonia, Monika; Agarwal, Hitesh; Manjuladevi, V.; Gupta, R. K.

    2016-05-01

    Detection of proteins and other biomolecules in liquid phase is the essence for the design of a biosensor. The sensitivity of a sensor can be enhanced by the appropriate functionalization of the sensing area so as to establish the molecular specific interaction. In the present work, we have studied the interaction of bovine serum albumin (BSA) protein with a chemically functionalized surface using a quartz crystal microbalance (QCM). The gold-coated quartz crystals (AT-cut/5 MHz) were functionalized by forming self-assembled monolayer (SAM) of 11-Mercaptoundecanoic acid (MUA). The adsorption characteristics of BSA onto SAM of MUA on quartz crystal are reported. BSA showed the highest affinity for SAM of MUA as compared to pure gold surface. The SAM of MUA provides carboxylated surface which enhances not only the adsorption of the BSA protein but also a very stable BSA-MUA complex in the liquid phase.

  15. Binding of estrogen receptor with estrogen conjugated to bovine serum albumin (BSA).

    PubMed

    Taguchi, Yasuto; Koslowski, Mirek; Bodenner, Donald L

    2004-08-19

    BACKGROUND: The classic model of estrogen action requires that the estrogen receptor (ER) activates gene expression by binding directly or indirectly to DNA. Recent studies, however, strongly suggest that ER can act through nongenomic signal transduction pathways and may be mediated by a membrane bound form of the ER. Estradiol covalently linked to membrane impermeable BSA (E2-BSA) has been widely used as an agent to study these novel membrane-associated ER events. However, a recent report suggests that E2-BSA does not compete for E2 binding to purified ER in vitro. To resolve this apparent discrepancy, we performed competition studies examining the binding of E2 and E2-BSA to both purified ER preparations and ER within intact cells. To eliminate potential artifacts due to contamination of commercially available E2-BSA preparations with unconjugated E2 (usually between 3-5%), the latter was carefully removed by ultrafiltration. RESULTS: As previously reported, a 10-to 1000-fold molar excess of E2-BSA was unable to compete with 3H-E2 binding to ER when added simultaneously. However, when ER was pre-incubated with the same concentrations of E2-BSA, the binding of 3H-E2 was significantly reduced. E2-BSA binding to a putative membrane-associated ER was directly visualized using fluorescein labeled E2-BSA (E2-BSA-FITC). Staining was restricted to the cell membrane when E2-BSA-FITC was incubated with stable transfectants of the murine ERalpha within ER-negative HeLa cells and with MC7 cells that endogenously produce ERalpha. This staining appeared highly specific since it was competed by pre-incubation with E2 in a dose dependent manner and with the competitor ICI-182,780. CONCLUSIONS: These results demonstrate that E2-BSA does bind to purified ER in vitro and to ER in intact cells. It seems likely that the size and structure of E2-BSA requires more energy for it to bind to the ER and consequently binds more slowly than E2. More importantly, these findings demonstrate

  16. Binding of estrogen receptor with estrogen conjugated to bovine serum albumin (BSA)

    PubMed Central

    Taguchi, Yasuto; Koslowski, Mirek; Bodenner, Donald L

    2004-01-01

    Background The classic model of estrogen action requires that the estrogen receptor (ER) activates gene expression by binding directly or indirectly to DNA. Recent studies, however, strongly suggest that ER can act through nongenomic signal transduction pathways and may be mediated by a membrane bound form of the ER. Estradiol covalently linked to membrane impermeable BSA (E2-BSA) has been widely used as an agent to study these novel membrane-associated ER events. However, a recent report suggests that E2-BSA does not compete for E2 binding to purified ER in vitro. To resolve this apparent discrepancy, we performed competition studies examining the binding of E2 and E2-BSA to both purified ER preparations and ER within intact cells. To eliminate potential artifacts due to contamination of commercially available E2-BSA preparations with unconjugated E2 (usually between 3–5%), the latter was carefully removed by ultrafiltration. Results As previously reported, a 10-to 1000-fold molar excess of E2-BSA was unable to compete with 3H-E2 binding to ER when added simultaneously. However, when ER was pre-incubated with the same concentrations of E2-BSA, the binding of 3H-E2 was significantly reduced. E2-BSA binding to a putative membrane-associated ER was directly visualized using fluorescein labeled E2-BSA (E2-BSA-FITC). Staining was restricted to the cell membrane when E2-BSA-FITC was incubated with stable transfectants of the murine ERα within ER-negative HeLa cells and with MC7 cells that endogenously produce ERα. This staining appeared highly specific since it was competed by pre-incubation with E2 in a dose dependent manner and with the competitor ICI-182,780. Conclusions These results demonstrate that E2-BSA does bind to purified ER in vitro and to ER in intact cells. It seems likely that the size and structure of E2-BSA requires more energy for it to bind to the ER and consequently binds more slowly than E2. More importantly, these findings demonstrate that in

  17. Buffers more than buffering agent: introducing a new class of stabilizers for the protein BSA.

    PubMed

    Gupta, Bhupender S; Taha, Mohamed; Lee, Ming-Jer

    2015-01-14

    In this study, we have analyzed the influence of four biological buffers on the thermal stability of bovine serum albumin (BSA) using dynamic light scattering (DLS). The investigated buffers include 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES), 4-(2-hydroxyethyl)-1-piperazine-propanesulfonic acid (EPPS), 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid sodium salt (HEPES-Na), and 4-morpholinepropanesulfonic acid sodium salt (MOPS-Na). These buffers behave as a potential stabilizer for the native structure of BSA against thermal denaturation. The stabilization tendency follows the order of MOPS-Na > HEPES-Na > HEPES ≫ EPPS. To obtain an insight into the role of hydration layers and peptide backbone in the stabilization of BSA by these buffers, we have also explored the phase transition of a thermoresponsive polymer, poly(N-isopropylacrylamide (PNIPAM)), a model compound for protein, in aqueous solutions of HEPES, EPPS, HEPES-Na, and MOPS-Na buffers at different concentrations. It was found that the lower critical solution temperatures (LCST) of PNIPAM in the aqueous buffer solutions substantially decrease with increase in buffer concentration. The mechanism of interactions between these buffers and protein BSA was probed by various techniques, including UV-visible, fluorescence, and FTIR. The results of this series of studies reveal that the interactions are mainly governed by the influence of the buffers on the hydration layers surrounding the protein. We have also explored the possible binding sites of BSA with these buffers using a molecular docking technique. Moreover, the activities of an industrially important enzyme α-chymotrypsin (α-CT) in 0.05 M, 0.5 M, and 1.0 M of HEPES, EPPS, HEPES-Na, and MOPS-Na buffer solutions were analyzed at pH = 8.0 and T = 25 °C. Interestingly, the activities of α-CT were found to be enhanced in the aqueous solutions of these investigated buffers. Based upon the Jones-Dole viscosity parameters, the

  18. One-step electrochemically co-assembled redox-active [Ru(bpy)2(tatp)]2+-BSA-SWCNTs hybrid film for non-redox protein biosensors.

    PubMed

    Ji, Shi-Bo; Yan, Zhi-Hong; Wu, Jun-Wen; Chen, Lin-Lin; Li, Hong

    2013-01-15

    A redox-active [Ru(bpy)(2)(tatp)](2+)-BSA-SWCNTs (bpy=2,2'-bipyridine, tatp=1,4,8,9-tetra-aza-triphenylene, BSA=bovine serum albumin, SWCNTs=single-walled carbon nanotubes) hybrid film is fabricated on an indium-tin oxide (ITO) electrode via one-step electrochemical co-assembly approach. BSA is inherently dispersive and therefore served as the linking mediator of SWCNTs, which facilitate the redox reactions of [Ru(bpy)(2)(tatp)](2+) employed as a reporter of BSA. The evidences from differential pulse voltammetry, cyclic voltammetry, scanning electron microscope, emission spectroscopy and fluorescence microscope reveal that the [Ru(bpy)(2)(tatp)](2+)-BSA-SWCNTs hybrid can be electrochemically co-assembled on the ITO electrode, showing two pairs of well-defined Ru(II)-based redox waves. Furthermore, the electrochemical co-assembly of the [Ru(bpy)(2)(tatp)](2+)-BSA-SWCNTs hybrid is found to be strongly dependent on the simultaneous presence of BSA and SWCNTs, indicating a good linear response to BSA in the range from 6 to 50mgL(-1). The results from this study provide an electrochemical co-assembly method for the development of non-redox protein biosensors.

  19. One-step electrochemically co-assembled redox-active [Ru(bpy)2(tatp)]2+-BSA-SWCNTs hybrid film for non-redox protein biosensors.

    PubMed

    Ji, Shi-Bo; Yan, Zhi-Hong; Wu, Jun-Wen; Chen, Lin-Lin; Li, Hong

    2013-01-15

    A redox-active [Ru(bpy)(2)(tatp)](2+)-BSA-SWCNTs (bpy=2,2'-bipyridine, tatp=1,4,8,9-tetra-aza-triphenylene, BSA=bovine serum albumin, SWCNTs=single-walled carbon nanotubes) hybrid film is fabricated on an indium-tin oxide (ITO) electrode via one-step electrochemical co-assembly approach. BSA is inherently dispersive and therefore served as the linking mediator of SWCNTs, which facilitate the redox reactions of [Ru(bpy)(2)(tatp)](2+) employed as a reporter of BSA. The evidences from differential pulse voltammetry, cyclic voltammetry, scanning electron microscope, emission spectroscopy and fluorescence microscope reveal that the [Ru(bpy)(2)(tatp)](2+)-BSA-SWCNTs hybrid can be electrochemically co-assembled on the ITO electrode, showing two pairs of well-defined Ru(II)-based redox waves. Furthermore, the electrochemical co-assembly of the [Ru(bpy)(2)(tatp)](2+)-BSA-SWCNTs hybrid is found to be strongly dependent on the simultaneous presence of BSA and SWCNTs, indicating a good linear response to BSA in the range from 6 to 50mgL(-1). The results from this study provide an electrochemical co-assembly method for the development of non-redox protein biosensors. PMID:22824544

  20. Interaction of bovine serum albumin (BSA) with novel gemini surfactants studied by synchrotron radiation scattering (SR-SAXS), circular dichroism (CD), and nuclear magnetic resonance (NMR).

    PubMed

    Gospodarczyk, W; Szutkowski, K; Kozak, M

    2014-07-24

    The interaction of three dicationic (gemini) surfactants-3,3'-[1,6-(2,5-dioxahexane)]bis(1-dodecylimidazolium) chloride (oxyC2), 3,3'-[1,16-(2,15-dioxahexadecane)]bis(1-dodecylimidazolium) chloride (oxyC12), and 1,4-bis(butane)imidazole-1-yl-3-dodecylimidazolium chloride (C4)--with bovine serum albumin (BSA) has been studied by the use of small-angle X-ray scattering (SAXS), circular dichroism (CD), and (1)H nuclear magnetic resonance diffusometry. The results of CD studies show that the conformation of BSA was changed dramatically in the presence of all studied surfactants. The greater decrease (from 56 to 24%) in the α-helical structure of BSA was observed for oxyC2 surfactant. The radii of gyration estimated from SAXS data varied between 3 and 26 nm for the BSA/oxyC2 and BSA/oxyC12 systems. The hydrodynamic radius of the BSA/surfactant system estimated from NMR diffusometry varies between 5 and 11 nm for BSA/oxyC2 and 5 and 8 nm for BSA/oxyC12.

  1. Total Protein and Albumin/Globulin Ratio Test

    MedlinePlus

    ... limited. Home Visit Global Sites Search Help? Total Protein and Albumin/Globulin (A/G) Ratio Share this ... Globulin Ratio; A/G Ratio Formal name: Total Protein; Albumin to Globulin Ratio Related tests: Albumin ; Liver ...

  2. Effect of protein thermo aggregation on the binding of BSA to gelatin type A.

    PubMed

    Antonov, Y A; Zhuravleva, I L

    2013-02-01

    We study the effect of limited heat-induced aggregation of BSA on structure development in the water-gelatin-thermally aggregated BSA (BSA(TA)) system. The pH is set at 5.4 and the temperature is higher than the conformation transition temperature of gelatin, but lower than the denaturation temperature of BSA. Dynamic light scattering, circular dichroism, and fluorescence measurements are used to monitor structure changes. Interaction of gelatin with BSA(TA) leads to formation of large complex particles with an average radius ∼1500 nm. BSA-gelatin complex formation accompanies partial destabilization of the secondary and tertiary structures of BSA and an additional exposure of hydrophobic tryptophan residues on the surface of the globule. It is shown that electrostatic interaction of the oppositely charged groups of BSA(TA) and gelatin is responsible for formation of such complex particles, whereas the secondary forces (hydrophobic interaction and hydrogen bonds) play an important role in stabilization of the complex particles. The zeta potentials of the native and the thermally aggregated BSA samples were determined, and the solvent quality has been quantified by determining the activity of the protein samples in their saturated solutions. It was shown that steric reasons (large size of the thermally aggregated BSA(TA) particles), and uncomplete charge compensation of the positively charged gelatin molecules by the negatively charged BSA(TA) particles are the main factors in determining structure formation, while the levels of the activity of the native BSA and BSA(TA) have a smaller effect on the structure of complex.

  3. Antimicrobial and cell viability measurement of bovine serum albumin capped silver nanoparticles (Ag/BSA) loaded collagen immobilized poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) film.

    PubMed

    Bakare, Rotimi; Hawthrone, Samantha; Vails, Carmen; Gugssa, Ayele; Karim, Alamgir; Stubbs, John; Raghavan, Dharmaraj

    2016-03-01

    Bacterial infection of orthopedic devices has been a major concern in joint replacement procedures. Therefore, this study is aimed at formulating collagen immobilized poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) film loaded with bovine serum albumin capped silver nanoparticles (Ag/BSA NPs) to inhibit bacterial growth while retaining/promoting osteoblast cells viability. The nanoparticles loaded collagen immobilized PHBV film was characterized for its composition by X-ray Photoelectron Spectroscopy and Anodic Stripping Voltammetry. The extent of loading of Ag/BSA NPs on collagen immobilized PHBV film was found to depend on the chemistry of the functionalized PHBV film and the concentration of Ag/BSA NPs solution used for loading nanoparticles. Our results showed that more Ag/BSA NPs were loaded on higher molecular weight collagen immobilized PHEMA-g-PHBV film. Maximum loading of Ag/BSA NPs on collagen immobilized PHBV film was observed when 16ppm solution was used for adsorption studies. Colony forming unit and optical density measurements showed broad antimicrobial activity towards Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa at significantly lower concentration i.e., 0.19 and 0.31μg/disc, compared to gentamicin and sulfamethoxazole trimethoprim while MTT assay showed that released nanoparticles from Ag/BSA NPs loaded collagen immobilized PHBV film has no impact on MCTC3-E1 cells viability.

  4. Trehalose limits BSA aggregation in spray-dried formulations at high temperatures: implications in preparing polymer implants for long-term protein delivery.

    PubMed

    Rajagopal, Karthikan; Wood, Joseph; Tran, Benjamin; Patapoff, Thomas W; Nivaggioli, Thierry

    2013-08-01

    Polymer implants are promising systems for sustained release applications but their utility for protein delivery has been hindered because of concerns over drug stability at elevated temperatures required for processing. Using bovine serum albumin (BSA) as a model, we have assessed whether proteins can be formulated for processing at elevated temperatures. Specifically, the effect of trehalose and histidine-HCl buffer on BSA stability in a spray-dried formulation has been investigated at temperatures ranging from 80°C to 110°C. When both the sugar and buffer are present, aggregation is suppressed even when exposed to 100°C, the extrusion temperature of poly(lactide-co-glycolide) (PLGA), a bioresorbable polymer. Estimation of aggregation rate constants (k) indicate that though both trehalose and histidine-HCl buffer contribute to BSA stability, the effect because of trehalose alone is more pronounced. BSA-loaded PLGA implants were prepared using hot-melt extrusion process and in vitro release was conducted in phosphate buffered saline at 37°C. Comparison of drug released from implants prepared using four different formulations confirmed that maximal release was achieved from the formulation in which BSA was least aggregated. These studies demonstrate that when trehalose and histidine-HCl buffer are included in spray-dried formulations, BSA stability is maintained both during processing at 100°C and long-term residence within implants.

  5. Cationic spin probe reporting on thermal denaturation and complexation-decomplexation of BSA with SDS. Potential applications in protein purification processes.

    PubMed

    Matei, Iulia; Ariciu, Ana Maria; Neacsu, Maria Victoria; Collauto, Alberto; Salifoglou, Athanasios; Ionita, Gabriela

    2014-09-25

    In this work, we present evidence on the suitability of spin probes to report on the thermal treatment of bovine serum albumin (BSA), in the temperature range 293-343 K, and indirectly monitor the release of sodium dodecyl sulfate (SDS) from its complex with BSA using a covalent gel with β-cyclodextrin (β-CD) in the network. The spin probes used, 5- and 7-doxyl-stearic acids (5-DSA, 7-DSA) or 4-(N,N'-dimethyl-N-hexadecyl)ammonium-2,2',6,6'-tetramethylpiperidine-1-oxyl iodide (CAT16), present similar, fatty acid-like structural features. Their continuous wave electron paramagnetic resonance (CW-EPR) spectra, however, reflect different dynamics when complexed with BSA: a restricted motion for 5-DSA, almost nonsensitive to the heating/cooling cycle, and a faster temperature-dependent dynamic motion for CAT16. Molecular docking allows us to rationalize these results by revealing the different binding modes of 5-DSA and CAT16. The EPR data on the temperature effect on BSA are supported by circular dichroism results projecting recovery, upon cooling, of the initial binding ability of BSA for samples heated to 323 K. The interactions occurring in BSA/SDS/β-CD systems are investigated by CW-EPR and FT-ESEEM spectroscopies. It is found that the covalent gel containing β-CD can efficiently remove SDS from the BSA/SDS complex. The gel is not permeable to BSA but it can encapsulate SDS, thus yielding the free protein in solution and allowing recovery of the native protein conformation. Collectively, the accrued knowledge supports potential applications in protein purification biotechnological processes.

  6. Molecular interactions between some non-steroidal anti-inflammatory drugs (NSAID's) and bovine (BSA) or human (HSA) serum albumin estimated by means of isothermal titration calorimetry (ITC) and frontal analysis capillary electrophoresis (FA/CE).

    PubMed

    Ràfols, Clara; Zarza, Sílvia; Bosch, Elisabeth

    2014-12-01

    The interactions between some non-steroidal anti-inflammatory drugs, NSAIDs, (naproxen, ibuprofen and flurbiprofen) and bovine (BSA) or human (HSA) serum albumin have been examined by means of two complementary techniques, isothermal titration calorimetry (ITC) and frontal analysis/capillary electrophoresis (FA/CE). It can be concluded that ITC is able to measure with high precision the strongest drug-albumin interactions but the higher order interactions can be better determined by means of FA/CE. Then, the combination of both techniques leads to a complete evaluation of the binding profiles between the selected NSAIDs and both kind of albumin proteins. When BSA is the binding protein, the NSAIDs show a strong primary interaction (binding constants: 1.5 × 10(7), 8 × 10(5) and 2 × 10(6) M(-1) for naproxen, ibuprofen and flurbiprofen, respectively), and also lower affinity interactions of the same order for the three anti-inflammatories (about 1.7 × 10(4) M(-1)). By contrast, when HSA is the binding protein two consecutive interactions can be observed by ITC for naproxen (9 × 10(5) and 7 × 10(4) M(-1)) and flurbiprofen (5 × 10(6) and 6 × 10(4) M(-1)) whereas only one is shown for ibuprofen (9 × 10(5) M(-1)). Measurements by FA/CE show a single interaction for each drug being the ones of naproxen and flurbiprofen the same that those evaluated by ITC as the second interaction events. Then, the ability of both techniques as suitable complementary tools to establish the whole interaction NSAIDs-albumin profile is experimentally demonstrated and allows foreseeing suitable strategies to establish the complete drug-protein binding profile. In addition, for the interactions analyzed by means of ITC, the thermodynamic signature is established and the relative contributions of the enthalpic and entropic terms discussed.

  7. Photo selective protein immobilization using bovine serum albumin

    NASA Astrophysics Data System (ADS)

    Kim, Wan-Joong; Kim, Ansoon; Huh, Chul; Park, Chan Woo; Ah, Chil Seong; Kim, Bong Kyu; Yang, Jong-Heon; Chung, Kwang Hyo; Choi, Yo Han; Hong, Jongcheol; Sung, Gun Yong

    2012-11-01

    A simple and selective technique which immobilizes protein onto a solid substrate by using UV illumination has been developed. In protein immobilization, a Bovine serum albumin (BSA) performed bifunctional role as a cross-linker between substrate and proteins and as a blocker inhibiting a nonspecific protein adsorption. A new photo-induced protein immobilization process has been investigated at each step by fluorescence microscopy, ellipsometry, and Fourier transform infrared (FT-IR) spectroscopy. A UV photomask has been used to induce selective protein immobilization on target regions of the surface of the SiO2 substrates under UV illumination with negligible nonspecific binding. The UV illumination also showed improved photostability than the conventional methods which employed bifunctional photo-crosslinker molecules of photo-reactive diazirine. This new UV illumination-based photo-addressable protein immobilization provides a new approach for developing novel protein microarrays for multiplexed sensing as well as other types of bio-immobilization in biomedical devices and biotechnologies.

  8. Intercalation of bovine serum albumin coated gold clusters between phospholipid bilayers: temperature-dependent behavior of lipid-AuQC@BSA assemblies with red emission and superlattice structure.

    PubMed

    Söptei, Balázs; Mihály, Judith; Visy, Júlia; Wacha, András; Bóta, Attila

    2014-04-10

    A method has been developed to encapsulate bovine serum albumin (BSA)-coated gold quantum clusters (AuQC@BSA) in a multilamellar system of dipalmitoylphosphatidylcholine (DPPC). Results have shown that intercalation of AuQC@BSA particles into lipid bilayers occurs in the presence of CaCl2. Intense red photoluminescence emission was observed after encapsulation of the clusters. A well-defined structure was found with periodic distances drastically larger than that in the pure DPPC/water system. Although Ca(2+) ions can change the dipole characteristics of the lipid bilayer surface, leading to unbinding between the bilayers of multilamellar DPPC/water system, the repulsion is shielded in the presence of AuQC@BSA particles. A coherent superlattice structure evolves due to mixed Ca(2+)-DPPC and Ca(2+)-AuQC@BSA interactions. Studies at different temperatures have suggested a correlation between the luminescence properties of the clusters and phase transition of the lipid layers. The temperature-dependent behavior assumes the connection between the coating and the lipid bilayer surface. Temperature-dependent features of lipid intercalated Au clusters provide new opportunities in their application.

  9. Probing deep into the interaction of a fluorescent chalcone derivative and bovine serum albumin (BSA): an experimental and computational study.

    PubMed

    Alvim, Haline G O; Fagg, Emma L; de Oliveira, Aline L; de Oliveira, Heibbe C B; Freitas, Sonia M; Xavier, Mary-Ann E; Soares, Thereza A; Gomes, Alexandre F; Gozzo, Fabio C; Silva, Wender A; Neto, Brenno A D

    2013-08-01

    In the present manuscript, a novel fluorescent chalcone derivative is synthesized and its photophysical properties are fully characterized. The designed fluorophore is applied as a probe to study protein-dye interactions with bovine serum albumin. Circular dichroism gave interesting results on the thermodynamics of the interaction. NMR spectroscopy, especially relaxation measurements, revealed the atoms in the chalcone derivative that interacts with the protein upon binding. Molecular docking calculations indicate that the most favourable binding sites are near the two tryptophan residues. Furthermore, ab initio and DFT calculations offer insights into the reactivity and physicochemical properties of this novel fluorophore. PMID:23680860

  10. Intestinal transmission of macromolecules (BSA and FITC-dextran) in the neonatal pig: enhancing effect of colostrum, proteins and proteinase inhibitors.

    PubMed

    Weström, B R; Ohlsson, B G; Svendsen, J; Tagesson, C; Karlsson, B W

    1985-01-01

    The effects of colostrum and constituents/factors in colostrum which may influence intestinal macromolecular transmission in the newborn preclosure pig were investigated. Unsuckled piglets were given, by use of a stomach tube, bovine serum albumin (BSA) and fluorescein-isothiocyanate (FITC)-labelled dextran 70,000 (FITC-D) as markers together with colostrum or the factors under study. The serum levels of BSA and FITC-D 4 h after feeding were then determined as a measure of the transfer. It was found that the two colostrums tested, bovine and especially porcine, markedly enhanced the transmission of both BSA and FITC-D. Furthermore, increasing amounts of the model proteins, BSA and bovine IgG (50-200 mg/ml), significantly increased the transfer of FITC-D, whereas unlabelled dextran 70,000 given in similar amounts did not. Proteinase inhibitors obtained from sow colostrum or soy bean also enhanced the transmission of both BSA and FITC-D while the inactive inhibitors, given as trypsin-inhibitor complexes, had no effect. On the other hand, addition of a proteinase, porcine trypsin, significantly decreased the transmission of FITC-D. These findings indicate that the intestinal transmission of macromolecules in the preclosure piglet is governed by the amount of protein available in the intestine. Therefore, feeding colostrum with a high protein content and proteinase inhibitors is likely to favour efficient intestinal transmission, although other colostrum factors may also be of importance.

  11. Osmotically unresponsive water fraction on proteins: non-ideal osmotic pressure of bovine serum albumin as a function of pH and salt concentration.

    PubMed

    Fullerton, Gary D; Kanal, Kalpana M; Cameron, Ivan L

    2006-01-01

    How much does protein-associated water differ in colligative properties (freezing point, boiling point, vapor pressure and osmotic behavior) from pure bulk water? This question was approached by studying the globular protein bovine serum albumin (BSA), using changes in pH and salt concentration to alter its native structural conformation and state of aggregation. BSA osmotic pressure was investigated experimentally and analyzed using the molecular model of Fullerton et al. [Biochem Cell Biol 1992;70(12):1325]. Analysis yielded both the extent of osmotically unresponsive water (OUW) and the effective molecular weight values of the membrane-impermeable BSA solute. Manipulation of BSA conformation and aggregation by membrane-penetrating cosolutes show that alterations in pH and salt concentration change the amount of bulk water that escapes into BSA from a minimum of 1.4 to a maximum of 11.7 g water per g dry mass BSA.

  12. Glutaraldehyde mediated conjugation of amino-coated magnetic nanoparticles with albumin protein for nanothermotherapy.

    PubMed

    Zhao, Lingyun; Yang, Bing; Dai, Xiaochen; Wang, Xiaowen; Gao, Fuping; Zhang, Xiaodong; Tang, Jintian

    2010-11-01

    A novel bioconjugation of amino saline capped Fe3O4 magnetic nanoparticles (MNPs) with bovine serum albumin (BSA) was developed by applying glutaraldehyde as activator. Briefly, Fe3O4 MNs were synthesized by the chemical co-precipitation method. Surface modification of the prepared MNPs was performed by employing amino saline as the coating agent. Glutaraldehyde was further applied as an activation agent through which BSA was conjugated to the amino-coated MNPs. The structure of the BSA-MNs was confirmed by FTIR analysis. Physico-chemical characterizations of the BSA-MNPs, such as surface morphology, surface charge and magnetic properties were investigated by Transmission Electron Microscopy (TEM), zeta-Potential and Vibrating Sample Magnetometer (VSM), etc. Magnetic inductive heating characteristics of the BSA-MNPs were analyzed by exposing the MNPs suspension (magnetic fluid) under alternative magnetic field (AMF). The results demonstrate that BSA was successfully conjugated with amino-coated MNs mediated through glutaraldehyde activation. The nanoparticles were spherical shaped with approximately 10 nm diameter. Possessing ideal magnetic inductive heating characteristics, which can generate very rapid and efficient heating while upon AMF exposure, BSA-MNPs can be applied as a novel candidature for magnetic nanothermotherapy for cancer treatment. In vitro cytotoxicity study on the human hepatocellular liver carcinoma cells (HepG-2) indicates that BSA-MNP is an efficient agent for cancer nanothermotherapy with satisfied biocompatibility, as rare cytotoxicity was observed in the absence of AMF. Moreover, our investigation provides a methodology for fabrication protein conjugated MNPs, for instance monoclonal antibody conjugated MNPs for targeting cancer nanothermotherapy. PMID:21137877

  13. Irradiation as an alternative route for protein crosslinking: Cosolvent free BSA nanoparticles

    NASA Astrophysics Data System (ADS)

    Varca, Gustavo H. C.; Queiroz, Rodrigo G.; Lugão, Ademar B.

    2016-07-01

    Recent studies reported the development of protein-based nanoparticles by the use of ɣ-irradiation for the production of advanced drug carriers and biomaterials at nanolevel. Basically, the technique combines protein aggregation by means of protein desolvation using a cosolvent, followed by crosslinking using irradiation. We hereby report the effect irradiation dose over the development of protein-based nanoparticles combined or not with cosolvents. BSA was used as a model protein and the samples were irradiated in phosphate buffer (pH=7.2) using a gammacell in absence or presence of ethanol or methanol at 30% and 40% (v/v) respectively. The irradiation dose effect was evaluated following the exposition of BSA to 2.5, 5, 7.5 and 10 kGy over particle size and protein crosslinking, as determined by photon correlation microscopy and fluorescence measurements. Optimized effects were achieved at 10 kGy, under the assayed dose range, with regard to higher particle size and protein crosslinking levels. The use of irradiation was suitable for the synthesis of BSA nanoparticles and tuning of particle size was achieved by controlling the absorbed dose. While the use of ethanol provided an additional control over BSA particle size if compared to the use of methanol at the concentrations assayed, the possibility to perform BSA crosslinking in absence of cosolvents unraveled a novel one-step procedure for the synthesis of protein nanoparticles with no toxicity generated by the use of cosolvents or monomers.

  14. (PCG) Protein Crystal Growth Human Serum Albumin

    NASA Technical Reports Server (NTRS)

    1989-01-01

    (PCG) Protein Crystal Growth Human Serum Albumin. Contributes to many transport and regulatory processes and has multifunctional binding properties which range from various metals, to fatty acids, hormones, and a wide spectrum of therapeutic drugs. The most abundant protein of the circulatory system. It binds and transports an incredible variety of biological and pharmaceutical ligands throughout the blood stream. Principal Investigator on STS-26 was Larry DeLucas.

  15. (PCG) Protein Crystal Growth Horse Serum Albumin

    NASA Technical Reports Server (NTRS)

    1995-01-01

    Horse Serum Albumin crystals grown during the USML-1 (STS-50) mission's Protein Crystal Growth Glovebox Experiment. These crystals were grown using a vapor diffusion technique at 22 degrees C. The crystals were allowed to grow for nine days while in orbit. Crystals of 1.0 mm in length were produced. The most abundant blood serum protein, regulates blood pressure and transports ions, metabolites, and therapeutic drugs. Principal Investigator was Edward Meehan.

  16. Investigation of Bovine Serum Albumin (BSA) Attachment onto Self-Assembled Monolayers (SAMs) Using Combinatorial Quartz Crystal Microbalance with Dissipation (QCM-D) and Spectroscopic Ellipsometry (SE)

    PubMed Central

    Phan, Hanh T. M.; Bartelt-Hunt, Shannon; Rodenhausen, Keith B.; Schubert, Mathias; Bartz, Jason C.

    2015-01-01

    Understanding protein adsorption kinetics to surfaces is of importance for various environmental and biomedical applications. Adsorption of bovine serum albumin to various self-assembled monolayer surfaces including neutral and charged hydrophilic and hydrophobic surfaces was investigated using in-situ combinatorial quartz crystal microbalance with dissipation and spectroscopic ellipsometry. Adsorption of bovine serum albumin varied as a function of surface properties, bovine serum albumin concentration and pH value. Charged surfaces exhibited a greater quantity of bovine serum albumin adsorption, a larger bovine serum albumin layer thickness, and increased density of bovine serum albumin protein compared to neutral surfaces at neutral pH value. The quantity of adsorbed bovine serum albumin protein increased with increasing bovine serum albumin concentration. After equilibrium sorption was reached at pH 7.0, desorption of bovine serum albumin occurred when pH was lowered to 2.0, which is below the isoelectric point of bovine serum albumin. Our data provide further evidence that combinatorial quartz crystal microbalance with dissipation and spectroscopic ellipsometry is a sensitive analytical tool to evaluate attachment and detachment of adsorbed proteins in systems with environmental implications. PMID:26505481

  17. Rapid detection of Cu(2+) by a paper-based microfluidic device coated with bovine serum albumin (BSA)-Au nanoclusters.

    PubMed

    Fang, Xueen; Zhao, Qianqian; Cao, Hongmei; Liu, Juan; Guan, Ming; Kong, Jilie

    2015-11-21

    In this work, bovine serum albumin (BSA)-Au nanoclusters were used to coat a paper-based microfluidic device. This device acted as a Cu(2+) biosensor that showed fluorescence quenching on detection of copper ions. The detection limit of this sensor could be adjusted by altering the water absorbing capacity of the device. Qualitative and semi-quantitative results could be obtained visually without the aid of any advanced instruments. This sensor could test Cu(2+) rapidly with high specificity and sensitivity, which would be useful for point-of-care testing (POCT). PMID:26462444

  18. Albumin-coated SPIONs: an experimental and theoretical evaluation of protein conformation, binding affinity and competition with serum proteins

    NASA Astrophysics Data System (ADS)

    Yu, Siming; Perálvarez-Marín, Alex; Minelli, Caterina; Faraudo, Jordi; Roig, Anna; Laromaine, Anna

    2016-07-01

    The variety of nanoparticles (NPs) used in biological applications is increasing and the study of their interaction with biological media is becoming more important. Proteins are commonly the first biomolecules that NPs encounter when they interact with biological systems either in vitro or in vivo. Among NPs, super-paramagnetic iron oxide nanoparticles (SPIONs) show great promise for medicine. In this work, we study in detail the formation, composition, and structure of a monolayer of bovine serum albumin (BSA) on SPIONs. We determine, both by molecular simulations and experimentally, that ten molecules of BSA form a monolayer around the outside of the SPIONs and their binding strength to the SPIONs is about 3.5 × 10-4 M, ten times higher than the adsorption of fetal bovine serum (FBS) on the same SPIONs. We elucidate a strong electrostatic interaction between BSA and the SPIONs, although the secondary structure of the protein is not affected. We present data that supports the strong binding of the BSA monolayer on SPIONs and the properties of the BSA layer as a protein-resistant coating. We believe that a complete understanding of the behavior and morphology of BSA-SPIONs and how the protein interacts with SPIONs is crucial for improving NP surface design and expanding the potential applications of SPIONs in nanomedicine.The variety of nanoparticles (NPs) used in biological applications is increasing and the study of their interaction with biological media is becoming more important. Proteins are commonly the first biomolecules that NPs encounter when they interact with biological systems either in vitro or in vivo. Among NPs, super-paramagnetic iron oxide nanoparticles (SPIONs) show great promise for medicine. In this work, we study in detail the formation, composition, and structure of a monolayer of bovine serum albumin (BSA) on SPIONs. We determine, both by molecular simulations and experimentally, that ten molecules of BSA form a monolayer around the

  19. Spectroscopic analyses on interaction of Amantadine-Salicylaldehyde, Amantadine-5-Chloro-Salicylaldehyde and Amantadine-o-Vanillin Schiff-Bases with bovine serum albumin (BSA)

    NASA Astrophysics Data System (ADS)

    Wang, Zhiqiu; Gao, Jingqun; Wang, Jun; Jin, Xudong; Zou, Mingming; Li, Kai; Kang, Pingli

    2011-12-01

    In this work, three Tricyclo [3.3.1.1(3,7)] decane-1-amine (Amantadine) Schiff-Bases, Amantadine-Salicylaldehyde (AS), Amantadine-5-Chloro-Salicylaldehyde (AS-5-C) and Amantadine-o-Vanillin (AS-o-V), were synthesized by direct heating reflux method in ethanol solution and characterized by infrared spectrum and elementary analysis. Fluorescence quenching was used to study the interaction of these Amantadine Schiff-Bases (AS, AS-5-C and AS-o-V) with bovine serum albumin (BSA). According to fluorescence quenching calculations the bimolecular quenching constant ( Kq), apparent quenching constant ( KSV), effective binding constant ( KA) and corresponding dissociation constant ( KD), binding site number ( n) and binding distance ( r) were obtained. The results show that these Amantadine Schiff-Bases can obviously bind to BSA molecules and the binding strength order is AS < AS-5-C = AS-o-V. Synchronous fluorescence spectroscopy reveals that these Amantadine Schiff-Bases adopt different way to bind with BSA molecules. That is, the AS and AS-5-C are accessibility to tryptophan (Trp) residues more than the tyrosine (Tyr) residues, while the AS-o-V is equally close to the Tyr and Trp residues.

  20. The Importance of Protein-Protein Interactions on the pH-Induced Conformational Changes of Bovine Serum Albumin: A Small-Angle X-Ray Scattering Study

    PubMed Central

    Barbosa, Leandro R.S.; Ortore, Maria Grazia; Spinozzi, Francesco; Mariani, Paolo; Bernstorff, Sigrid; Itri, Rosangela

    2010-01-01

    Abstract The combined effects of concentration and pH on the conformational states of bovine serum albumin (BSA) are investigated by small-angle x-ray scattering. Serum albumins, at physiological conditions, are found at concentrations of ∼35–45 mg/mL (42 mg/mL in the case of humans). In this work, BSA at three different concentrations (10, 25, and 50 mg/mL) and pH values (2.0–9.0) have been studied. Data were analyzed by means of the Global Fitting procedure, with the protein form factor calculated from human serum albumin (HSA) crystallographic structure and the interference function described, considering repulsive and attractive interaction potentials within a random phase approximation. Small-angle x-ray scattering data show that BSA maintains its native state from pH 4.0 up to 9.0 at all investigated concentrations. A pH-dependence of the absolute net protein charge is shown and the charge number per BSA is quantified to 10(2), 8(1), 13(2), 20(2), and 26(2) for pH values 4.0, 5.4, 7.0, 8.0, and 9.0, respectively. The attractive potential diminishes as BSA concentration increases. The coexistence of monomers and dimers is observed at 50 mg/mL and pH 5.4, near the BSA isoelectric point. Samples at pH 2.0 show a different behavior, because BSA overall shape changes as a function of concentration. At 10 mg/mL, BSA is partially unfolded and a strong repulsive protein-protein interaction occurs due to the high amount of exposed charge. At 25 and 50 mg/mL, BSA undergoes some re-folding, which likely results in a molten-globule state. This work concludes by confirming that the protein concentration plays an important role on the pH-unfolded BSA state, due to a delicate compromise between interaction forces and crowding effects. PMID:20085727

  1. Morphological Analysis and Interaction of Chlorophyll and BSA

    PubMed Central

    Gorza, Filipe D. S.; Pedro, Graciela C.; Trescher, Tarquin F.; da Silva, Romário J.; Silva, Josmary R.; de Souza, Nara C.

    2014-01-01

    Interactions between proteins and drugs, which can lead to formation of stable drug-protein complexes, have important implications on several processes related to human health. These interactions can affect, for instance, free concentration, biological activity, and metabolism of the drugs in the blood stream. Here, we report on the UV-Visible spectroscopic investigation on the interaction of bovine serum albumin (BSA) with chlorophyll (Chl) in aqueous solution under physiological conditions. Binding constants at different temperatures—obtained by using the Benesi-Hildebrand equation—were found to be of the same order of magnitude (~104 M−1) indicating low affinity of Chl with BSA. We have found a hyperchromism, which suggested an interaction between BSA and Chl occurring through conformational changes of BSA caused by exposition of tryptophan to solvent. Films from BSA and Chl obtained at different Chl concentrations showed fractal structures, which were characterized by fractal dimension calculated from microscopic image analysis. PMID:24963490

  2. Spectroscopic analyses on interaction of o-Vanillin- D-Phenylalanine, o-Vanillin- L-Tyrosine and o-Vanillin- L-Levodopa Schiff Bases with bovine serum albumin (BSA)

    NASA Astrophysics Data System (ADS)

    Gao, Jingqun; Guo, Yuwei; Wang, Jun; Wang, Zhiqiu; Jin, Xudong; Cheng, Chunping; Li, Ying; Li, Kai

    2011-04-01

    In this work, three o-Vanillin Schiff Bases (o-VSB: o-Vanillin- D-Phenylalanine (o-VDP), o-Vanillin- L-Tyrosine (o-VLT) and o-Vanillin- L-Levodopa (o-VLL)) with alanine constituent were synthesized by direct reflux method in ethanol solution, and then were used to study the interaction to bovine serum albumin (BSA) molecules by fluorescence spectroscopy. Based on the fluorescence quenching calculation, the bimolecular quenching constant ( Kq), apparent quenching constant ( Ksv), effective binding constant ( KA) and corresponding dissociation constant ( KD) as well as binding site number ( n) were obtained. In addition, the binding distance ( r) was also calculated according to Foster's non-radioactive energy transfer theory. The results show that these three o-VSB can efficiently bind to BSA molecules, but the binding array order is o-VDP-BSA > o-VLT-BSA > o-VLL-BSA. Synchronous fluorescence spectroscopy indicates that the o-VDP is more accessibility to tryptophan (Trp) residues of BSA molecules than to tyrosine (Tyr) residues. Nevertheless, the o-VLT and o-VLL are more accessibility to Tyr residues than to Trp residues.

  3. Functional significance of protein assemblies predicted by the crystal structure of the restriction endonuclease BsaWI.

    PubMed

    Tamulaitis, Gintautas; Rutkauskas, Marius; Zaremba, Mindaugas; Grazulis, Saulius; Tamulaitiene, Giedre; Siksnys, Virginijus

    2015-09-18

    Type II restriction endonuclease BsaWI recognizes a degenerated sequence 5'-W/CCGGW-3' (W stands for A or T, '/' denotes the cleavage site). It belongs to a large family of restriction enzymes that contain a conserved CCGG tetranucleotide in their target sites. These enzymes are arranged as dimers or tetramers, and require binding of one, two or three DNA targets for their optimal catalytic activity. Here, we present a crystal structure and biochemical characterization of the restriction endonuclease BsaWI. BsaWI is arranged as an 'open' configuration dimer and binds a single DNA copy through a minor groove contacts. In the crystal primary BsaWI dimers form an indefinite linear chain via the C-terminal domain contacts implying possible higher order aggregates. We show that in solution BsaWI protein exists in a dimer-tetramer-oligomer equilibrium, but in the presence of specific DNA forms a tetramer bound to two target sites. Site-directed mutagenesis and kinetic experiments show that BsaWI is active as a tetramer and requires two target sites for optimal activity. We propose BsaWI mechanism that shares common features both with dimeric Ecl18kI/SgrAI and bona fide tetrameric NgoMIV/SfiI enzymes. PMID:26240380

  4. Penetrable silica microspheres for immobilization of bovine serum albumin and their application to the study of the interaction between imatinib mesylate and protein by frontal affinity chromatography.

    PubMed

    Ma, Liyun; Li, Jing; Zhao, Juan; Liao, Han; Xu, Li; Shi, Zhi-guo

    2016-01-01

    In the current study, novel featured silica, named penetrable silica, simultaneously containing macropores and mesopores, was immobilized with bovine serum albumin (BSA) via Schiff base method. The obtained BSA-SiO2 was employed as the high-performance liquid chromatographic (HPLC) stationary phase. Firstly, D- and L-tryptophan were used as probes to investigate the chiral separation ability of the BSA-SiO2 stationary phase. An excellent enantioseparation factor was obtained up to 4.3 with acceptable stability within at least 1 month. Next, the BSA-SiO2 stationary phase was applied to study the interaction between imatinib mesylate (IM) and BSA by frontal affinity chromatography. A single type of binding site was found for IM with the immobilized BSA, and the hydrogen-bonding and van der Waals interactions were expected to be contributing interactions based on the thermodynamic studies, and this was a spontaneous process. Compared to the traditional silica for HPLC stationary phase, the proposed penetrable silica microsphere possessed a larger capacity to bond more BSA, minimizing column overloading effects and enhancing enantioseparation ability. In addition, the lower running column back pressure and fast mass transfer were meaningful for the column stability and lifetime. It was a good substrate to immobilize biomolecules for fast chiral resolution and screening drug-protein interactions.

  5. Penetrable silica microspheres for immobilization of bovine serum albumin and their application to the study of the interaction between imatinib mesylate and protein by frontal affinity chromatography.

    PubMed

    Ma, Liyun; Li, Jing; Zhao, Juan; Liao, Han; Xu, Li; Shi, Zhi-guo

    2016-01-01

    In the current study, novel featured silica, named penetrable silica, simultaneously containing macropores and mesopores, was immobilized with bovine serum albumin (BSA) via Schiff base method. The obtained BSA-SiO2 was employed as the high-performance liquid chromatographic (HPLC) stationary phase. Firstly, D- and L-tryptophan were used as probes to investigate the chiral separation ability of the BSA-SiO2 stationary phase. An excellent enantioseparation factor was obtained up to 4.3 with acceptable stability within at least 1 month. Next, the BSA-SiO2 stationary phase was applied to study the interaction between imatinib mesylate (IM) and BSA by frontal affinity chromatography. A single type of binding site was found for IM with the immobilized BSA, and the hydrogen-bonding and van der Waals interactions were expected to be contributing interactions based on the thermodynamic studies, and this was a spontaneous process. Compared to the traditional silica for HPLC stationary phase, the proposed penetrable silica microsphere possessed a larger capacity to bond more BSA, minimizing column overloading effects and enhancing enantioseparation ability. In addition, the lower running column back pressure and fast mass transfer were meaningful for the column stability and lifetime. It was a good substrate to immobilize biomolecules for fast chiral resolution and screening drug-protein interactions. PMID:26573171

  6. Highly sensitive bovine serum albumin biosensor based on liquid crystal

    NASA Astrophysics Data System (ADS)

    Sharma, Vikash; Kumar, Ajay; Ganguly, Prasun; Biradar, A. M.

    2014-01-01

    A highly sensitive liquid crystal (LC) based bovine serum albumin (BSA) protein biosensor is designed. A uniform homeotropic alignment of nematic LC was observed in BSA free substrate which changed into homogeneous in presence of BSA. The change in the LC orientation is found to depend strongly on BSA concentration. This change in the LC alignment is attributed to the modification in the surface conditions which is verified by contact angle measurements. We have detected an ultra low concentration (0.5 μg/ml) of BSA. The present study demonstrates the utilization of LC in the realization of high sensitivity biosensors.

  7. Comparative methyl linoleate and methyl linolenate oxidation in the presence of bovine serum albumin at several lipid/protein ratios.

    PubMed

    Zamora, Rosario; Hidalgo, Francisco J

    2003-07-30

    The oxidation of methyl linoleate (LMe) and methyl linolenate (LnMe) in the presence of bovine serum albumin (BSA) in the dark at 60 degrees C was studied to analyze the role of the type of fatty acid and the protein/lipid ratio on the relative progression of the processes involved when lipid oxidation occurs in the presence of proteins. The disappearance of the fatty acid, the formation of primary and secondary products of lipid peroxidation, the loss of amino acid residues, the production of oxidized lipid/amino acid reaction products, and the development of color and fluorescence were studied as a function of incubation time in protein/lipid samples at 10:1, 6:1, and 3:1 w/w ratios. The incubation of LMe and LnMe in the presence of BSA at 60 degrees C rapidly produced lipid peroxidation and protein damage. Although reaction rates were much faster for LnMe than for LMe, both fatty acids had similar behaviors, and LnMe seemed to be only slightly more reactive than LMe for BSA by producing a higher increase of protein pyrroles in the protein and the development of increased browning and fluorescence. The protein/lipid ratio also influenced the relative progress of the reactions implicated. Thus, a lower protein/lipid ratio increased sample oxidation and protein damage. This also produced an increased browning, in accordance with the mechanisms proposed for browning production by oxidized lipid/protein reactions. On the contrary, browning of extracted lipids increased at higher protein/lipid ratios. This opposite tendency allowed evaluation of the overall significance of the different browning processes implicated in the final colors observed, concluding that color changes observed in BSA/lipid samples were mostly a consequence of oxidized lipid/protein reactions. PMID:14705893

  8. Size-dependent interaction of silica nanoparticles with lysozyme and bovine serum albumin proteins.

    PubMed

    Yadav, Indresh; Aswal, Vinod K; Kohlbrecher, Joachim

    2016-05-01

    The interaction of three different sized (diameter 10, 18, and 28 nm) anionic silica nanoparticles with two model proteins-cationic lysozyme [molecular weight (MW) 14.7 kDa)] and anionic bovine serum albumin (BSA) (MW 66.4 kDa) has been studied by UV-vis spectroscopy, dynamic light scattering (DLS), and small-angle neutron scattering (SANS). The adsorption behavior of proteins on the nanoparticles, measured by UV-vis spectroscopy, is found to be very different for lysozyme and BSA. Lysozyme adsorbs strongly on the nanoparticles and shows exponential behavior as a function of lysozyme concentration irrespective of the nanoparticle size. The total amount of adsorbed lysozyme, as governed by the surface-to-volume ratio, increases on lowering the size of the nanoparticles for a fixed volume fraction of the nanoparticles. On the other hand, BSA does not show any adsorption for all the different sizes of the nanoparticles. Despite having different interactions, both proteins induce similar phase behavior where the nanoparticle-protein system transforms from one phase (clear) to two phase (turbid) as a function of protein concentration. The phase behavior is modified towards the lower concentrations for both proteins with increasing the nanoparticle size. DLS suggests that the phase behavior arises as a result of the nanoparticles' aggregation on the addition of proteins. The size-dependent modifications in the interaction potential, responsible for the phase behavior, have been determined by SANS data as modeled using the two-Yukawa potential accounting for the repulsive and attractive interactions in the systems. The protein-induced interaction between the nanoparticles is found to be short-range attraction for lysozyme and long-range attraction for BSA. The magnitude of attractive interaction irrespective of protein type is enhanced with increase in the size of the nanoparticles. The total (attractive+repulsive) potential leading to two-phase formation is found to be

  9. Solution structure of monomeric BsaL, the type III secretion needle protein of Burkholderia pseudomallei.

    PubMed

    Zhang, Lingling; Wang, Yu; Picking, Wendy L; Picking, William D; De Guzman, Roberto N

    2006-06-01

    Many gram-negative bacteria that are important human pathogens possess type III secretion systems as part of their required virulence factor repertoire. During the establishment of infection, these pathogens coordinately assemble greater than 20 different proteins into a macromolecular structure that spans the bacterial inner and outer membranes and, in many respects, resembles and functions like a syringe. This type III secretion apparatus (TTSA) is used to inject proteins into a host cell's membrane and cytoplasm to subvert normal cellular processes. The external portion of the TTSA is a needle that is composed of a single type of protein that is polymerized in a helical fashion to form an elongated tube with a central channel of 2-3 nm in diameter. TTSA needle proteins from a variety of bacterial pathogens share sequence conservation; however, no atomic structure for any TTSA needle protein is yet available. Here, we report the structure of a TTSA needle protein called BsaL from Burkholderia pseudomallei determined by nuclear magnetic resonance (NMR) spectroscopy. The central part of the protein assumes a helix-turn-helix core domain with two well-defined alpha-helices that are joined by an ordered, four-residue linker. This forms a two-helix bundle that is stabilized by interhelix hydrophobic contacts. Residues that flank this presumably exposed core region are not completely disordered, but adopt a partial helical conformation. The atomic structure of BsaL and its sequence homology with other TTSA needle proteins suggest potentially unique structural dynamics that could be linked with a universal mechanism for control of type III secretion in diverse gram-negative bacterial pathogens.

  10. Interaction between the Natural Components in Danhong Injection (DHI) with Serum Albumin (SA) and the Influence of the Coexisting Multi-Components on the SaB-BSA Binding System: Fluorescence and Molecular Docking Studies

    PubMed Central

    Hao, Jia; Zhang, Yingyue; Wang, Xingrui; Yan, Huo; Liu, Erwei; Gao, Xiumei

    2015-01-01

    Danhong injection (DHI) is a widely used Chinese Materia Medica standardized product for the clinical treatment of ischemic encephalopathy and coronary heart disease. The bindings of eight natural components in DHI between bovine serum albumin (BSA) were studied by fluorescence spectroscopy technology and molecular docking. According to the results, the quenching process of salvianolic acid B and hydroxysafflor yellow A was a static quenching procedure through the analysis of quenching data by the Stern-Volmer equation, the modified Stern-Volmer equation, and the modified Scatchard equation. Meanwhile, syringin (Syr) enhanced the fluorescence of BSA, and the data were analyzed using the Lineweaver-Burk equation. Molecular docking suggested that all of these natural components bind to serum albumin at the site I location. Further competitive experiments of SaB confirmed the result of molecular docking studies duo to the displacement of warfarin by SaB. Base on these studies, we selected SaB as a research target because it presented the strongest binding ability to BSA and investigated the influence of the multi-components coexisting in DHI on the interaction between the components of the SaB-BSA binding system. The participation of these natural components in DHI affected the interaction between the components of the SaB-BSA system. Therefore, when DHI is used in mammals, SaB is released from serum albumin more quickly than it is used alone. This work would provide a new experiment basis for revealing the scientific principle of compatibility for Traditional Chinese Medicine. PMID:26035712

  11. Interaction between the Natural Components in Danhong Injection (DHI) with Serum Albumin (SA) and the Influence of the Coexisting Multi-Components on the SaB-BSA Binding System: Fluorescence and Molecular Docking Studies.

    PubMed

    Hao, Jia; Zhang, Yingyue; Wang, Xingrui; Yan, Huo; Liu, Erwei; Gao, Xiumei

    2015-01-01

    Danhong injection (DHI) is a widely used Chinese Materia Medica standardized product for the clinical treatment of ischemic encephalopathy and coronary heart disease. The bindings of eight natural components in DHI between bovine serum albumin (BSA) were studied by fluorescence spectroscopy technology and molecular docking. According to the results, the quenching process of salvianolic acid B and hydroxysafflor yellow A was a static quenching procedure through the analysis of quenching data by the Stern-Volmer equation, the modified Stern-Volmer equation, and the modified Scatchard equation. Meanwhile, syringin (Syr) enhanced the fluorescence of BSA, and the data were analyzed using the Lineweaver-Burk equation. Molecular docking suggested that all of these natural components bind to serum albumin at the site I location. Further competitive experiments of SaB confirmed the result of molecular docking studies duo to the displacement of warfarin by SaB. Base on these studies, we selected SaB as a research target because it presented the strongest binding ability to BSA and investigated the influence of the multi-components coexisting in DHI on the interaction between the components of the SaB-BSA binding system. The participation of these natural components in DHI affected the interaction between the components of the SaB-BSA system. Therefore, when DHI is used in mammals, SaB is released from serum albumin more quickly than it is used alone. This work would provide a new experiment basis for revealing the scientific principle of compatibility for Traditional Chinese Medicine.

  12. Size-dependent interaction of silica nanoparticles with lysozyme and bovine serum albumin proteins

    NASA Astrophysics Data System (ADS)

    Yadav, Indresh; Aswal, Vinod K.; Kohlbrecher, Joachim

    2016-05-01

    The interaction of three different sized (diameter 10, 18, and 28 nm) anionic silica nanoparticles with two model proteins—cationic lysozyme [molecular weight (MW) 14.7 kDa)] and anionic bovine serum albumin (BSA) (MW 66.4 kDa) has been studied by UV-vis spectroscopy, dynamic light scattering (DLS), and small-angle neutron scattering (SANS). The adsorption behavior of proteins on the nanoparticles, measured by UV-vis spectroscopy, is found to be very different for lysozyme and BSA. Lysozyme adsorbs strongly on the nanoparticles and shows exponential behavior as a function of lysozyme concentration irrespective of the nanoparticle size. The total amount of adsorbed lysozyme, as governed by the surface-to-volume ratio, increases on lowering the size of the nanoparticles for a fixed volume fraction of the nanoparticles. On the other hand, BSA does not show any adsorption for all the different sizes of the nanoparticles. Despite having different interactions, both proteins induce similar phase behavior where the nanoparticle-protein system transforms from one phase (clear) to two phase (turbid) as a function of protein concentration. The phase behavior is modified towards the lower concentrations for both proteins with increasing the nanoparticle size. DLS suggests that the phase behavior arises as a result of the nanoparticles' aggregation on the addition of proteins. The size-dependent modifications in the interaction potential, responsible for the phase behavior, have been determined by SANS data as modeled using the two-Yukawa potential accounting for the repulsive and attractive interactions in the systems. The protein-induced interaction between the nanoparticles is found to be short-range attraction for lysozyme and long-range attraction for BSA. The magnitude of attractive interaction irrespective of protein type is enhanced with increase in the size of the nanoparticles. The total (attractive+repulsive) potential leading to two-phase formation is found to be

  13. Encapsulation of catechin and epicatechin on BSA NPS improved their stability and antioxidant potential.

    PubMed

    Yadav, Ramdhan; Kumar, Dharmesh; Kumari, Avnesh; Yadav, Sudesh Kumar

    2014-01-01

    Nanoencapsulation of antioxidant molecules on protein nanoparticles (NPs) could be an advanced approach for providing stable, better food nutraceuticals and anticancer drugs. The bioavailability and stability of catechin (CAT) and epicatechin (ECAT) were very poor. In the present study, the CAT and ECAT were loaded on bovine serum albumin (BSA) NPs following desolvation method. The transmission electron microscope (TEM) and atomic force microscope (AFM) recorded size of CAT-BSA NPs and ECAT-BSA NPs were 45 ± 5 nm and 48 ± 5 nm respectively. The encapsulation efficiency of CAT and ECAT on BSA NPs was found to be 60.5 and 54.5 % respectively. CAT-BSA NPs and ECAT-BSA NPs show slow and sustained in vitro release. The CAT-BSA NPs and ECAT-BSA NPs were stable in solution at various temperatures 37 °C, 47 °C and 57 °C. DPPH assay revealed that CAT and ECAT maintained their functional activity even after encapsulation on BSA NPs. Furthermore, the efficacy of CAT-BSA NPs and ECAT-BSA NPs determined against A549 cell lines was found to be improved. CAT and ECAT aptly encapsulated in BSA NPs, showed satisfactory sustained release, maintained antioxidant potential and found improved efficacy. This has thus suggested their more effective use in food and nutraceuticals as well as in medical field.

  14. Encapsulation of catechin and epicatechin on BSA NPS improved their stability and antioxidant potential

    PubMed Central

    Yadav, Ramdhan; Kumar, Dharmesh; Kumari, Avnesh; Yadav, Sudesh Kumar

    2014-01-01

    Nanoencapsulation of antioxidant molecules on protein nanoparticles (NPs) could be an advanced approach for providing stable, better food nutraceuticals and anticancer drugs. The bioavailability and stability of catechin (CAT) and epicatechin (ECAT) were very poor. In the present study, the CAT and ECAT were loaded on bovine serum albumin (BSA) NPs following desolvation method. The transmission electron microscope (TEM) and atomic force microscope (AFM) recorded size of CAT-BSA NPs and ECAT-BSA NPs were 45 ± 5 nm and 48 ± 5 nm respectively. The encapsulation efficiency of CAT and ECAT on BSA NPs was found to be 60.5 and 54.5 % respectively. CAT-BSA NPs and ECAT-BSA NPs show slow and sustained in vitro release. The CAT-BSA NPs and ECAT-BSA NPs were stable in solution at various temperatures 37 °C, 47 °C and 57 °C. DPPH assay revealed that CAT and ECAT maintained their functional activity even after encapsulation on BSA NPs. Furthermore, the efficacy of CAT-BSA NPs and ECAT-BSA NPs determined against A549 cell lines was found to be improved. CAT and ECAT aptly encapsulated in BSA NPs, showed satisfactory sustained release, maintained antioxidant potential and found improved efficacy. This has thus suggested their more effective use in food and nutraceuticals as well as in medical field. PMID:26417264

  15. Effect of temperature on the methotrexate BSA interaction: Spectroscopic study

    NASA Astrophysics Data System (ADS)

    Sułkowska, A.; Maciążek, M.; Równicka, J.; Bojko, B.; Pentak, D.; Sułkowski, W. W.

    2007-05-01

    Rheumatoid arthritis (RA) is an autoimmune and chronic inflammatory illness which affects about one percent of the world's population. Methotrexate (4-amino-10-methylfolic acid) (MTX) also known as amethopterin is commonly used to treat rheumatoid arthritis (RA). It is transported in the circulary system as a complex with serum albumin. The aim of this study was to investigate the interactions of MTX with transporting protein with the use of spectroscopic methods. The binding of MTX to bovine serum albumin (BSA) was studied by monitoring the changes in the emission fluorescence spectra of protein in the presence of MTX at excitation wavelength of 280 nm and 295 nm. The quenching of protein fluorescence at temperature range from 298 K to 316 K was observed. Energy transfer between methotrexate and fluorophores contained in the serum albumin structure was found at the molar ratio MTX:BSA 7.5:1. The relative fluorescence intensity of BSA decreases with increase of temperature. Similar results were observed for BSA excited with 280 nm and 295 nm at the same temperature range. The presence of MTX seems to prevent these changes. Temperature dependence of the binding constant has been presented. The binding and quenching constants for equilibrium complex were calculated using Scatchard and Stern-Volmer method, respectively. The results show that MTX forms π-π complex with aromatic amino acid residues of BSA. The binding site for MTX on BSA was found to be situated in the hydrophobic IIA or IB subdomain where the Trps were located. The spontaneity of MTX-BSA complex formation in the temperature range 298-316 K was ascertained.

  16. 2S Albumin Storage Proteins: What Makes them Food Allergens?

    PubMed Central

    Moreno, F. Javier; Clemente, Alfonso

    2008-01-01

    2S albumin storage proteins are becoming of increasing interest in nutritional and clinical studies as they have been reported as major food allergens in seeds of many mono- and di-cotyledonous plants. This review describes the main biochemical, structural and functional properties of these proteins thought to play a role in determining their potential allergenicity. 2S albumins are considered to sensitize directly via the gastrointestinal tract (GIT). The high stability of their intrinsic protein structure, dominated by a well-conserved skeleton of cysteine residues, to the harsh conditions present in the GIT suggests that these proteins are able to cross the gut mucosal barrier to sensitize the mucosal immune system and/or elicit an allergic response. The flexible and solvent-exposed hypervariable region of these proteins is immunodominant and has the ability to bind IgE from allergic patients´ sera. Several linear IgE-binding epitopes of 2S albumins spanning this region have been described to play a major role in allergenicity; the role of conformational epitopes of these proteins in food allergy is far from being understood and need to be investigated. Finally, the interaction of these proteins with other components of the food matrix might influence the absorption rates of immunologically reactive 2S albumins but also in their immune response. PMID:18949071

  17. Emergent membrane-affecting properties of BSA-gold nanoparticleconstructs

    NASA Astrophysics Data System (ADS)

    Lystvet, Sina M.; Volden, Sondre; Yasuda, Masahiro; Halskau, Øyvind, Jr.; Glomm, Wilhelm R.

    2011-04-01

    By adsorbing bovine serum albumin (BSA) on gold nanoparticles (Aunps) with diameters 30 nm and 80 nm, different degrees of protein unfolding were obtained. Adsorption and adlayer conformation were characterized by UV-vis spectroscopy, ζ-potential measurements, steady-state and time-resolved fluorescence. The unfolding was also studied using 1-anilino-8-naphthalene sulfonate (ANS) as an extrinsic probe, showing that BSA unfolds more on 80 nm Aunp than on 30 nm Aunp. Langmuir monolayer studies using two distinct methods of introducing the BSA and BSA-Aunp constructs accompanied with Brewster Angle Microscopy (BAM) and Digital Video Microscope (DVM) imaging demonstrated that BSA-Aunp constructs induce film miscibility with l-α-phosphatidylethanolamine not seen for BSA or Aunp alone. The changes induced by partial unfolding clearly give better film-penetration ability, as well as disruption of liquid crystalline domains in the film, thereby inducing film miscibility. Gold or protein only does not possess the nanoscale film-affecting properties of the protein-goldconstructs, and as such the surface-active and miscibility-affecting characteristics of the BSA-Aunp represent emergent qualities.

  18. Detailed scrutiny of the anion receptor pocket in subdomain IIA of serum proteins toward individual response to specific ligands: HSA-pocket resembles flexible biological slide-wrench unlike BSA.

    PubMed

    Datta, Shubhashis; Halder, Mintu

    2014-06-12

    Present study reveals that the subdomain IIA cavity of two homologous serum albumins (HSA, BSA) has inherent mutual structural and functional deviations which render noticeable difference in behavior toward specific ligands. The major drug binding site (subdomain IIA) of HSA is found to be largely hydrophobic while that of BSA is partially exposed to water. Larger shift in REE spectra and greater change in solvent reorganization energy of coumarin 343 (C343)-anion in HSA clearly reveals that binding pocket is relatively large and water molecules penetrate deeper into it unlike BSA. The individual response of proteins to perturbation by ligands is found to be way different. Although the subdomain IIA is primarily anion receptive (prefers anionic ligands), the present study suggests that HSA may also like to bind neutral guests due to its remarkable conformational features. Actually, HSA is capable of adopting favorable conformation like mechanical slide-wrench, when required, to accommodate neutral ligands [e.g., coumarin 314 (C314)], as well. But due to less flexible solution structure, BSA behaves like fixed mechanical spanners and hence is not very responsive to C314. Therefore, the generally speaking functional-structural similarities of homologous proteins can be apparent and needs to be analyzed exhaustively.

  19. Complexation of serum albumins and triton X-100: Quenching of tryptophan fluorescence and analysis of the rotational diffusion of complexes

    NASA Astrophysics Data System (ADS)

    Vlasova, I. M.; Vlasov, A. A.; Saletskii, A. M.

    2016-07-01

    The polarized and nonpolarized fluorescence of bovine serum albumin and human serum albumin in Triton X-100 solutions is studied at different pH values. Analysis of the constants of fluorescence quenching for BSA and HSA after adding Triton X-100 and the hydrodynamic radii of BSA/HSA-detergent complexes show that the most effective complexation between both serum albumins and Triton X-100 occurs at pH 5.0, which lies near the isoelectric points of the proteins. Complexation between albumin and Triton X-100 affects the fluorescence of the Trp-214 residing in the hydrophobic pockets of both BSA and HSA.

  20. Differing features of proteins in membranes may result in antioxidant or prooxidant action: opposite effects on lipid peroxidation of alcohol dehydrogenase and albumin in liposomal systems.

    PubMed

    Riedl, A; Shamsi, Z; Anderton, M; Goldfarb, P; Wiseman, A

    1996-02-01

    The influence of 3 thiol-containing compounds, bovine serum albumin (fatty acid free: BSA), glutathione (GSH) and yeast alcohol dehydrogenase (YADH) on lipid peroxidation in multilamellar liposomes, prepared from ox-brain phospholipid, was investigated. Thiol-compounds were added either before liposome formation, or after liposome formation; and their effects compared to a positive control. Bovine serum albumin (BSA), an acidic hydrophilic protein, displays a small, concentration dependent, antioxidant effect when added to preformed liposomes. A much larger antioxidant effect was observed when the BSA was entrapped inside the liposome, by adding BSA just prior to liposome preparation. In contrast, a Zn(2+) containing redox enzyme, YADH, a basic hydrophobic membrane-associating protein, displays a large pro-oxidant effect at much lower concentrations especially when entrapped inside the liposome. This was observed also with GSH; but per mole of -SH, YADH was about 18 times as powerful a pro-oxidant perhaps because of structural changes to the membrane. Oxidized glutathione and N-acetylcysteine were also pro-oxidant (cysteine and cystine showed little effect). Formation of thiyl radicals may occur in the presence of iron ions with these pro-oxidant sulphur-containing compounds. Partial protection against lipid peroxidation was observed with EDTA, desferrioxamine and protoporphyrin (IX), potent iron-chelating agents.

  1. Evaluating interaction forces between BSA and rabbit anti-BSA in sulphathiazole sodium, tylosin and levofloxacin solution by AFM

    PubMed Central

    2011-01-01

    Protein-protein interactions play crucial roles in numerous biological processes. However, it is still challenging to evaluate the protein-protein interactions, such as antigen and antibody, in the presence of drug molecules in physiological liquid. In this study, the interaction between bovine serum albumin (BSA) and rabbit anti-BSA was investigated using atomic force microscopy (AFM) in the presence of various antimicrobial drugs (sulphathiazole sodium, tylosin and levofloxacin) under physiological condition. The results show that increasing the concentration of tylosin decreased the single-molecule-specific force between BSA and rabbit anti-BSA. As for sulphathiazole sodium, it dramatically decreased the specific force at a certain critical concentration, but increased the nonspecific force as its concentration increasing. In addition, the presence of levofloxacin did not greatly influence either the specific or nonspecific force. Collectively, these results suggest that these three drugs may adopt different mechanisms to affect the interaction force between BSA and rabbit anti-BSA. These findings may enhance our understanding of antigen/antibody binding processes in the presence of drug molecules, and hence indicate that AFM could be helpful in the design and screening of drugs-modulating protein-protein interaction processes. PMID:22053876

  2. [Binding mechanism of traditional Chinese medicine active component 5-hydroxymethyl-furfural and HSA or BSA].

    PubMed

    Guo, Ming; He, Ling; Lu, Xiao-Wang

    2012-03-01

    A combination of spectral experiment and molecular modeling techniques has been used to characterize the binding mechanism between an active component 5-hydroxymethyl-furfural (5-HMF) of traditional Chinese medicine and human serum albumin (HSA) or bovine serum albumin (BSA). The interaction mechanism of 5-HMF binding with HSA/BSA is analyzed. Although the drug can bind with HSA/BSA to form stable complexes, there are some differences in the bond strength. The values of binding distances (r) are different and low, which indicated the occurrence of energy transfer. The drug had conformational effect on HSA/BSA, which resulted in different changes of hydrophobic environment of the binding domain in HSA/BSA. The 'phase diagram' of fluorescence revealed that the changes on the conformational pattern of proteins have been affected by drug conformed to the "all-or-none" pattern. The interactions between drug and protein influenced by Co(II) were also discussed. Its effects acting on 5-HMF-HSA/BSA interactions are different. The computational modeling method was used to study the interaction between 5-HMF and HSA/BSA. The results of molecular model studies revealed that the binding modes for drug-serum albumin systems are mainly hydrophobic interactions and hydrogen bonding. These results are in accordance with spectral results. The research results have given a better theoretical reference for the study of pharmacological mechanism of 5-hydroxymethyl-furfural.

  3. Impact of surface modification in BSA nanoparticles for uptake in cancer cells.

    PubMed

    Choi, Jin-Seok; Meghani, Nilesh

    2016-09-01

    Recent studies have shown that cellular uptake of nanoparticles are strongly affected by the presence and physicochemical characteristics of protein on the surface of these nanoparticles. Hence, We developed surface-modified bovine serum albumin (BSA) nanoparticles (NPs) and evaluated the effect of surface modification on cellular uptake in two types of cancer cells, MCF-7 and A549. BSA NPs were prepared by desolvation method and their surface was modified with apo-transferrin, hyaluronic acid, and Poly(allylamine hydrochloride) (PAH). Morphology of surface-modified BSA NPs was characterized by field emission scanning electron microscopy and differential scanning calorimetry. In vitro-fluorescence release study was performed in phosphate buffered saline with trypsin (100μL/mL (v/v)) for 24h. Confocal microscopy was performed to evaluate cellular uptake followed by fluorescence analysis to evaluate the quantitative uptake of nanoparticles at 0.5, 1, and 2h. Different types of BSA NPs with a mean size of ∼100nm were successfully prepared. In vitro-fluorescent release showed sustained release pattern in surface-modified BSA NPs compared to unmodified BSA NPs. Surface-modified BSA NPs showed more cellular internalization than unmodified BSA NPs. The uptake of PAH-BSA NPs was about 2 times higher than that of unmodified BSA NPs in both cell types. In conclusion, surface-modified BSA NPs showed enhanced cellular uptake, and PAH-BSA NPs are more effective compared to ligand-specific surface-modified BSA NPs (HA-BSA NPs and Tf-BSA NPs).

  4. Development of a sandwich enzyme-linked immunosorbent assay (ELISA) for determining of bovine serum albumin (BSA) in trivalent measles-mump-rubella (MMR) vaccines.

    PubMed

    Khamehchian, Sedigheh; Madani, Rasool; Golchinfar, Fariba; Taghavian, Mohammad

    2008-01-01

    A sandwich enzyme-linked immunosorbent assay (ELISA), using polyclonal antibody, was developed and compared with the commercial kit for detecting and estimating of BSA content in Measles-Mump-Rubella (MMR) vaccine samples in detection limit of nanogram level. The test depends on the capturing and detecting of BSA antigen by the polyclonal antibody. Initially, a detection range of 0-64 ng/ml was established, could be used for estimation of BSA content according to WHO requirement (50 ng/ml) in MMR vaccines. Comparative analysis of the test results for 85 MMR vaccine samples obtained with the commercial kit gave a sensitivity of 58.8% and a specificity of 97%. A high correlation (r = 0.94) was observed between BSA sandwich ELISA and commercial kit for BSA content in MMR samples. However, variations in values also were observed for the two assays. These variations may have been due to difference of upper limit of detection range of BSA content in commercial kit (32 ng/ml) and new sandwich ELISA (64 ng/ml) as well as the use of a different polyclonal antibody. In concerning the cutoff value for the WHO requirement and employment of standard solution of 64 ng/ml in developing assay, it would be adequate to use this test for assessing BSA content in viral vaccines same as MMR vaccines.

  5. A selective, long-lived deep-red emissive ruthenium(II) polypyridine complexes for the detection of BSA.

    PubMed

    Babu, Eththilu; Muthu Mareeswaran, Paulpandian; Singaravadivel, Subramanian; Bhuvaneswari, Jayaraman; Rajagopal, Seenivasan

    2014-09-15

    A selective, label free luminescence sensor for bovine serum albumin (BSA) is investigated using ruthenium(II) complexes over the other proteins. Interaction between BSA and ruthenium(II) complexes has been studied using absorption, emission, excited state lifetime and circular dichroism (CD) spectral techniques. The luminescence intensity of ruthenium(II) complexes (I and II), has enhanced at 602 and 613 nm with a large hypsochromic shift of 18 and 5 nm respectively upon addition of BSA. The mode of binding of ruthenium(II) complexes with BSA has analyzed using computational docking studies.

  6. Biologically active protein fragments containing specific binding regions of serum albumin or related proteins

    NASA Technical Reports Server (NTRS)

    Carter, Daniel C. (Inventor)

    1998-01-01

    In accordance with the present invention, biologically active protein fragments can be constructed which contain only those specific portions of the serum albumin family of proteins such as regions known as subdomains IIA and IIIA which are primarily responsible for the binding properties of the serum albumins. The artificial serums that can be prepared from these biologically active protein fragments are advantageous in that they can be produced much more easily than serums containing the whole albumin, yet still retain all or most of the original binding potential of the full albumin proteins. In addition, since the protein fragment serums of the present invention can be made from non-natural sources using conventional recombinant DNA techniques, they are far safer than serums containing natural albumin because they do not carry the potentially harmful viruses and other contaminants that will be found in the natural substances.

  7. 21 CFR 862.1645 - Urinary protein or albumin (nonquantitative) test system.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Urinary protein or albumin (nonquantitative) test... Chemistry Test Systems § 862.1645 Urinary protein or albumin (nonquantitative) test system. (a) Identification. A urinary protein or albumin (nonquantitative) test system is a device intended to...

  8. 21 CFR 862.1645 - Urinary protein or albumin (nonquantitative) test system.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Urinary protein or albumin (nonquantitative) test... Chemistry Test Systems § 862.1645 Urinary protein or albumin (nonquantitative) test system. (a) Identification. A urinary protein or albumin (nonquantitative) test system is a device intended to...

  9. 21 CFR 862.1645 - Urinary protein or albumin (nonquantitative) test system.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Urinary protein or albumin (nonquantitative) test... Chemistry Test Systems § 862.1645 Urinary protein or albumin (nonquantitative) test system. (a) Identification. A urinary protein or albumin (nonquantitative) test system is a device intended to...

  10. 21 CFR 862.1645 - Urinary protein or albumin (nonquantitative) test system.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Urinary protein or albumin (nonquantitative) test... Chemistry Test Systems § 862.1645 Urinary protein or albumin (nonquantitative) test system. (a) Identification. A urinary protein or albumin (nonquantitative) test system is a device intended to...

  11. Behaviors of bovine serum albumin and rapeseed proteins at the air/water interface after grafting aliphatic or aromatic chains.

    PubMed

    Gerbanowski, Alice; Rabiller, Claude; Guéguen, Jacques

    2003-06-15

    The influence of grafting aliphatic or aromatic groups on the behaviors of bovine serum albumin (BSA) and rapeseed proteins (napin and cruciferin) at the air/water interface is studied. From compression isotherms, it is shown that the chemical modification induces an increase in the interfacial molecular areas of the three proteins. The more hydrophobic the groups grafted, the more important this increase is. The dilatational modulus clearly emphasized that the grafting of hydrophobic groups also leads to an increase of the collapse pressure, demonstrating a higher cohesiveness and resistance to pressure of the interfacial films. These results are discussed on the basis of the physicochemical changes due to these chemical modifications, especially the conformation, the surface hydrophobicity, and the flexibility of the modified proteins. The improvement of surface properties obtained by grafting aliphatic or aromatic chains onto these proteins looks very promising in regard to emulsifying and foaming properties.

  12. Effect of BSA-induced ER stress on SGLT protein expression levels and alpha-MG uptake in renal proximal tubule cells.

    PubMed

    Lee, Yu Jin; Suh, Han Na; Han, Ho Jae

    2009-06-01

    Recent studies demonstrated that endoplasmic reticulum (ER) stress regulates glucose homeostasis and that ER stress preconditioning which induces an adaptive, protective unfolded protein response (UPR) offers cytoprotection against nephrotoxins. Thus the aim of the present study was to use renal proximal tubule cells (PTCs) to further elucidate the link between the BSA-induced ER stress and alpha-methyl-d-glucopyranoside (alpha-MG) uptake and to identify related signaling pathways. Among ER stress inducers such as high glucose, BSA, H2O2, or tumicamycin, BSA pretreatment ameliorated the reduction of Na(+)-glucose cotransporter (SGLT) expression and alpha-MG uptake by gentamicin or cyclosporine A. Immunofluorescence studies revealed that BSA (10 mg/ml) stimulated the expression of glucose-regulated protein 78 (GRP78), an ER stress biomarker. In addition, BSA increased levels of GRP78 protein expression and eukaryotic initiation factor 2alpha (eIF2alpha) phosphorylation in a time-dependent manner. Furthermore, transfection with a GRP78-specific small interfering RNA (siRNA) inhibited BSA-stimulated SGLT expression and alpha-MG uptake. In experiments designed to unravel the mechanisms underlying BSA-induced ER stress, BSA stimulated the production of cellular reactive oxygen species (ROS), and antioxidants such as ascorbic acid or N-acetylcysteine (NAC) blocked BSA-induced increases in GRP78 activation, eIF2alpha phosphorylation, SGLT expression, and alpha-MG uptake. Moreover, the cells upregulated peroxisome proliferator-activated receptor-gamma (PPARgamma) mRNA levels in response to BSA or troglitazone (a PPARgamma agonist), but BSA was ineffective in the presence of GW9662 (a PPARgamma antagonist). In addition, both BSA and troglitazone stimulated GRP78 and eIF2alpha activation, SGLT expression, and alpha-MG uptake, whereas GW9662 inhibited the effects of BSA. BSA also stimulated phosphorylation of JNK and NF-kappaB, and GW9662 or GRP78 siRNA attenuated this

  13. Synthesis of nano-bioactive glass-ceramic powders and its in vitro bioactivity study in bovine serum albumin protein

    NASA Astrophysics Data System (ADS)

    Nabian, Nima; Jahanshahi, Mohsen; Rabiee, Sayed Mahmood

    2011-07-01

    Bioactive glasses and ceramics have proved to be able to chemically bond to living bone due to the formation of an apatite-like layer on its surface. The aim of this work was preparation and characterization of bioactive glass-ceramic by sol-gel method. Nano-bioglass-ceramic material was crushed into powder and its bioactivity was examined in vitro with respect to the ability of hydroxyapatite layer to form on the surface as a result of contact with bovine serum albumin (BSA) protein. The obtained nano-bioactive glass-ceramic was analyzed before and after contact with BSA solution. This study used scanning electron microscope (SEM), transmission electron microscope (TEM), X-ray powder diffraction (XRD) and Fourier transform infrared spectroscopy (FTIR) analysis to examine its morphology, crystallinity and composition. The TEM images showed that the NBG particles size were 10-40 nm. Bioactivity of nanopowder was confirmed by SEM and XRD due to the presence of a rich bone-like apatite layer. Therefore, this nano-BSA-bioglass-ceramic composite material is promising for medical applications such as bone substitutes and drug carriers.

  14. Peroxidase mediated conjugation of corn fibeer gum and bovine serum albumin to improve emulsifying properties

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The emulsifying properties of corn fiber gum (CFG), a naturally-occurring polysaccharide protein complex, were improved by kinetically controlled formation of hetero-covalent linkages with bovine serum albumin (BSA), using horseradish peroxidase. The formation of hetero-crosslinked CFG-BSA conjugate...

  15. Chromatographic and traditional albumin isotherms on cellulose: a model for wound protein adsorption on modified cotton

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Albumin is the most abundant protein found in healing wounds. Traditional and chromatogrpahic protein isotherms of albumin binding on modified cotton fibers are useful in understanding albumin binding to cellulose wound dressings. An important consideration in the design of cellulosic wound dressin...

  16. Small-angle neutron scattering study of differences in phase behavior of silica nanoparticles in the presence of lysozyme and bovine serum albumin proteins

    NASA Astrophysics Data System (ADS)

    Yadav, Indresh; Kumar, Sugam; Aswal, V. K.; Kohlbrecher, J.

    2014-03-01

    The differences in phase behavior of anionic silica nanoparticles (88 Å) in the presence of two globular proteins [cationic lysozyme (molecular weight (MW) 14.7 kD) and anionic bovine serum albumin (BSA) (MW 66.4 kD)] have been studied by small-angle neutron scattering. The measurements were carried out on a fixed concentration (1 wt %) of Ludox silica nanoparticles with varying concentrations of proteins (0-5 wt %) at pH = 7. It is found that, despite having different natures (opposite charges), both proteins can render to the same kind of aggregation of silica nanoparticles. However, the concentration regions over which the aggregation is observed are widely different for the two proteins. Lysozyme with very small amounts (e.g., 0.01 wt %) leads to the aggregation of silica nanoparticles. On the other hand, silica nanoparticles coexist with BSA as independent entities at low protein concentrations and turn to aggregates at high protein concentrations (>1 wt %). In the case of lysozyme, the charge neutralization by the protein on the nanoparticles gives rise to the protein-mediated aggregation of the nanoparticles. The nanoparticle aggregates coexist with unaggregated nanoparticles at low protein concentrations, whereas, they coexist with a free protein at higher protein concentrations. For BSA, the nonadsorbing nature of the protein produces the depletion force that causes the aggregation of the nanoparticles at higher protein concentrations. The evolution of the interaction is modeled by the two Yukawa potential, taking account of both attractive and repulsive terms of the interaction in these systems. The nanoparticle aggregation is found to be governed by the short-range attraction for lysozyme and the long-range attraction for BSA. The aggregates are characterized by the diffusion limited aggregate type of mass fractal morphology.

  17. Solution combustion synthesis of calcium phosphate particles for controlled release of bovine serum albumin.

    PubMed

    Zhao, Junfeng; Zhao, Junjie; Qian, Yu; Zhang, Xiali; Zhou, Feifei; Zhang, Hong; Lu, Hongbin; Chen, JianHua; Wang, XuHong; Yu, Wencong

    2015-05-01

    Four different phase compositions of calcium phosphate (CaP) particles were prepared via a solution combustion method. X-ray diffraction (XRD) and Rietveld analysis results revealed that the variations in the nominal Ca/P (molar) ratios were found to provide a favorable control in the different proportions of CaP materials. Bovine serum albumin (BSA) was used as a model protein to study the loading and release behavior. The release profile indicated that the BSA release rates depended on the phase compositions of the CaP particles, and showed an order of TCP-BSA>BCP-1-BSA>BCP-2-BSA>HA-BSA. The results suggested that the BSA protein release rate can be controlled by varying the phase compositions of CaP carriers. Moreover, the release process involved two stages: firstly surface diffusion via ion exchange and secondly intraparticle diffusion.

  18. Binding of Sulpiride to Seric Albumins.

    PubMed

    da Silva Fragoso, Viviane Muniz; de Morais Coura, Carla Patrícia; Hoppe, Luanda Yanaan; Soares, Marília Amável Gomes; Silva, Dilson; Cortez, Celia Martins

    2016-01-04

    The aim of this work was to study the interaction of sulpiride with human serum albumin (HSA) and bovine serum albumin (BSA) through the fluorescence quenching technique. As sulpiride molecules emit fluorescence, we have developed a simple mathematical model to discriminate the quencher fluorescence from the albumin fluorescence in the solution where they interact. Sulpiride is an antipsychotic used in the treatment of several psychiatric disorders. We selectively excited the fluorescence of tryptophan residues with 290 nm wavelength and observed the quenching by titrating HSA and BSA solutions with sulpiride. Stern-Volmer graphs were plotted and quenching constants were estimated. Results showed that sulpiride form complexes with both albumins. Estimated association constants for the interaction sulpiride-HSA were 2.20 (±0.08) × 10⁴ M(-1), at 37 °C, and 5.46 (±0.20) × 10⁴ M(-1), at 25 °C. Those for the interaction sulpiride-BSA are 0.44 (±0.01) × 10⁴ M(-1), at 37 °C and 2.17 (±0.04) × 10⁴ M(-1), at 25 °C. The quenching intensity of BSA, which contains two tryptophan residues in the peptide chain, was found to be higher than that of HSA, what suggests that the primary binding site for sulpiride in albumin should be located next to the sub domain IB of the protein structure.

  19. Binding of Sulpiride to Seric Albumins

    PubMed Central

    da Silva Fragoso, Viviane Muniz; de Morais Coura, Carla Patrícia; Hoppe, Luanda Yanaan; Soares, Marília Amável Gomes; Silva, Dilson; Cortez, Celia Martins

    2016-01-01

    The aim of this work was to study the interaction of sulpiride with human serum albumin (HSA) and bovine serum albumin (BSA) through the fluorescence quenching technique. As sulpiride molecules emit fluorescence, we have developed a simple mathematical model to discriminate the quencher fluorescence from the albumin fluorescence in the solution where they interact. Sulpiride is an antipsychotic used in the treatment of several psychiatric disorders. We selectively excited the fluorescence of tryptophan residues with 290 nm wavelength and observed the quenching by titrating HSA and BSA solutions with sulpiride. Stern-Volmer graphs were plotted and quenching constants were estimated. Results showed that sulpiride form complexes with both albumins. Estimated association constants for the interaction sulpiride–HSA were 2.20 (±0.08) × 104 M−1, at 37 °C, and 5.46 (±0.20) × 104 M−1, at 25 °C. Those for the interaction sulpiride-BSA are 0.44 (±0.01) × 104 M−1, at 37 °C and 2.17 (±0.04) × 104 M−1, at 25 °C. The quenching intensity of BSA, which contains two tryptophan residues in the peptide chain, was found to be higher than that of HSA, what suggests that the primary binding site for sulpiride in albumin should be located next to the sub domain IB of the protein structure. PMID:26742031

  20. Binding of Sulpiride to Seric Albumins.

    PubMed

    da Silva Fragoso, Viviane Muniz; de Morais Coura, Carla Patrícia; Hoppe, Luanda Yanaan; Soares, Marília Amável Gomes; Silva, Dilson; Cortez, Celia Martins

    2016-01-01

    The aim of this work was to study the interaction of sulpiride with human serum albumin (HSA) and bovine serum albumin (BSA) through the fluorescence quenching technique. As sulpiride molecules emit fluorescence, we have developed a simple mathematical model to discriminate the quencher fluorescence from the albumin fluorescence in the solution where they interact. Sulpiride is an antipsychotic used in the treatment of several psychiatric disorders. We selectively excited the fluorescence of tryptophan residues with 290 nm wavelength and observed the quenching by titrating HSA and BSA solutions with sulpiride. Stern-Volmer graphs were plotted and quenching constants were estimated. Results showed that sulpiride form complexes with both albumins. Estimated association constants for the interaction sulpiride-HSA were 2.20 (±0.08) × 10⁴ M(-1), at 37 °C, and 5.46 (±0.20) × 10⁴ M(-1), at 25 °C. Those for the interaction sulpiride-BSA are 0.44 (±0.01) × 10⁴ M(-1), at 37 °C and 2.17 (±0.04) × 10⁴ M(-1), at 25 °C. The quenching intensity of BSA, which contains two tryptophan residues in the peptide chain, was found to be higher than that of HSA, what suggests that the primary binding site for sulpiride in albumin should be located next to the sub domain IB of the protein structure. PMID:26742031

  1. Production of BSA-poly(ethyl cyanoacrylate) nanoparticles as a coating material that improves wetting property

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Alkyl cyanoacrylates have long been used for the synthesis of colloidal nanoparticles. In the involved polymerization reaction, OH- ions derived from dissociation of water have been used as an initiator. In the current research, an animal protein, bovine serum albumin (BSA) molecules were utilized a...

  2. Exploring the affinity binding of alkylmaltoside surfactants to bovine serum albumin and their effect on the protein stability: A spectroscopic approach.

    PubMed

    Hierrezuelo, J M; Carnero Ruiz, C

    2015-08-01

    Steady-state and time-resolved fluorescence together with circular dichroism (CD) spectroscopic studies was performed to examine the interactions between bovine serum albumin (BSA) and two alkylmaltoside surfactants, i.e. n-decyl-β-D-maltoside (β-C10G2) and n-dodecyl-β-D-maltoside (β-C12G2), having identical structures but different tail lengths. Changes in the intrinsic fluorescence of BSA from static as well as dynamic measurements revealed a weak protein-surfactant interaction and gave the corresponding binding curves, suggesting that the binding mechanism of surfactants to protein is essentially cooperative in nature. The behavior of both surfactants is similar, so that the differences detected were attributed to the more hydrophobic nature of β-C12G2, which favors the adsorption of micelle-like aggregates onto the protein surface. These observations were substantially demonstrated by data derived from synchronous, three-dimensional and anisotropy fluorescence experiments. Changes in the secondary structure of the protein induced by the interaction with surfactants were analyzed by CD to determine the contents of α-helix and β-strand. It was noted that whereas the addition of β-C10G2 appears to stabilize the secondary structure of the protein, β-C12G2 causes a marginal denaturation of BSA for a protein:surfactant molar ratio as high as 1 to 100.

  3. Preparation and Characterization of 125I Labeled Bovine Serum Albumin

    PubMed Central

    Ashwitha Rai, K. S.; Jyothi; Rasmi, R. R.; Sarnaik, Jayula; Kadwad, V. B.; Shenoy, K. B.; Somashekarappa, H. M.

    2015-01-01

    Bovine serum albumin is a model protein, which has been conventionally used as protein standard and in many areas of biochemistry, pharmacology and medicine. Radioiodination procedure for bovine serum albumin employing chloramine-T as an oxidant with slight modification was evaluated critically to establish the optimal conditions for the preparation of radiolabeled tracer (125I-BSA) with required specific activity without impairing the immune reactivity and biological activity. Optimized radioiodination procedure involving 10 µg of chloramine-T along with 20 µg of sodium metabisulphite with 60 seconds incubation at 2° yielded 125I-BSA with high integrity. PMID:25767326

  4. Gastric acid output and circulating anti-bovine serum albumin in adults

    PubMed Central

    Kraft, S. C.; Rothberg, R. M.; Knauer, C. M.; Svoboda, A. C.; Monroe, L. S.; Farr, R. S.

    1967-01-01

    Gastric acid output and circulating antibodies to a dietary protein, bovine serum albumin (BSA), were studied in 241 adult human subjects. Among individuals 41 years of age and older there was a higher than expected incidence of circulating anti-BSA which correlated with a high stimulated gastric acid output. When other possible contributing factors such as the site of gastroduodenal disease or a history of allergy were considered, the relationship between high gastric acid and an increased incidence of anti-BSA again was demonstrated. The quantity and immunoglobulin classes of anti-BSA were not correlated with differences in gastric acid output or dietary histories, except that none of the sixteen patients who denied recent BSA intake had demonstrable anti-BSA activity. These data suggest that for a given individual the capacity to maintain or reinitiate anti-BSA production is a multifaceted phenomenon subject to many variables, one of which is gastric acidity. PMID:6035195

  5. Insulin Is Required to Maintain Albumin Expression by Inhibiting Forkhead Box O1 Protein.

    PubMed

    Chen, Qing; Lu, Mingjian; Monks, Bobby R; Birnbaum, Morris J

    2016-01-29

    Diabetes is accompanied by dysregulation of glucose, lipid, and protein metabolism. In recent years, much effort has been spent on understanding how insulin regulates glucose and lipid metabolism, whereas the effect of insulin on protein metabolism has received less attention. In diabetes, hepatic production of serum albumin decreases, and it has been long established that insulin positively controls albumin gene expression. In this study, we used a genetic approach in mice to identify the mechanism by which insulin regulates albumin gene transcription. Albumin expression was decreased significantly in livers with insulin signaling disrupted by ablation of the insulin receptor or Akt. Concomitant deletion of Forkhead Box O1 (Foxo1) in these livers rescued the decreased albumin secretion. Furthermore, activation of Foxo1 in the liver is sufficient to suppress albumin expression. These results suggest that Foxo1 acts as a repressor of albumin expression.

  6. Insulin Is Required to Maintain Albumin Expression by Inhibiting Forkhead Box O1 Protein.

    PubMed

    Chen, Qing; Lu, Mingjian; Monks, Bobby R; Birnbaum, Morris J

    2016-01-29

    Diabetes is accompanied by dysregulation of glucose, lipid, and protein metabolism. In recent years, much effort has been spent on understanding how insulin regulates glucose and lipid metabolism, whereas the effect of insulin on protein metabolism has received less attention. In diabetes, hepatic production of serum albumin decreases, and it has been long established that insulin positively controls albumin gene expression. In this study, we used a genetic approach in mice to identify the mechanism by which insulin regulates albumin gene transcription. Albumin expression was decreased significantly in livers with insulin signaling disrupted by ablation of the insulin receptor or Akt. Concomitant deletion of Forkhead Box O1 (Foxo1) in these livers rescued the decreased albumin secretion. Furthermore, activation of Foxo1 in the liver is sufficient to suppress albumin expression. These results suggest that Foxo1 acts as a repressor of albumin expression. PMID:26668316

  7. Stereoselective interaction of cinchona alkaloid isomers with bovine serum albumin.

    PubMed

    Liu, Yan; Chen, Mingmao; Jiang, Longguang; Song, Ling

    2015-08-15

    The dependence of the interaction between bovine serum albumin (BSA) and two cinchona alkaloids, quinine (QN) and quinidine (QD), on the absolute configuration of these stereoisomers has been comprehensively studied. The FTIR spectra showed that QN and QD interacted with both CO and C-N groups of BSA, resulting in changes to the secondary structure of the protein. Fluorescence quenching of BSA by the stereoisomers revealed lower efficiency for QD in quenching the Trp emission of BSA when compared to QN. Further analysis accurately described the different binding behaviors and recognition discrepancies of QN and QD towards BSA, which was reflected through binding affinities, driving forces, energy changes and conformational changes during the ligand-protein interactions. Synchronous fluorescence further proved that QD was farther from Trp and Tyr than that of QN. This work could provide basic data for clarifying the binding interaction, metabolism and distribution of cinchona alkaloid stereoisomers in vivo.

  8. Spectroscopic investigation of interaction between mangiferin and bovine serum albumin

    NASA Astrophysics Data System (ADS)

    Lin, Hui; Lan, Jingfeng; Guan, Min; Sheng, Fenling; Zhang, Haixia

    2009-09-01

    The mechanism of interaction between mangiferin (MA) and bovine serum albumin (BSA) in aqueous solution was investigated by fluorescence spectra, synchronous fluorescence spectra, absorbance spectra and Fourier transform infrared (FT-IR) spectroscopy. The binding constants and binding sites of MA to BSA at different reaction times were calculated. And the distance between MA and BSA was estimated to be 5.20 nm based on Föster's theory. In addition, synchronous fluorescence and FT-IR measurements revealed that the secondary structures of the protein changed after the interaction of MA with BSA. As a conclusion, the interaction between the anti-diabetes Chinese medicine MA and BSA may provide some significant information for the mechanism of the traditional chinese medicine MA on the protein level to cure diabetes or other diseases.

  9. Competitive interactions between glucose and lactose with BSA: which sugar is better for children?

    PubMed

    Zhang, Qiulan; Ni, Yongnian; Kokot, Serge

    2016-04-01

    The interactions of the sugars glucose and lactose with the transport protein bovine serum albumin (BSA) were investigated using fluorescence, FT-IR and circular dichroism (CD) techniques. The results indicated that glucose could be bonded and transported by BSA, mainly involving hydrogen bonds and van der Waals interactions (ΔH = -86.13 kJ mol(-1)). The obtained fluorescence data from the binding of sugar and BSA were processed by the multivariate curve resolution-alternating least squares (MCR-ALS) method, and the extracted concentration profiles showed that the equilibrium constant, rglucose:BSA, was about 7. However, the binding of lactose to BSA did not quench the fluorescence significantly, and this indicated that lactose could not be directly transported by BSA. The binding experiments were further performed using the fluorescence titration method in the presence of calcium and BSA. Calcium was added so that the calcium/BSA reactions could be studied in the presence or absence of glucose, lactose or hydrolysis products. The results showed that hydrolyzed lactose seemed to enhance calcium absorption in bovine animals. It would also appear that for children, lactose provides better nutrition; however, glucose is better for adults.

  10. Metal chelate affinity precipitation: purification of BSA using poly(N-vinylcaprolactam-co-methacrylic acid) copolymers.

    PubMed

    Ling, Yuan-Qing; Nie, Hua-Li; Brandford-White, Christopher; Williams, Gareth R; Zhu, Li-Min

    2012-06-01

    This investigation involves the metal chelate affinity precipitation of bovine serum albumin (BSA) using a copper ion loaded thermo-sensitive copolymer. The copolymer of N-vinylcaprolactam with methacrylic acid PNVCL-co-MAA was synthesized by free radical polymerization in aqueous solution, and Cu(II) ions were attached to provide affinity properties for BSA. A maximum loading of 48.1mg Cu(2+) per gram of polymer was attained. The influence of pH, temperature, BSA and NaCl concentrations on BSA precipitation and of pH, ethylenediaminetetraacetic acid (EDTA) and NaCl concentrations on elution were systematically probed. The optimum conditions for BSA precipitation occurred when pH, temperature and BSA concentration were 6.0, 10°C and 1.0 mg/ml, respectively and the most favorable elution conditions were at pH 4.0, with 0.2M NaCl and 0.06 M EDTA. The maximum amounts of BSA precipitation and elution were 37.5 and 33.7 mg BSA/g polymer, respectively. It proved possible to perform multiple precipitation/elution cycles with a minimal loss of polymer efficacy. The results show that PNVCL-co-MAA is a suitable matrix for the purification of target proteins from unfractionated materials.

  11. Fusion protein of retinol-binding protein and albumin domain III reduces liver fibrosis.

    PubMed

    Lee, Hongsik; Jeong, Hyeyeun; Park, Sangeun; Yoo, Wonbaek; Choi, Soyoung; Choi, Kyungmin; Lee, Min-Goo; Lee, Mihwa; Cha, DaeRyong; Kim, Young-Sik; Han, Jeeyoung; Kim, Wonkon; Park, Sun-Hwa; Oh, Junseo

    2015-04-11

    Activated hepatic stellate cells (HSCs) play a key role in liver fibrosis, and inactivating HSCs has been considered a promising therapeutic approach. We previously showed that albumin and its derivative designed for stellate cell-targeting, retinol-binding protein-albumin domain III fusion protein (referred to as R-III), inactivate cultured HSCs. Here, we investigated the mechanism of action of albumin/R-III in HSCs and examined the anti-fibrotic potential of R-III in vivo. R-III treatment and albumin expression downregulated retinoic acid (RA) signaling which was involved in HSC activation. RA receptor agonist and retinaldehyde dehydrogenase overexpression abolished the anti-fibrotic effect of R-III and albumin, respectively. R-III uptake into cultured HSCs was significantly decreased by siRNA-STRA6, and injected R-III was localized predominantly in HSCs in liver. Importantly, R-III administration reduced CCl4- and bile duct ligation-induced liver fibrosis. R-III also exhibited a preventive effect against CCl4-inducd liver fibrosis. These findings suggest that the anti-fibrotic effect of albumin/R-III is, at least in part, mediated by downregulation of RA signaling and that R-III is a good candidate as a novel anti-fibrotic drug.

  12. Recombinant fusion protein of albumin-retinol binding protein inactivates stellate cells

    SciTech Connect

    Choi, Soyoung; Park, Sangeun; Kim, Suhyun; Lim, Chaeseung; Kim, Jungho; Cha, Dae Ryong; Oh, Junseo

    2012-02-03

    Highlights: Black-Right-Pointing-Pointer We designed novel recombinant albumin-RBP fusion proteins. Black-Right-Pointing-Pointer Expression of fusion proteins inactivates pancreatic stellate cells (PSCs). Black-Right-Pointing-Pointer Fusion proteins are successfully internalized into and inactivate PSCs. Black-Right-Pointing-Pointer RBP moiety mediates cell specific uptake of fusion protein. -- Abstract: Quiescent pancreatic- (PSCs) and hepatic- (HSCs) stellate cells store vitamin A (retinol) in lipid droplets via retinol binding protein (RBP) receptor and, when activated by profibrogenic stimuli, they transform into myofibroblast-like cells which play a key role in the fibrogenesis. Despite extensive investigations, there is, however, currently no appropriate therapy available for tissue fibrosis. We previously showed that the expression of albumin, composed of three homologous domains (I-III), inhibits stellate cell activation, which requires its high-affinity fatty acid-binding sites asymmetrically distributed in domain I and III. To attain stellate cell-specific uptake, albumin (domain I/III) was coupled to RBP; RBP-albumin{sup domain} {sup III} (R-III) and albumin{sup domain} {sup I}-RBP-albumin{sup III} (I-R-III). To assess the biological activity of fusion proteins, cultured PSCs were used. Like wild type albumin, expression of R-III or I-R-III in PSCs after passage 2 (activated PSCs) induced phenotypic reversal from activated to fat-storing cells. On the other hand, R-III and I-R-III, but not albumin, secreted from transfected 293 cells were successfully internalized into and inactivated PSCs. FPLC-purified R-III was found to be internalized into PSCs via caveolae-mediated endocytosis, and its efficient cellular uptake was also observed in HSCs and podocytes among several cell lines tested. Moreover, tissue distribution of intravenously injected R-III was closely similar to that of RBP. Therefore, our data suggest that albumin-RBP fusion protein comprises

  13. Photophysical investigations of squaraine and cyanine dyes and their interaction with bovine serum albumin

    NASA Astrophysics Data System (ADS)

    Saikiran, M.; Sato, D.; Pandey, S. S.; Kato, T.

    2016-04-01

    A model far-red sensitive symmetrical squaraine dye (SQ-3) and unsymmetrical near infra-red sensitive cyanine dye (UCD-1) bearing direct-COOH functionalized indole ring were synthesized, characterized and subjected to photophysical investigations including their interaction with bovine serum albumin (BSA) as a model protein in phosphate buffer solution (PBS). Both of the dyes exhibit strong interaction with BSA in phosphate buffer with high apparent binding constant. A judicious tuning of hydrophobic main backbone with reactive functionality for associative interaction with active site of BSA has been found to be necessary for BSA detection in PBS.

  14. Photophysical investigations of squaraine and cyanine dyes and their interaction with bovine serum albumin

    NASA Astrophysics Data System (ADS)

    Saikiran, M.; Sato, D.; Pandey, S. S.; Kato, T.

    2016-04-01

    A model far-red sensitive symmetrical squaraine dye (SQ-3) and unsymmetrical near infra-red sensitive cyanine dye (UCD-1) bearing direct–COOH functionalized indole ring were synthesized, characterized and subjected to photophysical investigations including their interaction with bovine serum albumin (BSA) as a model protein in phosphate buffer solution (PBS). Both of the dyes exhibit strong interaction with BSA in phosphate buffer with high apparent binding constant. A judicious tuning of hydrophobic main backbone with reactive functionality for associative interaction with active site of BSA has been found to be necessary for BSA detection in PBS.

  15. Molecular Aspects of the Interaction of Iminium and Alkanolamine Forms of the Anticancer Alkaloid Chelerythrine with Plasma Protein Bovine Serum Albumin.

    PubMed

    Bhuiya, Sutanwi; Pradhan, Ankur Bikash; Haque, Lucy; Das, Suman

    2016-01-14

    The interaction between a quaternary benzophenanthridine alkaloid chelerythrine (herein after, CHL) and bovine serum albumin (herein after, BSA) was probed by employing various spectroscopic tools and isothermal titration calorimetry (ITC). Fluorescence studies revealed that the binding affinity of the alkanolamine form of the CHL is higher compared to the iminium counterpart. This was further established by fluorescence polarization anisotropy measurement and ITC. Fluorescence quenching study along with time-resolved fluorescence measurements establish that both forms of CHL quenched the fluorescence intensity of BSA through the mechanism of static quenching. Site selective binding and molecular modeling studies revealed that the alkaloid binds predominantly in the BSA subdomain IIA by electrostatic and hydrophobic forces. From Forster resonance energy transfer (FRET) studies, the average distances between the protein donor and the alkaloid acceptor were found to be 2.71 and 2.30 nm between tryptophan (Trp) 212 (donor) and iminium and alkanolamine forms (acceptor), respectively. Circular dichroism (CD) study demonstrated that the α-helical organization of the protein is reduced due to binding with CHL along with an increase in the coiled structure. This is indicative of a small but definitive partial unfolding of the protein. Thermodynamic parameters obtained from ITC experiments revealed that the interaction is favored by negative enthalpy change and positive entropy change. PMID:26653994

  16. Rotational diffusion of bovine serum albumin denaturated by sodium dodecylsulfate, According to data from tryptophan fluorescence

    NASA Astrophysics Data System (ADS)

    Vlasova, I. M.; Zhuravleva, V. V.; Saletskii, A. M.

    2014-03-01

    The rotational diffusion of bovine serum albumin (BSA) molecules in solutions with different concentrations of the anionic detergent sodium dodecylsulfate (SDS) at different pH values is investigated, yielding information on the denaturation of BSA under the action of SDS. It is found from the increased degree of polarization in the tryptophan fluorescence of BSA and the registered parameters for the rotational diffusion of BSA molecules that the denaturation of BSA under the action of SDS at pH values less than the isoelectric point (pI) of BSA (4-9) is a two-stage process. It is shown that the first stage of BSA denaturation common for all pH values is the decondensation of BSA globules, while the second stage of BSA denaturation at pH greater than the pI of BSA is the unfolding of the protein's amino acid chain. It is concluded that the denaturation of BSA under the action of SDS proceeds more deeply at pH values greater than the pI of BSA.

  17. Complexes of photosensitizer and CdSe/ZnS quantum dots passivated with BSA: optical properties and intracomplex energy transfer

    NASA Astrophysics Data System (ADS)

    Kuznetsova, Vera; Orlova, Anna; Martynenko, Irina; Kundelev, Evgeny; Maslov, Vladimir; Fedorov, Anatoly; Baranov, Alexander; Gun'ko, Yurii

    2016-04-01

    Here we report our investigations of the formation conditions and photophysical properties of complexes between luminescent semiconducting nanoparticles (quantum dots, QDs) and the photosensitizer chlorin e6, which is widely used for the photodynamic therapy. In our complexes, bovine serum albumin (BSA), the most abundant protein in blood serum, was used as a linker between QDs and chlorin e6 molecules. The influence of BSA on the optical properties of Ce6 and QDs in complexes was properly examined using spectral-luminescent methods. It was found that BSA passivated QD surface and substantially QD quantum yield of luminescence was increased. In addition, BSA prevented the aggregation of chlorin e6 molecules in complexes with QDs. We demonstrated that the use of BSA as a linker allows to create functional QD-chlorin e6 complexes with effective photoexcitation energy transfer from QDs to the molecules.

  18. 5alpha-Reduced androgens block estradiol-BSA-stimulated release of oxytocin.

    PubMed

    Caldwell, Jack D; Song, Yan; Englöf, Ila; Höfle, Simone; Key, Mary; Morris, Mariana

    2003-06-27

    In this study we test the postulate that estradiol conjugated to bovine serum albumin (E-BSA) acts via receptors for the steroid-binding protein sex hormone binding globulin (SHBG) by attempting to block E-BSA-stimulated release of oxytocin with two antagonists of SHBG receptor actions: the 5alpha-reduced androgens dihydrotestosterone (DHT) and 3alpha-diol. Simultaneous superfusion with either DHT or 3alpha-diol significantly blocked E-BSA-stimulated release of oxytocin. We also found that a wide range of free 17beta-estradiol was unable to stimulate oxytocin release, suggesting that E-BSA stimulates receptors other than those for free estradiol to release oxytocin, perhaps SHBG receptors.

  19. Spectroscopic studies of the interaction between tetra-substituted aluminum phthalocyanines and bovine serum albumin

    NASA Astrophysics Data System (ADS)

    He, Yipeng; Zheng, Liqin; Huang, Yide; Lin, Pingping; Yang, Hongqin; Peng, Yiru

    2014-11-01

    Serum albumin, the most abundant plasma protein in mammalian blood, shows significant effects on delivery and therapeutic efficacy of drugs, therefore, the investigation of binding interaction between serum albumin and drugs is vital and necessary. In the present study, the binding interaction of two aluminum (III) phthalocyanine (AlPc) derivatives, tetrasulfonate- and tetra-(p-sulfoazophenyl-4-aminosulfonyl)-substituted AlPc (complexes 1 and 2), with bovine serum albumin (BSA) was investigated by UV-Vis and fluorescence spectroscopy. Adding BSA to the Pc complexes in water caused remarkable changes in the Q-band of the Pc complexes, indicating an altered aggregation behavior. When titrating these AlPcs with BSA in PBS, the intrinsic fluorescence of BSA was significantly quenched through a static quenching process. The binding of Pc complexes to BSA might change its conformation, evidenced by the red shift of maximum emission wavelength. Furthermore, binding constants and binding sites were obtained and binding ability between the Pc complexes and BSA was assessed. Our results suggest that complexes 1 and 2 readily interact with BSA whereas the latter shows more affinity (with higher binding constant value) to BSA, implying the stretched amphiphilic substituents of complex 2 may contribute to their transportation in the blood.

  20. Fatty acids exacerbate tubulointerstitial injury in protein-overload proteinuria.

    PubMed

    Thomas, Mark E; Harris, Kevin P G; Walls, John; Furness, Peter N; Brunskill, Nigel J

    2002-10-01

    The role of the albumin-carried fatty acids in the induction of tubulointerstitial injury was studied in protein-overload proteinuria. Rats were injected with fatty acid-carrying BSA [FA(+)BSA], fatty acid-depleted BSA [FA(-)BSA], or saline. Macrophage infiltration was measured by immunohistochemical staining, apoptotic cells were detected by in situ end labeling, and proliferating cells were identified by in situ hybridization for histone mRNA. Macrophage infiltration was significantly greater in the FA(+)BSA group than in the FA(-)BSA and saline groups. The infiltrate was largely restricted to the outer cortex. Apoptosis was greater in the FA(+)BSA group than in the FA(-)BSA and saline groups. Compared with the saline group, apoptosis was significantly increased in the FA(+)BSA group but not in the FA(-)BSA group. Cortical cells proliferated significantly more in the FA(+)BSA and FA(-)BSA groups than in the saline group. FA(+)BSA is therefore a more potent inducer of macrophage infiltration and cell death than FA(-)BSA. The fatty acids carried on albumin may be the chief instigators of tubulointerstitial injury in protein-overload proteinuria. PMID:12217854

  1. Competitive binding of phenylbutazone and colchicine to serum albumin in multidrug therapy: A spectroscopic study

    NASA Astrophysics Data System (ADS)

    Sułkowska, A.; Maciążek-Jurczyk, M.; Bojko, B.; Równicka, J.; Zubik-Skupień, I.; Temba, E.; Pentak, D.; Sułkowski, W. W.

    2008-06-01

    The binding sites for phenylbutazone and colchicine were identified in tertiary structure of bovine and human serum albumin with the use of spectrofluorescence analysis. It was found that phenylbutazone has two binding sites in both sera albumins (HSA and BSA), while colchicine has one binding site in BSA as well as in HSA. The comparison of the quenching effect of BSA and HSA fluorescence by phenylbutazone and colchicine allows us to identify subdomain IIA in protein as the binding site for these two drugs. In this subdomain tryptophan 214 is located. The participation of tyrosyl and tryptophanyl residues of protein was also estimated in the drug-albumin complex. The comparison of quenching of fluorescence of HSA and BSA excited at 280 nm with that at 295 nm allowed us to state that the participation of tyrosyl residues of albumin in the phenylbutazone-serum albumin interaction is significant. The analysis of quenching of fluorescence of BSA in the binary and ternary systems showed that phenylbutazone does not affect the complex formed between colchicine and BSA. Similarly, colchicine has no effect on the Phe-BSA complex. However marked differences were observed for the complex with HSA. On the basis of Ka and KQ values it was concluded that colchicine may probably cause displacement of phenylbutazone from its complex with serum albumin (SA). Static and dynamic quenching for the binary and ternary systems is also discussed. The competition of phenylbutazone and colchicine in binding to serum albumin should be taken into account in the multi-drug therapy.

  2. Impairment of human keratinocyte mobility and proliferation by advanced glycation end products-modified BSA.

    PubMed

    Zhu, Ping; Yang, Chuan; Chen, Li-Hong; Ren, Meng; Lao, Guo-Juan; Yan, Li

    2011-07-01

    The migration and proliferation of keratinocytes is critical to wound re-epithelialization and defects in this function are associated with the clinical phenomenon of chronic non-healing wounds. Advanced glycation end products (AGEs) occur through non-enzymatic glycation of long-lived proteins in diabetes and play important roles in diabetic complications. However, specific roles for AGEs in keratinocyte migration and proliferation, and the underlying molecular mechanisms, have not been fully established. The aim of the current study was to elucidate the interaction between AGE-modified bovine serum albumin (AGE-BSA) and keratinocytes. As a result, we found that AGE-BSA had no effect on the viability of keratinocytes for up to 48 h of incubation with 50 μg/ml of AGE-BSA. AGE-BSA (but not non-glycated BSA) exerted a concentration-dependent suppression of keratinocyte migration at a range of concentrations. The expression of matrix metalloproteinase-9 (MMP-9) was significantly up-regulated in keratinocytes incubated with increasing AGE-BSA, but tissue inhibitor of metalloproteinases-1 (TIMP-1) expression was down-regulated. AGE-BSA also profoundly depressed phospho-focal adhesion kinase-Tyr397 (p-FAK) and α2β1 integrin expression, while total-FAK expression levels remained constant, in keratinocytes. The proliferative capacity of keratinocytes was diminished after 72 h AGE-BSA incubation. Taken together, these findings suggested that in the presence of AGE-BSA, keratinocytes lose their migratory and proliferation abilities. These data also indicated that, in the context of the chronic hyperglycemia in diabetes, the effects of AGE-BSA on keratinocyte migration might be mediated through MMP-9/TIMP-1, p-FAK and α2β1 integrin.

  3. Larger red-shift in optical emissions obtained from the thin films of globular proteins (BSA, lysozyme) - polyelectrolyte (PAA) complexes

    NASA Astrophysics Data System (ADS)

    Talukdar, Hrishikesh; Kundu, Sarathi; Basu, Saibal

    2016-09-01

    Globular proteins (lysozyme and BSA) and polyelectrolyte (sodium polyacrylic acid) are used to form protein-polyelectrolyte complexes (PPC). Out-of-plane structures of ≈30-60 nm thick PPC films and their surface morphologies have been studied by using X-ray reflectivity and atomic force microscopy, whereas optical behaviors of PPC and protein conformations have been studied by using UV-vis, photoluminescence and FTIR spectroscopy respectively. Our study reveals that thin films of PPC show a larger red-shift of 23 and 16 nm in the optical emissions in comparison to that of pure protein whereas bulk PPC show a small blue-shift of ≈3 nm. A small amount of peak-shift is found to occur due to the heat treatment or concentration variation of the polyelectrolyte/protein in bulk solution but cannot produce such film thickness independent larger red-shift. Position of the emission peak remains nearly unchanged with the film thickness. Mechanism for such larger red-shift has been proposed.

  4. Humoral and cell-mediated immunity following vaccination with synthetic Candida cell wall mannan derived heptamannoside-protein conjugate: immunomodulatory properties of heptamannoside-BSA conjugate.

    PubMed

    Paulovičová, Lucia; Paulovičová, Ema; Karelin, Alexander A; Tsvetkov, Yury E; Nifantiev, Nikolay E; Bystrický, Slavomír

    2012-10-01

    Chemically defined glycoprotein conjugate composed of synthetically prepared mannan-derived heptamannoside with terminal β-1,2-linked mannose residue attached to the α-1,3-linked mannose residues and BSA as carrier protein (M7-BSA conjugate) was analysed for the capacity to induce protective humoral immunity and appropriate alteration cellular immunity. To identify protective antigenic structure of Candida cell wall mannan M7-BSA conjugate was used for BALB/c mice immunization. The obtained results were compared with placebo group and with heat-inactivated C. albicans whole cells immunization. The administration route of M7-BSA conjugate secondary booster injection significantly affected the intensity of humoral immune response and the specificity of produced antibodies. All prepared sera were able to elevate candidacidal activity of polymorphonuclear leukocytes (PMN) in cooperation with complement. Moreover, polyclonal sera obtained after secondary subcutaneous (s.c.) booster injection of M7-BSA conjugate were able to induce candidacidal activity of PMN also in complement independent manner. M7-BSA conjugate immunization induced increases of phagocytic activity and respiratory burst of granulocytes, caused a raise of the proportion of CD3(+) T lymphocytes and increased the CD4(+)/CD8(+) T lymphocyte ratio. We observed also an increasing proportion of CD4(+)CD25(+) T cells compared to immunization with heat inactivated whole C. albicans cells, which in turn promoted an increase of the CD8(+)CD25(+) cell proportion. Immunization with M7-BSA conjugate induced Th1, Th2 and Th17 immune responses as indicated by the elevation of relevant cytokines levels. These data provide some insights on the immunomodulatory properties of oligomannosides and contribute to the development of synthetic oligosaccharide vaccines against fungal diseases.

  5. Is the pre-Tg DSC endotherm observed with solid state proteins associated with the protein internal dynamics? Investigation of bovine serum albumin by solid state hydrogen/deuterium exchange.

    PubMed

    Mizuno, Masayasu; Pikal, Michael J

    2013-10-01

    DSC thermograms of solid state pure proteins often show a distinct endotherm at a temperature far below the glass transition temperature of the system (Tg). We hypothesized this endotherm represents enthalpy recovery associated with an internal mobility transition of the protein molecule. Although the existence of an internal transition has been postulated, whether this endotherm is associated with such a transition has not previously been discussed. The purpose of this study was to investigate the origin of the pre-Tg endotherm in lyophilized bovine serum albumin (BSA). Due to strong glass behavior, the system Tg was determined by extrapolating Tg data of disaccharide/BSA formulations to zero saccharide. A small pre-Tg endotherm around 40-60 °C was observed in amorphous BSA equilibrated at 11%RH. The apparent activation energy suggested the endotherm was "α-mobility"-related. A solid state hydrogen/deuterium exchange study using FTIR was conducted over a temperature range spanning the endotherm. We found a fast phase, followed by essentially a plateau level which is highly temperature dependent in the 40-60 °C range, suggesting enhanced internal protein motion as the system passes through the temperature range of the endotherm. These results suggest the pre-Tg endotherm is associated with a protein internal mobility transition.

  6. Study on the interaction of sodium morin-5-sulfonate with bovine serum albumin by spectroscopic techniques

    NASA Astrophysics Data System (ADS)

    Shahabadi, Nahid; Mohammadpour, Mahnaz

    2012-02-01

    In the present investigation, an attempt has been made to study the interaction of sodium morin-5-sulfonate (NaMSA) with the transport proteins, bovine serum albumin (BSA) employing UV-vis, fluorometric and circular dichroism (CD) techniques. The experimental results indicated that the quenching mechanism of BSA by the compound was a static procedure. Various binding parameters were evaluated. The negative value of Δ H, positive value of Δ S and the negative value of Δ G indicated that electrostatic interactions and hydrogen bonding play major roles in the binding of the NaMSA and BSA. Based on the Forster's theory of non-radiation energy transfer, the binding distance, r, between the donor (BSA) and acceptor (NaMSA) was evaluated. The results of CD and UV-vis spectroscopy showed that the binding of this complex to BSA induces some conformational changes in BSA.

  7. Study on interaction between a new fluorescent probe 2-methylbenzo[b][1,10]phenanthrolin-7(12H)-one and BSA.

    PubMed

    Qiu, Bin; Guo, Longhua; Chen, Mingluan; Lin, Zhenyu; Chen, Guonan

    2011-03-01

    A new fluorescence reagent, 2-methylbenzo[b][1,10]phenanthrolin-7(12H)-one (mBPO), synthesized in our laboratory was used as the probe for protein and its interaction with Bovine Serum Albumin (BSA) was investigated in detail in this paper. It was found that BSA had the ability to quench the fluorescence of mBPO at 411 nm (λ(ex) = 286 nm), and the quenched intensity of fluorescence was proportional to the concentration of BSA. Based on this fact, mBPO has been used as a fluorescence probe for the detection of BSA. Under the optimal conditions, the calibration graph is linear up to 0.5 mg L(-1) for BSA and the limit of detection (LOD) was 0.06 mg L(-1). The regression equation is y = 1048.8x + 7.2093 with R(2) = 0.9913. The mechanism for the interaction of mBPO with BSA was also studied, while the binding constant and the number of binding sites were calculated. According to the thermodynamics parameter, the binding mode between mBPO and BSA was deduced. The results suggested the interaction between mBPO and BSA to be hydrophobic force in nature. It also proved that the fluorescence quenching reaction was affected by the tryptophan residue of BSA. For there are two tryptophan (Trp) residues, in site 134 and site 212 of BSA, and mBPO maybe has interaction with them respectively.

  8. Purification, identification and preliminary crystallographic studies of a 2S albumin seed protein from Lens culinaris

    SciTech Connect

    Gupta, Pankaj; Gaur, Vineet; Salunke, Dinakar M.

    2008-08-01

    A 2S albumin from L. culinaris was purified and crystallized and preliminary crystallographic studies were carried out. Lens culinaris (lentil) is a widely consumed high-protein-content leguminous crop. A 2S albumin protein (26.5 kDa) has been identified using NH{sub 2}-terminal sequencing from a 90% ammonium sulfate saturation fraction of total L. culinaris seed protein extract. The NH{sub 2}-terminal sequence shows very high homology to PA2, an allergy-related protein from Pisum sativum. The 2S albumin protein was purified using a combination of size-exclusion and ion-exchange chromatography. Crystals of the 2S seed albumin obtained using the hanging-drop vapour-diffusion method diffracted to 2.5 Å resolution and were indexed in space group P4{sub 1} (or P4{sub 3}), with unit-cell parameters a = b = 78.6, c = 135.2 Å.

  9. Protein adsorption and cell adhesion on nanoscale bioactive coatings formed from poly(ethylene glycol) and albumin microgels

    PubMed Central

    Scott, Evan A.; Nichols, Michael D.; Cordova, Lee H.; George, Brandon J.; Jun, Young-Shin; Elbert, Donald L.

    2008-01-01

    Late-term thrombosis on drug-eluting stents is an emerging problem that might be addressed using extremely thin, biologically-active hydrogel coatings. We report a dip-coating strategy to covalently link poly(ethylene glycol) (PEG) to substrates, producing coatings with <≈100 nm thickness. Gelation of PEG-octavinylsulfone with amines in either bovine serum albumin (BSA) or PEG-octaamine was monitored by dynamic light scattering (DLS), revealing the presence of microgels before macrogelation. NMR also revealed extremely high end group conversions prior to macrogelation, consistent with the formation of highly crosslinked microgels and deviation from Flory-Stockmayer theory. Before macrogelation, the reacting solutions were diluted and incubated with nucleophile-functionalized surfaces. Using optical waveguide lightmode spectroscopy (OWLS) and quartz crystal microbalance with dissipation (QCM-D), we identified a highly hydrated, protein-resistant layer with a thickness of approximately 75 nm. Atomic force microscopy in buffered water revealed the presence of coalesced spheres of various sizes but with diameters less than about 100 nm. Microgel-coated glass or poly(ethylene terephthalate) exhibited reduced protein adsorption and cell adhesion. Cellular interactions with the surface could be controlled by using different proteins to cap unreacted vinylsulfone groups within the coating. PMID:18771802

  10. 21 CFR 862.1645 - Urinary protein or albumin (nonquantitative) test system.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry Test Systems § 862.1645 Urinary protein or albumin (nonquantitative) test system. (a... are characterized by proteinuria or albuminuria. (b) Classification. Class I (general controls)....

  11. The Effect of Albumin on MRP2 and BCRP in the Vesicular Transport Assay

    PubMed Central

    Kidron, Heidi

    2016-01-01

    The ABC transporters multidrug resistance associated protein 2 (MRP2) and breast cancer resistance protein (BCRP) are of interest in drug development, since they affect the pharmacokinetics of several drugs. Membrane vesicle transport assays are widely used to study interactions with these proteins. Since albumin has been found to affect the kinetics of metabolic enzymes in similar membrane preparations, we investigated whether albumin affects the kinetic parameters of efflux transport. We found that albumin increased the Vmax of 5(6)-carboxy-2’,7’-dichlorofluorescein (CDCF) and estradiol-17-β-D-glucuronide uptake into MRP2 vesicles in the presence of 0.1% bovine serum albumin (BSA) by 2 and 1.5-fold, respectively, while BSA increased Lucifer yellow uptake by 30% in BCRP vesicles. Km values increased slightly, but the change was not statistically significant. The effect of BSA on substrate uptake was dependent on the vesicle amount, while increasing BSA concentration did not significantly improve substrate uptake. These results indicate a minor effect of albumin on MRP2 and BCRP, but it should be considered if albumin is added to transporter assays for example as a solubilizer, since the effect may be substrate or transporter specific. PMID:27706255

  12. Synthesis and Characterization of BSA Conjugated Silver Nanoparticles (Ag/BSA Nanoparticles) and Evaluation of Biological Properties of Ag/BSA Nanoparticles and Ag/BSA Nanoparticles Loaded Poly(hydroxy butyrate valerate) PHBV Films

    NASA Astrophysics Data System (ADS)

    Ambaye, Almaz

    Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa are the etiological agents of several infectious diseases. Antibiotic resistance by these three microbes has emerged as a prevalent problem due in part to the misuse of existing antibiotics and the lack of novel antibiotics. Nanoparticles have emerged as an alternative antibacterial agents to conventional antibiotics owing to their high surface area to volume ratio and their unique chemical and physical properties. Among the nanoparticles, silver nanoparticles have gained increasing attention because silver nanoparticles exhibit antibacterial activity against a range of gram positive and gram negative bacteria. Nanoparticles of well-defined chemistry and morphology can be used in broad biomedical applications, especially in bone tissue engineering applications, where bone infection by bacteria can be acute and lethal. It is commonly noted in the literature that the activity of nanoparticles against microorganisms is dependent upon the size and concentration of the nanoparticles as well as the chemistry of stabilizing agent. To the best of our knowledge, a comprehensive study that evaluates the antibacterial activity of well characterized silver nanoparticles in particular Bovine Serum Albumin (BSA) stabilized against S. aureus and E. coli and cytotoxicity level of BSA stabilized silver nanoparticles towards osteoblast cells (MC3T3-E1) is currently lacking. Therefore, the primary objective of this study was to characterize protein conjugated silver nanoparticles prepared by chemical reduction of AgNO3 and BSA mixture. The formation of Ag/BSA nanoparticles was studied by UV-Vis spectroscopy. The molar ratio of silver to BSA in the Ag/BSA nanoparticles was established to be 27+/- 3: 1, based on Thermogravimetric Analysis and Atomic Absorption Spectroscopy. Based on atomic force microscopy, dynamic light scattering,and transmission electron microscopy(TEM) measurements, the particle size (diameter) of

  13. Protein degradation by ruminal microorganisms from sheep fed dietary supplements of urea, casein, or albumin.

    PubMed Central

    Wallace, R J; Broderick, G A; Brammall, M L

    1987-01-01

    Ruminal fluid from sheep fed hay plus concentrate diets containing 1.8% urea, 6% casein, or 6% egg albumin had proteolytic activities of 4.12, 3.02, or 4.00 mg of [14C]casein hydrolyzed ml-1 h-1, respectively. Dietary albumin had no effect on the rate of albumin breakdown relative to that of casein (0.06). Greater numbers of highly proteolytic bacteria, mainly Butyrivibrio spp., were isolated from the rumens of sheep receiving albumin. Albumin hydrolysis by these isolates was even slower relative to that of casein (0.03) than in ruminal fluid and was similar to that found in isolates from urea- and casein-fed sheep. Hence, there appears to be no mechanism by which ruminal bacteria can alter their proteolytic activity to utilize resistant soluble protein more effectively. PMID:3579280

  14. Changes of protein solutions during storage: a study of albumin pharmaceutical preparations.

    PubMed

    Christiansen, Cathrine; Skotland, Tore

    2010-03-05

    During the production of air-filled albumin microspheres, to be used as an ultrasound contrast agent, it was observed that some albumin lots could not be used owing to albumin precipitation. In order to understand the reason for these lot-to-lot variations, 24 lots of 5% (w/v) human albumin pharmaceutical preparations were analysed. The results revealed that the good albumin lots all contained <0.03 mol of free SH groups per mol of albumin. The precipitation observed with other lots was most probably due to higher amounts of free SH groups. The lower amount of free SH groups in the good lots correlated with: (i) a yellow colour of the solutions and a UV-visible spectrum similar to that observed for non-enzymatic glycosylation; (ii) a decreased fructosamine content; (iii) an increased mobility against the anode in isoelectric focusing; and (iv) an increased truncation of the two N-terminal amino acids. No, or only small, differences were observed for the amounts of albumin dimer, albumin aggregates and protein impurities, and these could not account for the albumin precipitation. The differences observed between the albumin lots were most probably due to varying storage times and/or storage conditions, and incubation experiments revealed changes in all parameters that differed between the good and bad lots. Increasing the storage temperature or exposing the solutions to light resulted in a faster decrease of free SH groups and increase of the yellow colouration. It is likely that at least some of the changes observed were due to reactive degradation products formed from the stabilizer N-acetyl-L-tryptophan. The results presented should also be of interest regarding the storage of monoclonal antibodies and other proteins used in pharmaceuticals.

  15. Albumin-fatty acid interactions at monolayer interface

    NASA Astrophysics Data System (ADS)

    Gew, Lai Ti; Misran, Misni

    2014-05-01

    The fluid mosaic model of Singer and Nicolson in 1972 shows how proteins are embedded in membranes. To elucidate the interactions between proteins and the surrounding lipids, stearic acid (SA) and bovine serum albumin (BSA) were used as lipid-protein components to mimic the normal membrane bilayer environment using the Langmuir-Blodgett technique. Surface pressure ( π)-molecular area ( A) isotherms were recorded for the SA monolayer in the presence of BSA on water. The mixed monolayer was successfully transferred onto an oxidized silicon wafer and imaged by tapping mode atomic force microscopy (AFM). Miscibility, compressibility and thermodynamic stability of the mixed system were examined. A large negative deviation of A ex, together with the minimum value of Δ G ex, was observed when the mole fraction of BSA ( X BSA) was 0.8, indicating this to be the most stable mixture. In a compressibility analysis, X BSA was observed at below 50 mN m-1, denoting a liquid-expanded phase and showing the occurrence of a strong interaction of SA with BSA molecules in this phase. AFM observations supported the quantitative data indicating that BSA was strongly attracted onto the membrane surface as predicted.

  16. A comparison study on the binding of hesperetin and luteolin to bovine serum albumin by spectroscopy

    NASA Astrophysics Data System (ADS)

    Tang, Lin; Jia, Wanteng

    2013-02-01

    Binding mechanism of luteolin (LUT) and hesperetin (HES) to bovine serum albumin (BSA) was investigated at 288,298,310 K and pH = 7.40 by UV absorption spectroscopy, fluorescence quenching and synchronous fluorescence spectroscopy. Under simulated physiological conditions, the fluorescence data indicated that hesperetin binding to BSA mainly occurs through a static mechanism. In contrast, binding of luteolin to BSA is a combined quenching process while static quenching is prevailing. Linear interval of the Stern-Volmer plot of LUT-BSA for the concentration ratio of LUT to BSA ranged from 0.5 to 1.25 was obtained. The thermodynamic parameters obtained from the Van't Hoff equation indicated that electrostatic force was the predominant force in the LUT-BSA and HES-BSA complex. The inner filter effect was eliminated to get accurate data. The conformational changes of BSA caused by LUT and HES were observed in the UV absorption. Results of fluorescence quenching and synchronous fluorescence showed that degree of luteolin-BSA quenching was higher than hesperetin-BSA quenching, which indicated that the 4'-hydroxide radical was more helpful to the ligand binding to proteins than 4'-methoxyl group for flavones.

  17. Culture of ovine embryos in the absence of bovine serum albumin.

    PubMed

    Russler-Long, J A; Dickey, J F; Richardson, M E; Ivey, K W

    1991-02-01

    Bovine serum albumin (BSA), a relatively impure protein, is routinely used as a component of embryo culture media. Since media containing BSA are chemically undefined, it would be desirable to replace BSA with substitutes of similar activity which are either chemically better defined and/or better standardized than BSA. Two commercial products, Ultroser((R)) G (USG) and Solcoseryl((R)) (SOL), were evaluated as replacements for BSA in culture with respect to the development of ovine embryos in vitro. A total of 126 late 8-cell and early 16-cell embryos were distributed among modified Brinster's medium for ovum culture (BMOC-2) containing either 1.5% BSA, 2.0% USG or 2.0% SOL. All three culture media supported development of ovine embryos. Results indicate that 8- and 16-cell embryos will develop into blastocysts in a BSA-free medium containing either USG or SOL. A higher number of embryos developed into blastocysts in media containing BSA than in media containing USG or SOL, and more blastocysts hatched in media containing BSA. Although the overall degree of embryonic development was more advanced in BSA-supplemented media, the concentrations of USG and SOL that were used in this study may not have been optimal for ovine embryo culture. PMID:16726908

  18. New insight into the binding interaction of hydroxylated carbon nanotubes with bovine serum albumin.

    PubMed

    Guan, Yonghui; Zhang, Hongmei; Wang, Yanqing

    2014-04-24

    In order to understand the effects of carbon nanotubes on the structural stability of proteins, the ligand-binding ability, fibrillation, and chemical denaturation of bovine serum albumin in the presence of a multi-walled hydroxylated carbon nanotubes (HO-MWCNTs) was characterized by UV-vis, circular dichroism, fluorescence spectroscopy and molecule modeling methods at the molecular level. The experiment results indicated that the fluorescence intensity of BSA was decreased obviously in presence of HO-MWCNTs. The binding interaction of HO-MWCNTs with BSA led to the secondary structure changes of BSA. This interaction could not only affect the ligand-binding ability of BSA, but also change the rate of fibrillation and denaturation of BSA. This work gave us some important information about the structures and properties of protein induced by carbon nanotubes.

  19. Study on the interaction between bovine serum albumin and starch nanoparticles prepared by isoamylolysis and recrystallization.

    PubMed

    Ji, Na; Qiu, Chao; Li, Xiaojing; Xiong, Liu; Sun, Qingjie

    2015-04-01

    The current study primarily investigated the interaction of bovine serum albumin (BSA) with starch nanoparticles (SNPs) prepared by isoamylolysis and recrystallization using UV-vis, fluorescence, transmission electron microscopy (TEM), Fourier transform infrared (FTIR) and circular dichroism (CD). The enhanced absorbance observed by UV-vis spectroscopy and decreased intensity of fluorescence spectroscopy suggested that BSA could bind to SNPs and form a BSA-SNP complex. The synchronous fluorescence spectra revealed that the emission maximum of Tyr residue (at Δλ=15nm) was red-shifted at the investigated concentrations range, indicating that the conformation of BSA was changed. Quenching parameters showed that the quenching effect of SNPs was static quenching. TEM images showed that the SNPs were surrounded by protein coronae, indicating that nanoparticle-protein complexes had formed. The FTIR and CD characterization indicated that the SNPs induced structural changes in the secondary structure of BSA.

  20. Influence of diets with different levels of protein and energy on liver albumin content in the rat.

    PubMed

    Maurice, M; Lardeux, B; De Saint-Steban, C; Bourdel, G; Feldmann, G

    1986-11-01

    The influence of protein ingestion on liver albumin synthesis and albumin content was investigated in rats fed protein as a meal (90% casein) given apart from the other dietary components provided ad libitum. In this condition, protein ingestion rapidly stimulates liver total protein synthesis. Separately fed rats were studied 6 and 20 h after the protein meal. Control rats fed mixed diets containing 13 or 80% casein were killed either during the absorptive (night) or postabsorptive (light) periods. The ratio of hepatic albumin synthesis to total protein synthesis remained fairly constant (12-15%) in all groups, indicating that albumin synthesis paralleled total protein synthesis. Liver albumin content measured in microsomes by immunonephelometry was significantly higher in separately fed rats killed 6 h postmeal than in those killed after 20 h. In rats fed 13% casein, the liver albumin content remained high regardless of the time of killing. In rats fed 80% casein, the albumin content was higher during the absorptive period than during the postabsorptive period. Immunoperoxidase staining of the hepatocyte organelles involved in albumin synthesis, especially the Golgi apparatus, was more intense for separately fed rats killed 6 h postmeal than for those killed after 20 h. Livers of rats fed 13% casein also exhibited a pattern indicative of high hepatocyte albumin content, whereas livers of rats fed 80% casein contained less. These results show that, in separate feeding, wide circadian variations of albumin synthesis run parallel to changes in liver albumin content.

  1. Noninvasive real-time fluorescence imaging of the lymphatic uptake of BSA-IRDye 680 conjugate administered subcutaneously in mice.

    PubMed

    Wu, Fang; Bhansali, Suraj G; Tamhane, Mitalee; Kumar, Rajiv; Vathy, Lisa A; Ding, Hong; Yong, Ken-Tye; Bergey, Earl J; Prasad, Paras N; Morris, Marilyn E

    2012-05-01

    The goal of our studies was to determine lymphatic uptake of bovine serum albumin (BSA) using real-time noninvasive fluorescence imaging. BSA labeled with near-infrared dye (IRDye) 680 was used as a model protein-dye conjugate. The conjugation of BSA with IRDye 680 was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Size-exclusion high-performance liquid chromatography and SDS-PAGE demonstrated that the IRDye 680-labeled BSA conjugate in the lymph node (LN) homogenate samples was stable at physiological temperature (37°C) for at least 5 days. Whole-body noninvasive optical imaging of hairless SKH-1 mice was performed after subcutaneous (s.c.) injection (dose = 0.1 mg/kg) into the front footpad. Noninvasive fluorescence imaging demonstrated that BSA-IRDye 680 conjugates were dynamically taken up by the lymphatic system, accumulated in the axillary LNs and then cleared, indicating that lymphatic transport plays a role in the absorption of BSA. Ex vivo tissue imaging of LN homogenates provided confirmatory data with respect to the uptake of fluorescent-labeled BSA determined by in vivo imaging. Noninvasive real-time imaging of LNs provides a novel tool for evaluating uptake and accumulation of fluorescent-labeled proteins by the lymphatic system after s.c. injection in a mouse model.

  2. A comparison of the physical properties of ultrasonically synthesized lysozyme- and BSA-shelled microbubbles.

    PubMed

    Vong, Fiona; Son, Younggyu; Bhuiyan, Sadia; Zhou, Meifang; Cavalieri, Francesca; Ashokkumar, Muthupandian

    2014-01-01

    Ultrasonic technique has been used for synthesising protein microspheres possessing specific physical and functional properties. Various proteins have been used as shell materials under different experimental conditions. In previous studies, thermal or chemical denaturation of the proteins was used to obtain stable bovine-serum albumin (BSA) and lysozyme microbubbles (MBs), respectively. It is ideal to establish a generic procedure to synthesise microspheres irrespective of the nature of the protein. In order to see if a generic procedure can be established, ultrasonic synthesis of lysozyme and BSA MBs was carried out under similar experimental conditions and their properties were evaluated. The size, size distribution and the stability of the MBs were significantly different for the lysozyme and BSA MBs. The size and size distribution of the lysozyme coated MBs were larger than BSA bubbles. The mechanical strength of MBs against the shear forces, generated when irradiated by high frequency ultrasound, was studied using pulsed-sonoluminescence (SL). This study indicated that lysozyme MBs were significantly more stable than BSA MBs. An increase in mechanical strength of the MBs may lead to an increase in their storage lifetime and stability against gas diffusion. Possible reasons for such observations have been discussed.

  3. Protein-protein binding before and after photo-modification of albumin

    NASA Astrophysics Data System (ADS)

    Rozinek, Sarah C.; Glickman, Randolph D.; Thomas, Robert J.; Brancaleon, Lorenzo

    2016-03-01

    Bioeffects of directed-optical-energy encompass a wide range of applications. One aspect of photochemical interactions involves irradiating a photosensitizer with visible light in order to induce protein unfolding and consequent changes in function. In the past, irradiation of several dye-protein combinations has revealed effects on protein structure. Beta lactoglobulin, human serum albumin (HSA) and tubulin have all been photo-modified with meso-tetrakis(4- sulfonatophenyl)porphyrin (TSPP) bound, but only in the case of tubulin has binding caused a verified loss of biological function (loss of ability to form microtubules) as a result of this light-induced structural change. The current work questions if the photo-induced structural changes that occur to HSA, are sufficient to disable its biological function of binding to osteonectin. The albumin-binding protein, osteonectin, is about half the molecular weight of HSA, so the two proteins and their bound product can be separated and quantified by size exclusion high performance liquid chromatography. TSPP was first bound to HSA and irradiated, photo-modifying the structure of HSA. Then native HSA or photo-modified HSA (both with TSPP bound) were compared, to assess loss in HSA's innate binding ability as a result of light-induced structure modification.

  4. A novel affinity disks for bovine serum albumin purification.

    PubMed

    Tuzmen, Nalan; Kalburcu, Tülden; Uygun, Deniz Aktaş; Akgol, Sinan; Denizli, Adil

    2015-01-01

    The adsorption characteristics of bovine serum albumin (BSA) onto the supermacroporous poly(hydroxyethylmethacrylate)-Reactive Green 19 [p(HEMA)-RG] cryogel disks have been investigated in this paper. p(HEMA) cryogel disks were prepared by radical polymerization initiated by N,N,N',N'-tetramethylene diamine (TEMED) and ammonium persulfate (APS) pair in an ice bath. Reactive Green (RG) 19 was covalently attached to the p(HEMA) cryogel disks. These disks were used in BSA adsorption studies to interrogate the effects of pH, initial protein concentration, ionic strength, and temperature. BSA adsorption capacity of the p(HEMA)-RG cryogel disk was significantly improved after the incorporation of RG. Adsorption capacity reached a plateau value at about 0.8 mg/mL at pH 4.0. The amount of adsorbed BSA decreased from 37.7 to 13.9 mg/g with increasing NaCl concentration. The enthalpy of BSA adsorption onto the p(HEMA)-RG cryogel disk was calculated as -58.4 kJ/mol. The adsorption equilibrium isotherm was fitted well by the Freundlich model. BSA was desorbed from cryogel disks (over 90 %) using 0.5 M NaSCN, and the purity of desorbed BSA was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The experimental results showed that the p(HEMA)-RG cryogel disks have potential for the quick protein separation and purification process. PMID:25308615

  5. BSA-boronic acid conjugate as lectin mimetics.

    PubMed

    Narla, Satya Nandana; Pinnamaneni, Poornima; Nie, Huan; Li, Yu; Sun, Xue-Long

    2014-01-10

    We report bovine serum albumin (BSA)-boronic acid (BA) conjugates as lectin mimetics and their glyco-capturing capacity. The BSA-BA conjugates were synthesized by amidation of carboxylic acid groups in BSA with aminophenyl boronic acid in the presence of EDC, and were characterized by Alizarin Red S (ARS) assay and SDS-PAGE gel. The BSA-BA conjugates were immobilized onto maleimide-functionalized silica beads and their sugar capturing capacity and specificity were confirmed by ARS displacement assay. Further, surface plasmon resonance (SPR) analysis of the glyco-capturing activity of the BSA-BA conjugates was conducted by immobilizing BSA-BA onto SPR gold chip. Overall, we demonstrated a BSA-BA-based lectin mimetics for glyco-capturing applications. These lectin mimetics are expected to provide an important tool for glycomics and biosensor research and applications.

  6. Isolation and characterization of serum albumin from Camelus dromedarius

    PubMed Central

    MALIK, AJAMALUDDIN; AL-SENAIDY, ABDULRAHMAN; SKRZYPCZAK-JANKUN, EWA; JANKUN, JERZY

    2013-01-01

    Serum albumin constitutes 35–50 mg/ml of plasma proteins and performs various physiological activities including the regulation of osmotic pressure on blood, maintaining buffering of the blood pH, carrying different fatty acids and other small molecules, such as bilirubin, hormones, drugs and metal ions, as well as participating in immunological responses. Serum albumin is an extensively used protein in biotechnological and pharmaceutical industries. The camel (Camelus dromedarius) is well tailored to successfully survive in extremely hot and dry climates. Plasma osmolality in the camel increases during water-deprived conditions. In such circumstances serum albumin is crucial in the regulation of blood pressure. The study of biochemical, biophysical and immunological aspects of camel serum albumin (CSA) are likely to provide molecular insights into camel physiology and may render it an alternative to human serum albumin (HSA) and bovine serum albumin (BSA) in all cases. However, these proteins are currently not available or cannot be utilized due to a variety of considerations. In this study, 12 mg of highly pure CSA was obtained from 1 ml plasma. Coomassie Brilliant Blue staining of SDS-PAGE yielded one band and RP-HPLC results revealed a single sharp peak, indicating homogenous preparation of the CSA. The charge/mass ratio and surface hydrophobicity of the CSA was similar to that of BSA. Mass spectrometry analysis of the purified protein confirmed the identity of CSA. PMID:24137219

  7. Bovine serum albumin as the dominant form of dietary protein reduces subcutaneous fat mass, plasma leptin and plasma corticosterone in high fat-fed C57/BL6J mice.

    PubMed

    McManus, Bettina L; Korpela, Riitta; Speakman, John R; Cryan, John F; Cotter, Paul D; Nilaweera, Kanishka N

    2015-08-28

    Increasing evidence suggests that the source of dietary protein can have an impact on weight gain and fat mass during high-fat feeding in both humans and rodents. The present study examined whether dietary bovine serum albumin (BSA) as the dominant source of protein alters energy balance and adiposity associated with high-fat feeding. C57/BL6J mice were given a diet with 10 % of energy from fat and 20 % of energy from casein or a diet with 45 % of energy from fat and either 20 % of energy from casein (HFD) or BSA (HFD+BSA) for 13 weeks. The HFD+BSA diet did not significantly alter daily energy expenditure, locomotor activity and RER, but did increase cumulative energy intake and percentage of lean mass while reducing feed efficiency and percentage of fat mass when compared with the HFD (P< 0·05). In subcutaneous adipose tissue (SAT), the HFD+BSA diet increased the mRNA levels of PPARα (PPARA), carnitine palmitoyltransferase 1b (CPT1b) and uncoupling protein 3 (UCP3), but reduced the mRNA level of leptin when compared with the HFD (P< 0·05). The SAT mRNA levels of PPARA, CPT1b and UCP3 were negatively correlated (P< 0·05) with SAT mass, which was reduced in HFD+BSA mice compared with HFD controls (P< 0·01). No differences in epididymal fat mass existed between the groups. The HFD+BSA diet normalised plasma leptin and corticosterone levels compared with the HFD (P< 0·05). While differences in leptin levels were associated with the percentage of fat mass (P< 0·01), changes in corticosterone concentrations were independent of the percentage of fat mass (P< 0·05). The data suggest that the HFD+BSA diet influences plasma leptin levels via SAT mass reduction where mRNA levels of genes linked to β-oxidation were increased, whereas differences in plasma corticosterone levels were not related to fat mass reduction. PMID:26189974

  8. Characterization and analytical application of Morin - Bovine serum albumin system by spectroscopic approaches

    NASA Astrophysics Data System (ADS)

    Wang, Feng; Huang, Wei; Miao, Xiaowen; Tang, Bo

    2012-12-01

    It is found that the fluorescence intensity of Morin can be strongly quenched by proteins. Based on this, a new fluorimetric method for the determination of protein was developed. Under optimum conditions, the quenchment of Morin fluorescence was in proportion to the concentration of proteins in the range 0.0001-0.1000 g · L-1 for bovine serum albumin (BSA) and 0.0005-0.1000 g · L-1 for human serum albumin (HSA). The reaction mechanism indicates that proteins can bind with Morin at the 3-hydroxyl and the 4-carbonyl and form a non-fluorescence complex 4:1 molar ratio of Morin/BSA, which results in the fluorescence of Morin and BSA are all quenched.

  9. Differences in the electrostatic surfaces of the type III secretion needle proteins PrgI, BsaL, and MxiH.

    PubMed

    Wang, Yu; Ouellette, Andrew N; Egan, Chet W; Rathinavelan, Thenmalarchelvi; Im, Wonpil; De Guzman, Roberto N

    2007-08-31

    Gram-negative bacteria use a needle-like protein assembly, the type III secretion apparatus, to inject virulence factors into target cells to initiate human disease. The needle is formed by the polymerization of approximately 120 copies of a small acidic protein that is conserved among diverse pathogens. We previously reported the structure of the BsaL needle monomer from Burkholderia pseudomallei by nuclear magnetic resonance (NMR) spectroscopy and others have determined the crystal structure of the Shigella flexneri MxiH needle. Here, we report the NMR structure of the PrgI needle protein of Salmonella typhimurium, a human pathogen associated with food poisoning. PrgI, BsaL, and MxiH form similar two helix bundles, however, the electrostatic surfaces of PrgI differ radically from those of BsaL or MxiH. In BsaL and MxiH, a large negative area is on a face formed by the helix alpha1-alpha2 interface. In PrgI, the major negatively charged surface is not on the "face" but instead is on the "side" of the two-helix bundle, and only residues from helix alpha1 contribute to this negative region. Despite being highly acidic proteins, these molecules contain large basic regions, suggesting that electrostatic contacts are important in needle assembly. Our results also suggest that needle-packing interactions may be different among these bacteria and provide the structural basis for why PrgI and MxiH, despite 63% sequence identity, are not interchangeable in S. typhimurium and S. flexneri.

  10. Differences in the electrostatic surfaces of the type III secretion needle proteins PrgI, BsaL, and MxiH

    PubMed Central

    Wang, Yu; Ouellette, Andrew N.; Egan, Chet W.; Rathinavelan, Thenmalarchelvi; Im, Wonpil; De Guzman, Roberto N.

    2007-01-01

    Gram-negative bacteria use a needle-like protein assembly, the type III secretion apparatus, to inject virulence factors into target cells to initiate human disease. The needle is formed by the polymerization of ~120 copies of a small acidic protein that is conserved among diverse pathogens. We previously reported the structure of the BsaL needle monomer from Burkholderia pseudomallei by nuclear magnetic resonance (NMR) spectroscopy and others have determined the crystal structure of the Shigella flexneri MxiH needle. Here, we report the NMR structure of the PrgI needle protein of Salmonella typhimurium, a human pathogen associated with food poisoning. PrgI, BsaL, and MxiH form similar two helix bundles, however, the electrostatic surfaces of PrgI differ radically from those of BsaL or MxiH. In BsaL and MxiH, a large negative area is on a face formed by the helix α1-α2 interface. In PrgI, the major negatively charged surface is not on the “face” but instead is on the “side” of the two helix bundle, and only residues from helix α1 contribute to this negative region. Despite being highly acidic proteins, these molecules contain large basic regions, suggesting that electrostatic contacts are important in needle assembly. Our results also suggest that needle packing interactions may be different among these bacteria and provide the structural basis for why PrgI and MxiH, despite 63% sequence identity, are not interchangeable in S. typhimurium and S. flexneri. PMID:17617421

  11. Kinetic investigation on the oxidation of tris(1,10-phenanthroline)iron(II) by oxone: the effect of BSA-SDS interaction.

    PubMed

    Mandal, Harasit Kumar; Kundu, Arjama; Balti, Subrata; Mahapatra, Ambikesh

    2012-07-15

    The kinetic investigations of oxidation of tris(1,10-phenanthroline)iron(II) by oxone have been studied spectrophotometrically in phosphate buffer medium of pH 6.8, temperature 308 K, and ionic strength 0.25 mol L(-1). The reactions were also carried out in presence of globular transport protein, bovine serum albumin (BSA) having isoelectric point 4.9, anionic surfactant sodium dodecyl sulfate (SDS), and their mixtures. The critical aggregation concentration (CAC) and critical micelle concentration (CMC) of SDS in presence of BSA have been determined using conductivity and kinetic measurement techniques. The secondary structure of BSA was examined by Circular Dichroism (CD) measurement at 308 K. The helix nature of BSA decreases with increase of SDS concentration. The effect of pH on rate in presence of BSA is opposite to its absence, and the effect of urea on rate in presence of BSA indicates the denaturation of BSA. The results depict that amphiphile SDS interacts with BSA and different molecular events, for example, specific binding, cooperative binding, protein unfolding, and micelle formation act. Activation parameters of the reaction in different environments have been determined.

  12. Caveolae may enable albumin to enter human renal glomerular endothelial cells.

    PubMed

    Moriyama, Takahito; Takei, Takashi; Itabashi, Mitsuyo; Uchida, Keiko; Tsuchiya, Ken; Nitta, Kosaku

    2015-06-01

    Caveolae on human renal glomerular endothelial cells (HRGECs) are increased in glomerular disease and correlate with the degree of albuminuria. To assess the mechanism by which caveolae contribute to albuminuria, we investigated whether albumin enters into HRGECs through caveolae. HRGECs were incubated with Alexa Fluor 488 labeled BSA or transferrin, followed by immunofluorescence localization with antibody to caveolin-1 (Cav-1), the main structural protein of caveolae, or clathrin, the major structural protein of clathrin coated pits, to assess whether BSA colocalized with Cav-1. HRGECs were also incubated with albumin and caveolae disrupting agents, including methyl beta cyclodextrin (MBCD) and nystatin, to determine whether disrupting caveolae interfered with albumin endocytosis into HRGECs. HRGECs were also incubated with albumin after transfection with Cav-1 small interfering RNAs (siRNAs). Labeled BSA colocalized with Cav-1, but not with clathrin. In contrast, labeled transferrin colocalized with clathrin, but not with Cav-1. Incubation of HRGECs with MBCD or nystatin, or transfection with Cav-1 siRNA, significantly reduced the intracellular amounts of albumin and Cav-1, relative to normal HRGECs, as shown by western blotting and immunofluorescence. These findings indicate that albumin enters HRGECs through the caveolae, suggesting that caveolae play an important role in the pathogenesis of albuminuria by providing a pathway through which albumin can enter glomerular endothelial cells.

  13. Study of the effect of total serum protein and albumin concentrations on canine fructosamine concentration.

    PubMed Central

    Loste, A; Marca, M C

    1999-01-01

    The relationship among serum fructosamine concentration and total serum protein and albumin concentrations were evaluated in healthy and sick dogs (diabetics and dogs with insulinoma were not included). Fructosamine was determined using a commercial colorimetric nitroblue tetrazolium method applied to the Technicon RA-500 (Bayer). Serum fructosamine concentration was not correlated to total protein in normoproteinemic (r = 0.03) and hyperproteinemic dogs (r = 0.29), but there was a high correlation (r = 0.73) in hypoproteinemic dogs. Similar comparison between serum fructosamine and albumin concentrations showed middle correlation (r = 0.49) in normoalbuminemic dogs and high degree of correlation (r = 0.67) in hypoalbuminemic dogs. These results showed the importance of recognizing serum glucose concentration as well as total serum protein and albumin concentrations in the assay of canine serum fructosamine concentration. PMID:10369572

  14. Paeoniflorin protects HUVECs from AGE-BSA-induced injury via an autophagic pathway by acting on the RAGE.

    PubMed

    Chen, Yufang; Du, Xing; Zhou, Yande; Zhang, Yanlin; Yang, Yaping; Liu, Zhihua; Liu, Chunfeng; Xie, Ying

    2015-01-01

    The aim of our study was to investigate the protective effects of Paeoniflorin (PF) against injury induced by AGE-modified bovine serum albumin (AGE-BSA) in human umbilical vein endothelial cells (HUVECs), and to examine the underlying mechanisms of these effects. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay was used to determine cell viability. Protein expression levels were determined by western blotting. For function-blocking experiments, we used small interfering RNA molecules (siRNA) for function-blocking experiments. At 6 h, we found that 100 μg/mL AGE-BSA reduced the viability of HUVECs. However, pretreatment with PF restored cell viability in a dose-dependent manner. AGE-BSA increased the levels of microtubule-associated protein light chain 3-II (LC3-II) and the receptor for advanced glycation end products (RAGE). Expression of p62 protein was also increased, but not at a statistically significant level. Pretreatment with PF further increased levels of LC3-II and RAGE, but reduced the expression of p62. In cells transfected with Atg5 and RAGE siRNA, cell viability and expression of LC3-II decreased in both the AGE-BSA and PF + AGE-BSA treatments. PF can protect HUVECs from AGE-BSA-induced injury by upregulating autophagy and promoting the completion of autophagy flux. RAGE plays an important role in this autophagic protection effect.

  15. Interaction of triprolidine hydrochloride with serum albumins: thermodynamic and binding characteristics, and influence of site probes.

    PubMed

    Sandhya, B; Hegde, Ashwini H; Kalanur, Shankara S; Katrahalli, Umesha; Seetharamappa, J

    2011-04-01

    The interaction between triprolidine hydrochloride (TRP) to serum albumins viz. bovine serum albumin (BSA) and human serum albumin (HSA) has been studied by spectroscopic methods. The experimental results revealed the static quenching mechanism in the interaction of TRP with protein. The number of binding sites close to unity for both TRP-BSA and TRP-HSA indicated the presence of single class of binding site for the drug in protein. The binding constant values of TRP-BSA and TRP-HSA were observed to be 4.75 ± 0.018 × 10(3) and 2.42 ± 0.024 × 10(4)M(-1) at 294 K, respectively. Thermodynamic parameters indicated that the hydrogen bond and van der Waals forces played the major role in the binding of TRP to proteins. The distance of separation between the serum albumin and TRP was obtained from the Förster's theory of non-radioactive energy transfer. The metal ions viz., K(+), Ca(2+), Co(2+), Cu(2+), Ni(2+), Mn(2+) and Zn(2+) were found to influence the binding of the drug to protein. Displacement experiments indicated the binding of TRP to Sudlow's site I on both BSA and HSA. The CD, 3D fluorescence spectra and FT-IR spectral results revealed the changes in the secondary structure of protein upon interaction with TRP.

  16. Interaction of coffee compounds with serum albumins. Part II: Diterpenes.

    PubMed

    Guercia, Elena; Forzato, Cristina; Navarini, Luciano; Berti, Federico

    2016-05-15

    Cafestol and 16-O-methylcafestol are diterpenes present in coffee, but whilst cafestol is found in both Coffea canephora and Coffea arabica, 16-O-methylcafestol (16-OMC) was reported to be specific of only C. canephora. The interactions of such compounds, with serum albumins, have been studied. Three albumins have been considered, namely human serum albumin (HSA), fatty acid free HSA (ffHSA) and bovine serum albumin (BSA). The proteins interact with the diterpenes at the interface between Sudlow site I and the fatty acid binding site 6 in a very peculiar way, leading to a significant change in the secondary structure. The diterpenes do not displace reference binding drugs of site 2, but rather they enhance the affinity of the site for the drugs. They, therefore, may alter the pharmacokinetic profile of albumin - bound drugs.

  17. Nucleolin is a receptor for maleylated-bovine serum albumin on macrophages.

    PubMed

    Miki, Yuichi; Koyama, Keisuke; Kurusu, Haruna; Hirano, Kazuya; Beppu, Masatoshi; Fujiwara, Yasuyuki

    2015-01-01

    Scavenger receptors have a broad range of functions that include pathogen clearance, and identification of the scavenger receptor family has been of great benefit to the field of physiology. The shuttling-protein nucleolin has recently been shown to possess scavenger receptor-like activity. We therefore investigated whether or not nucleolin is a receptor for maleylated-bovine serum albumin (maleylated-BSA), which is a common ligand for scavenger receptors. Binding and phagocytosis of native control-BSA by thioglycollate-elicited mouse peritoneal macrophages was weak, but that of maleylated-BSA was strong. Surface plasmon-resonance analysis revealed that nucleolin strongly associated with maleylated-BSA but not control-BSA or maleic anhydride. Further, co-treatment of macrophages with anti-nucleolin antibody, but not control-immunoglobulin G, inhibited binding of maleylated-BSA. In addition, antineoplastic guanine rich oligonucleotide (AGRO), a nucleolin-specific oligonucleotide aptamer, inhibited binding of maleylated-BSA. Further, binding of maleylated-BSA to nucleolin-transfected HEK293 cells was higher than that by control HEK cells. These results indicate that nucleolin is a receptor that enables macrophages to recognize maleylated-BSA.

  18. Zinc oxide nanoparticle and bovine serum albumin interaction and nanoparticles influence on cytotoxicity in vitro.

    PubMed

    Žūkienė, Rasa; Snitka, Valentinas

    2015-11-01

    Bovine serum albumin (BSA) and zinc oxide nanoparticles (ZnO NPs) are chosen as a model system to investigate NPs-protein corona complex formation. ZnO NPs with average size of ∼ 20 nm are coated with BSA using covalent and non-covalent conjugation at temperatures of 4 °C and 20 °C. The interaction mechanism between ZnO NPs and BSA is studied by using UV-vis absorption, fluorescence, synchronous fluorescence and Raman spectroscopy. Raman spectra of BSA in the presence of ZnO NPs are registered for the first time and confirm decreased α-helix content, increased unstructured folding and β-sheet content in BSA structure. The synchronous fluorescence spectra revealed that the hydrophobicity of the tyrosine residue is decreased and that of the tryptophan is increased. The relation of elucidated changes in BSA structure of BSA-coated ZnO NPs cytotoxicity is tested for CHO cell viability and reactive oxygen species (ROS) generation in vitro. Covalent and non-covalent binding of BSA to ZnO NPs reduces ZnO NPs cytotoxicity and ROS generation, however changes in BSA conformation makes corona less protective against ZnO NPs.

  19. Functional improvements in bovine serum albumin-fucoidan conjugate through the Maillard reaction.

    PubMed

    Kim, Do-Yeong; Shin, Weon-Sun

    2016-01-01

    The solubility, thermal stability, surface activity and emulsifying properties of native bovine serum albumin (BSA), heat-treated BSA, a BSA-fucoidan mixture, and a BSA-fucoidan conjugate were assessed. Covalent linkage of BSA with fucoidan resulted in significantly (p < 0.05) high solubility after heating at 90 °C for 15 min, particularly at pH 5. The BSA-fucoidan conjugate had a high melting temperature (97.09 ± 1.45 °C), as found by differential scanning calorimetry, indicating strong heat stability and high resistance to denaturation. Although the attachment of fucoidan, a non-surface-active hydrophilic polysaccharide, gave no change in the surface activity, the emulsifying activity and the emulsion stability of the conjugate at pH 5 were superior to those of native BSA, heat-treated BSA, and the BSA-fucoidan mixture. Conclusively, fucoidan attachment enhanced the solubility, thermal stability and emulsifying properties of the protein molecules with negative charge distribution and steric stabilization.

  20. Systematic study on the preparation of BSA nanoparticles.

    PubMed

    Galisteo-González, F; Molina-Bolívar, J A

    2014-11-01

    Albumins, in the form of nanoparticles, are increasingly used as drug carriers in the medical field, and the size effect of these nanomaterials is of major importance since it may affect their bioavailability and the in vivo behaviour after intravenous injection. This research provides a comprehensive study on the preparation of BSA nanoparticles, based on a simple coacervation method, with suitable size, size distribution, and surface charge for drug-delivery applications. Numerous experimental variables were examined in order to characterize their impact on nanoparticle size, distribution, electrophoretic mobility, and yield. Particle size was controlled by adjusting self-assembly phenomena of the protein molecules, which was affected by preparation conditions including BSA content, pH, and ionic strength (a parameter that strongly influences nanoparticle formation but surprisingly has not been previously studied in detail). Small particles with a narrow size distribution were obtained under experimental conditions where the repulsion between BSA molecules was high, i.e. at pH values far from the isoelectric point of the protein and low salt concentration. Changes in temperature, volume, and rate of addition of the dehydrating agent (ethanol) also affect nanoparticle characteristics, as they influence the nucleation rate and particle growth. The effect of these experimental conditions on the quantity of protein still dissolved in the aqueous phase after desolvation (i.e. the yield of BSA nanoparticles) was also studied. Nanoparticles surface charge was modulated with the extension of cross-linking. Finally, long-term colloidal stability of samples was evaluated after 2 months of storage.

  1. Stability of the complex BSA-6-propyl-2-thiouracil in the presence of Gu·HCl and urea

    NASA Astrophysics Data System (ADS)

    Równicka, Joanna; Sułkowska, Anna; Pożycka, Jadwiga; Bojko, Barbara; Sułkowski, Wiesław W.

    2006-07-01

    Pathologic structure of albumin is present in blood of uremic or diabetic patients. The defective binding of albumin can cause, among others, inhibition of the metabolism of amphipathic hormones and the enhancement of the toxicity of drugs. We aim to show the alteration of the binding site of 6-propyl-2-thiouracil (PTU) in bovine serum albumin (BSA) in the presence of urea and guanidine hydrochloride. This study shows that PTU has the tendency to interact within the hydrophobic domain IIA or IB of serum albumin and to quench the fluorescence of tryptophanyl side chains by the static and collisional quenching mechanism. For the binding to the native protein, two classes of binding sites are seen. In the first one, the binding constant is equal to K bI-9.6×10 4 M -1, and in the second class, the binding constant is K bII-7.1×10 3 M -1. For the binding to the denaturated serum albumin, only one class of the binding sites can be observed. The binding constants for BSA denatured with urea and Gu·HCl are lower and are equal to 2.2×10 4 M -1 and 3.5×10 4 M -1, respectively. Evaluation of the binding ability of serum albumin is also important in case of older people because weaker drug-protein interaction can result in the increase of drug concentration in the blood serum.

  2. Tracing the conformational changes in BSA using FRET with environmentally-sensitive squaraine probes

    NASA Astrophysics Data System (ADS)

    Govor, Iryna V.; Tatarets, Anatoliy L.; Obukhova, Olena M.; Terpetschnig, Ewald A.; Gellerman, Gary; Patsenker, Leonid D.

    2016-06-01

    A new potential method of detecting the conformational changes in hydrophobic proteins such as bovine serum albumin (BSA) is introduced. The method is based on the change in the Förster resonance energy transfer (FRET) efficiency between protein-sensitive fluorescent probes. As compared to conventional FRET based methods, in this new approach the donor and acceptor dyes are not covalently linked to protein molecules. Performance of the new method is demonstrated using the protein-sensitive squaraine probes Square-634 (donor) and Square-685 (acceptor) to detect the urea-induced conformational changes of BSA. The FRET efficiency between these probes can be considered a more sensitive parameter to trace protein unfolding as compared to the changes in fluorescence intensity of each of these probes. Addition of urea followed by BSA unfolding causes a noticeable decrease in the emission intensities of these probes (factor of 5.6 for Square-634 and 3.0 for Square-685), and the FRET efficiency changes by a factor of up to 17. Compared to the conventional method the new approach therefore demonstrates to be a more sensitive way to detect the conformational changes in BSA.

  3. Electrochemical deposition of mineralized BSA/collagen coating.

    PubMed

    Zhuang, Junjun; Lin, Jun; Li, Juan; Wang, Huiming; Cheng, Kui; Weng, Wenjian

    2016-09-01

    In this work, mineralized collagen coatings with different loading quantity of bovine serum albumin (BSA) were prepared via in situ electrochemical deposition on titanium substrate. The microstructure and BSA loading quantity of the coatings could be controlled by the electrochemical deposition parameters, such as deposition potential, BSA concentration and its adding sequence in the electrolyte. The BSA loading quantity in the coatings was obtained in the range of 0.0170-0.173mg/cm(2), enhancing the cell adhesion and proliferation of the coatings with the simultaneous release. The distinct release behaviors of BSA were attributed to their gradient distribution with different mineralization degrees, which could be adjusted by the deposition process. These results suggest that in situ electrochemical deposition is a promising way to incorporate functional molecules into the mineralized collagen coatings and the mineralized BSA/collagen coatings are highly promising for improving the rhBMP-2 loading capability (1.8-fold).

  4. The impact of skin viability on drug metabolism and permeation -- BSA toxicity on primary keratinocytes.

    PubMed

    Haberland, A; Schreiber, S; Maia, C Santos; Rübbelke, M K; Schaller, M; Korting, H C; Kleuser, B; Schimke, I; Schäfer-Korting, M

    2006-04-01

    For testing cutaneous absorption of drugs, ingredients of cosmetics and also for risk assessment of industrial compounds predictable in vitro test protocols are under investigation using excised skin or reconstructed human epidermis. Since the metabolizing enzymes expressed by viable skin can influence the absorption behaviour of substances by changing their structure and thereby their physicochemical characteristics, the metabolic capacity should be considered in the design of the test protocols of compounds susceptible to metabolism. Then data, generated using viable reconstructed epidermis may reflect the in vivo situation. Interestingly, bovine serum albumin (BSA) commonly used in receptor media in permeation studies to facilitate solubility of highly lipophilic substances strongly inhibited the metabolism of topically applied prednicarbate in reconstructed epidermis. Here, we show that 5% BSA is toxic to reconstructed epidermis and keratinocytes which was consistent with the earlier findings. While media toxicity (deficiency media) was at least partly the cause of both apoptotic and necrotic processes in keratinocytes, BSA only slightly increased the rate of necrotic cells. Moreover, caspase inhibitors did not reduce BSA toxicity. Yet, the results show that BSA toxicity on keratinocytes has to be carefully considered if this protein is used in permeation studies with reconstructed epidermis.

  5. Albumin extravasation rates in tissues of anesthetized and unanesthetized rats

    SciTech Connect

    Renkin, E.M.; Joyner, W.L.; Gustafson-Sgro, M.; Plopper, G.; Sibley, L.

    1989-05-01

    Bovine serum albumin (BSA) labeled with /sup 131/I was injected intravenously in chronically prepared, unanesthetized rats and into pentobarbital-anesthetized rats that had received 2 ml 5% BSA to help sustain plasma volume. Initial uptake rates (clearances) in skin, skeletal muscles, diaphragm, and heart (left ventricle) were measured over 1 h. BSA labeled with /sup 125/I was injected terminally to correct for intravascular /sup 131/I-BSA. Observed clearances were in the following order in both groups of animals: heart much greater than diaphragm approximately equal to skin greater than resting skeletal muscles. Differences between unanesthetized and anesthetized animals were small and inconsistently directed. Our results suggest that the lower albumin clearances reported in the literature for anesthetized rats are not the result of their immobility or any direct effect of anesthesia on albumin transport in these tissues. The lower transport rates appear to result indirectly from changes produced by anesthesia and/or surgery in controllable parameters such as plasma volume and intravascular protein mass.

  6. Concentration-dependent reversible self-oligomerization of serum albumins through intermolecular β-sheet formation.

    PubMed

    Bhattacharya, Arpan; Prajapati, Roopali; Chatterjee, Surajit; Mukherjee, Tushar Kanti

    2014-12-16

    Proteins inside a cell remain in highly crowded environments, and this often affects their structure and activity. However, most of the earlier studies involving serum albumins were performed under dilute conditions, which lack biological relevance. The effect of protein-protein interactions on the structure and properties of serum albumins at physiological conditions have not yet been explored. Here, we report for the first time the effect of protein-protein and protein-crowder interactions on the structure and stability of two homologous serum albumins, namely, human serum albumin (HSA) and bovine serum albumin (BSA), at physiological conditions by using spectroscopic techniques and scanning electron microscopy (SEM). Concentration-dependent self-oligomerization and subsequent structural alteration of serum albumins have been explored by means of fluorescence and circular dichroism spectroscopy at pH 7.4. The excitation wavelength (λex) dependence of the intrinsic fluorescence and the corresponding excitation spectra at each emission wavelength indicate the presence of various ground state oligomers of serum albumins in the concentration range 10-150 μM. Circular dichroism and thioflavin T binding assay revealed formation of intermolecular β-sheet rich interfaces at high protein concentration. Excellent correlations have been observed between β-sheet content of both the albumins and fluorescence enhancement of ThT with protein concentrations. SEM images at a concentration of 150 μM revealed large dispersed self-oligomeric states with sizes vary from 330 to 924 nm and 260 to 520 nm for BSA and HSA, respectively. The self-oligomerization of serum albumins is found to be a reversible process; upon dilution, these oligomers dissociate into a native monomeric state. It has also been observed that synthetic macromolecular crowder polyethylene glycol (PEG 200) stabilizes the self-associated state of both the albumins which is contrary to expectations that the

  7. Role of single-walled carbon nanotubes on ester hydrolysis and topography of electrospun bovine serum albumin/poly(vinyl alcohol) membranes.

    PubMed

    Ford, Ericka N J; Suthiwangcharoen, Nisaraporn; D'Angelo, Paola A; Nagarajan, Ramanathan

    2014-07-23

    Electrospun membranes were studied for the chemical deactivation of threat agents by means of enzymatic proteins. Protein loading and the surface chemistry of hybrid nanofibers influenced the efficacy by which embedded enzymes could digest the substrate of interest. Bovine serum albumin (BSA), selected as a model protein, was electrospun into biologically active fibers of poly(vinyl alcohol), PVA. Single-walled carbon nanotubes (SWNTs) were blended within these mixtures to promote protein assembly during the process of electrospinning and subsequently the ester hydrolysis of the substrates. The SWNT incorporation was shown to influence the topography of PVA/BSA nanofibers and enzymatic activity against paraoxon, a simulant for organophosphate agents and a phosphorus analogue of p-nitrophenyl acetate (PNA). The esterase activity of BSA against PNA was uncompromised upon its inclusion within nanofibrous membranes because similar amounts of PNA were hydrolyzed by BSA in solution and the electrospun BSA. However, the availability of BSA along the fiber surface was shown to affect the ester hydrolysis of paraoxon. Atomic force microscopy images of nanofibers implicated the surface migration of BSA during the electrospinning of SWNT filled dispersions, especially as greater weight fractions of protein were added to the spinning mixtures. In turn, the PVA/SWNT/BSA nanofibers outperformed the nanotube free PVA/BSA membranes in terms of paraoxon digestion. The results support the development of electrospun polymer nanofiber platforms, modulated by SWNTs for enzyme catalytic applications relevant to soldier protective ensembles. PMID:25007411

  8. Structural influence of graft and block polycations on the adsorption of BSA.

    PubMed

    Zhang, Li; Jin, Fengmin; Zhang, Tingbin; Zhang, Ling; Xing, Jinfeng

    2016-04-01

    Protein adsorption is considered as an important factor for the low transfection efficiency of polycations in vivo. In this study, two typical polycations of equal molecular weight with different structures were chosen to investigate their adsorption on bovine serum albumin (BSA), including the block copolymer named poly (N-vinylpyrrolidone)-b-poly (2-dimethylaminoethyl methacrylate) (PVP-b-PDMAEMA, i.e. PbP) and graft copolymer named PVP-g-PDMAEMA (PgP), respectively. Fluorescence spectroscopy was used to confirm the binding constants and binding sites between polycations and BSA in static state. The binding constants were 4.1×10(4)M(-1) vs 8.3×10(4)M(-1) and binding sites were 0.3 vs 1.1 for PbP and PgP, respectively, indicating PgP had stronger binding affinity with BSA. Surface plasmon resonance (SPR) was used to study the dynamical non-specific interaction between BSA and polycations as well as the polyplexes. The numbers of both PbP and PgP adsorbed on BSA increased with concentration of polycations increasing, and the number of PgP adsorbed on BSA is higher compared with PbP when their concentration is low. When their concentration is high, the number of PbP adsorbed on BSA is more than that of PgP. However, PgP/DNA polyplexes showed higher adsorption amount compared with PbP/DNA polyplexes at different N/P ratios.

  9. Ion-Mobility-Based Quantification of Surface-Coating-Dependent Binding of Serum Albumin to Superparamagnetic Iron Oxide Nanoparticles.

    PubMed

    Jeon, Seongho; Oberreit, Derek R; Van Schooneveld, Gary; Gao, Zhe; Bischof, John C; Haynes, Christy L; Hogan, Christopher J

    2016-09-21

    Protein binding and protein-induced nanoparticle aggregation are known to occur for a variety of nanomaterials, with the extent of binding and aggregation highly dependent on nanoparticle surface properties. However, often lacking are techniques that enable quantification of the extent of protein binding and aggregation, particularly for nanoparticles with polydisperse size distributions. In this study, we adapt ion mobility spectrometry (IMS) to examine the binding of bovine serum albumin to commercially available anionic-surfactant-coated superparamagnetic iron oxide nanoparticles (SPIONs), which are initially ∼21 nm in mean mobility diameter and have a polydisperse size distribution function (geometric standard deviation near 1.4). IMS, carried out with a hydrosol-to-aerosol converting nebulizer, a differential mobility analyzer, and a condensation particle counter, enables measurements of SPION size distribution functions for varying BSA/SPION number concentration ratios. IMS measurements suggest that initially (at BSA concentrations below 50 nM) BSA binds reversibly to SPION surfaces with a binding site density in the 0.05-0.08 nm(-2) range. However, at higher BSA concentrations, BSA induces SPION-SPION aggregation, evidenced by larger shifts in SPION size distribution functions (mean diameters beyond 40 nm for BSA concentrations near 100 nM) and geometric standard deviations (near 1.3) consistent with self-preserving aggregation theories. The onset of BSA aggregation is correlated with a modest but statistically significant decrease in the specific absorption rate (SAR) of SPIONs placed within an alternating magnetic field. The coating of SPIONs with mesoporous silica (MS-SPIONs) as well as PEGylation (MS-SPIONs-PEG) is found to completely mitigate BSA binding and BSA-induced aggregation; IMS-inferred size distribution functions are insensitive to BSA concentration for MS-SPIONs and MS-SPIONs-PEG. The SARs of MS-SPIONs are additionally insensitive to BSA

  10. Multilayer Capsules of Bovine Serum Albumin and Tannic Acid for Controlled Release by Enzymatic Degradation.

    PubMed

    Lomova, Maria V; Brichkina, Anna I; Kiryukhin, Maxim V; Vasina, Elena N; Pavlov, Anton M; Gorin, Dmitry A; Sukhorukov, Gleb B; Antipina, Maria N

    2015-06-10

    With the purpose to replace expensive and significantly cytotoxic positively charged polypeptides in biodegradable capsules formed via Layer-by-Layer (LbL) assembly, multilayers of bovine serum albumin (BSA) and tannic acid (TA) are obtained and employed for encapsulation and release of model drugs with different solubility in water: hydrophilic-tetramethylrhodamine-isothiocyanate-labeled BSA (TRITC-BSA) and hydrophobic 3,4,9,10-tetra-(hectoxy-carbonyl)-perylene (THCP). Hydrogen bonding is proposed to be predominant within thus formed BSA/TA films. The TRITC-BSA-loaded capsules comprising 6 bilayers of the protein and polyphenol are benchmarked against the shells composed of dextran sulfate (DS) and poly-l-arginine (PARG) on degradability by two proteolytic enzymes with different cleavage site specificity (i.e., α-chymotrypsin and trypsin) and toxicity for murine RAW264.7 macrophage cells. Capsules of both types possess low cytotoxicity taken at concentrations equal or below 50 capsules per cell, and evident susceptibility to α-chymotrypsin resulted in release of TRITC-BSA. While the BSA/TA-based capsules clearly display resistance to treatment with trypsin, the assemblies of DS/PARG extensively degrade. Successful encapsulation of THCP in the TRITC-BSA/TA/BSA multilayer is confirmed, and the release of the model drug is observed in response to treatment with α-chymotrypsin. The thickness, surface morphology, and enzyme-catalyzed degradation process of the BSA/TA-based films are investigated on a planar multilayer comprising 40 bilayers of the protein and polyphenol deposited on a silicon wafer. The developed BSA/TA-based capsules with a protease-specific degradation mechanism are proposed to find applications in personal care, pharmacology, and the development of drug delivery systems including those intravenous injectable and having site-specific release capability.

  11. Spectroscopic study of TPPS4 nanostructures in the presence of bovine serum albumin.

    PubMed

    Valanciunaite, Jurga; Bagdonas, Saulius; Streckyte, Giedre; Rotomskis, Ricardas

    2006-04-01

    The influence of bovine serum albumin (BSA) on the formation of J-aggregates of meso-tetra(4-sulfonatophenyl)porphine (TPPS4) in aqueous acid solution (pH 1.3) has been investigated by means of absorption and fluorescence spectroscopy. TPPS4 concentration was kept constant at 2 microM while BSA concentration was varied to get TPPS4 : BSA molar ratios from 1 : 0.005 to 1 : 20. In the presence of protein at all used concentrations the intensity of J-aggregates absorption band was higher than that in the pure solution. Spectral changes indicated that the dynamic equilibrium of the aggregated TPPS4 species was highly dependent on the molar ratio between TPPS4 and BSA. Small relative concentrations of BSA (TPPS4 : BSA, 1 : 0.005-1 : 0.1) had a stimulating effect on formation of J-aggregates. Several fractions of J-aggregates located in protein and aqueous moieties were detected in mixed solutions at intermediate BSA concentrations (TPPS4 : BSA, 1 : 0.5-1 : 8), when the absorbance intensity of the J-aggregates was the highest. At the highest used BSA concentrations (TPPS4 : BSA, 1 : 10-1 : 20) the spectral properties of the remaining J-aggregates were similar to those typical for pure porphyrin solution. Additionally, the split of the Soret band into two with peaks at 440 nm and 423 nm was followed by the simultaneous appearance of Q bands and reflected the formation of TPPS4-BSA complexes including both protonated and deprotonated TPPS4 forms.

  12. Scavenger receptor-mediated recognition of maleyl bovine plasma albumin and the demaleylated protein in human monocyte macrophages.

    PubMed Central

    Haberland, M E; Fogelman, A M

    1985-01-01

    Maleyl bovine plasma albumin competed on an equimolar basis with malondialdehyde low density lipoprotein (LDL) in suppressing the lysosomal hydrolysis of 125I-labeled malondialdehyde LDL mediated by the scavenger receptor of human monocyte macrophages. Maleyl bovine plasma albumin, in which 94% of the amino groups were modified, exhibited an anodic mobility in agarose electrophoresis 1.7 times that of the native protein. Incubation of maleyl bovine plasma albumin at pH 3.5 regenerated the free amino groups and restored the protein to the same electrophoretic mobility as native albumin. The demaleylated protein suppressed 75% of the hydrolysis of 125I-labeled malondialdehyde LDL and greater than 80% of 125I-labeled maleyl bovine plasma albumin. The ability of the demaleylated protein to compete was abolished after treatment with guanidine hydrochloride. Although ligands recognized by the scavenger receptor typically are anionic, we propose that addition of new negative charge achieved by maleylation, rather than directly forming the receptor binding site(s), induces conformational changes in albumin as a prerequisite to expression of the recognition domain(s). The altered conformation of the modified protein apparently persists after removal of the maleyl groups. We conclude that the primary sequence of albumin, rather than addition of new negative charge, provides the recognition determinant(s) essential for interaction of maleyl bovine plasma albumin with the scavenger receptor. PMID:3857610

  13. Spectroscopic studies on the interaction between tetrandrine and two serum albumins by chemometrics methods

    NASA Astrophysics Data System (ADS)

    Cheng, Zhengjun; Liu, Rong; jiang, Xiaohui

    2013-11-01

    The binding interactions of tetrandrine (TETD) with bovine serum albumin (BSA) and human serum albumin (HSA) have been investigated by spectroscopic methods. These experimental data were further analyzed using multivariate curve resolution-alternating least squares (MCR-ALS) method, and the concentration profiles and pure spectra for three species (BSA/HSA, TETD and TETD-BSA/HSA) existed in the interaction procedure, as well as, the apparent equilibrium constants Kapp were evaluated. The binding sites number n and the binding constants K were obtained at various temperatures. The binding distance between TETD and BSA/HSA was 1.455/1.451 nm. The site markers competitive experiments indicated that TETD primarily bound to the tryptophan residue of BSA/HSA within site I. The thermodynamic parameters (ΔG, ΔH and ΔS) calculated on the basis of different temperatures revealed that the binding of TETD-BSA was mainly depended on the hydrophobic interaction strongly and electrostatic interaction, and yet the binding of TETD-HSA was strongly relied on the hydrophobic interaction. The results of synchronous fluorescence, 3D fluorescence and FT-IR spectra show that the conformation of proteins has altered in the presence of TETD. In addition, the effect of some common ions on the binding constants between TETD and proteins were also discussed.

  14. Significance of Total Protein, Albumin, Globulin, Serum Effusion Albumin Gradient and LDH in the Differential Diagnosis of Pleural Effusion Secondary to Tuberculosis and Cancer

    PubMed Central

    Sharma, Ashish; Das, Biswajit; Mallick, Ayaz K; Kumar, Amit

    2016-01-01

    Introduction Lung cancer and Pulmonary tuberculosis are two major public health problems associated with significant morbidity and mortality worldwide particularly in low and middle income countries like India. Wrong diagnosis of lung cancer cases as pulmonary tuberculosis in primary health care system delays the onset of anti-cancer chemotherapy as well as initiation of DOTS thus increasing complication and mortality rate in malignancy patients. In this context easy, cost effective diagnostic tool at primary level must be the priority and need of hour. Aim To study and evaluate any significance of biochemical parameters (total protein, albumin, globulin, serum effusion albumin gradient, LDH) in serum and pleural effusion secondary to tuberculosis and lung cancer. Materials and Methods A case control study was carried out on patients attending OPD and IPD, Department of Pulmonary Medicine, RMCH. Hundred cases of Tuberculosis effusion, 50 cases of Malignant effusion and 100 age and sex matched apparently healthy controls were taken for correlation of biochemical parameters (total protein, albumin, globulin, serum effusion albumin gradient, LDH) and statistically evaluated to find any significance between tuberculosis, lung cancer and control group. Blood and pleural fluid samples were collected and then subjected to assessment of parameters (total protein, albumin, LDH) by using EM360 Autoanalyser and kits were supplied by Transasia diagnostics. Globulin and Serum Effusion Albumin Gradient (SEAG) was calculated mathematically. Statistical Analysis Data is presented as mean ± SD. Comparison of serum and pleural fluid levels (of taken parameters) were done in TB, Lung Cancer and Control groups by ANOVA and students t-test. The p-value <0.05 were considered as statistically significant. Results We found serum-total protein, albumin, globulin to be significantly higher in TB group than lung cancer group but serum LDH was higher in lung cancer group (in all parameters p

  15. Acute metabolic acidosis decreases muscle protein synthesis but not albumin synthesis in humans.

    PubMed

    Kleger, G R; Turgay, M; Imoberdorf, R; McNurlan, M A; Garlick, P J; Ballmer, P E

    2001-12-01

    Chronic metabolic acidosis induces negative nitrogen balance by either increased protein breakdown or decreased protein synthesis. Few data exist regarding effects of acute metabolic acidosis on protein synthesis. We investigated fractional synthesis rates (FSRs) of muscle protein and albumin, plasma concentrations of insulin-like growth factor-I (IGF-I), thyroid-stimulating hormone (TSH), and thyroid hormones (free thyroxin [fT(4)] and triiodothyronine [fT(3)]) in seven healthy human volunteers after a stable controlled metabolic period of 5 days and again 48 hours later after inducing metabolic acidosis by oral ammonium chloride intake (4.2 mmol/kg/d divided in six daily doses). Muscle and albumin FSRs were obtained by the [(2)H(5)ring]phenylalanine flooding technique. Ammonium chloride induced a significant decrease in pH (7.43 +/- 0.02 versus 7.32 +/- 0.04; P < 0.0001) and bicarbonate concentration (24.6 +/- 1.6 versus 16.0 +/- 2.7 mmol/L; P < 0.0001) within 48 hours. Nitrogen balance decreased significantly on the second day of acidosis. The FSR of muscle protein decreased (1.94 +/- 0.25 versus 1.30 +/- 0.39; P < 0.02), whereas the FSR of albumin remained constant. TSH levels increased significantly (1.1 +/- 0.5 versus 1.9 +/- 1.1 mU/L; P = 0.03), whereas IGF-I, fT(4), and fT(3) levels showed no significant change. We conclude that acute metabolic acidosis for 48 hours in humans induces a decrease in muscle protein synthesis, which contributes substantially to a negative nitrogen balance. In contrast to prolonged metabolic acidosis of 7 days, a short period of acidosis in the present study did not downregulate albumin synthesis.

  16. Exploring the binding mechanism of ondansetron hydrochloride to serum albumins: spectroscopic approach.

    PubMed

    B, Sandhya; Hegde, Ashwini H; K C, Ramesh; J, Seetharamappa

    2012-02-01

    The mechanism of interaction of ondansetron hydrochloride (OND) to serum albumins [bovine serum albumin (BSA) and human serum albumin (HSA)] was studied for the first time employing fluorimetric, circular dichroism, FTIR and UV-vis absorption techniques under the simulated physiological conditions. Fluorimetric results were utilized to investigate the binding and conformational characteristics of protein upon interaction with varying concentrations of the drug. Higher binding constant values revealed the strong interaction between the drug and protein while the number of binding sites close to unity indicated single class of binding site for OND in protein. Thermodynamic results revealed that both hydrogen bond and hydrophobic interactions played a major role in stabilizing drug-protein complex. Site marker competitive experiments indicated that the OND bound to albumins at subdomin II A (Sudlow's site I). Further, the binding distance between OND and serum albumin was calculated based on the Förster's theory of non-radioactive energy transfer and found to be 2.30 and 3.41 nm, respectively for OND-BSA and OND-HSA. The circular dichroism data revealed that the presence of OND decreased the α-helix content of serum albumins. 3D-fluorescence results also indicated the conformational changes in protein upon interaction with OND. Further, the effects of some cations have been investigated in the interaction of drug to protein.

  17. Site-specific albumination of a therapeutic protein with multi-subunit to prolong activity in vivo

    PubMed Central

    Lim, Sung In; Hahn, Young S.; Kwon, Inchan

    2015-01-01

    Albumin fusion/conjugation (albumination) has been an effective method to prolong in vivo half-life of therapeutic proteins. However, its broader application to proteins with complex folding pathway or multi-subunit is restricted by incorrect folding, poor expression, heterogeneity, and loss of native activity of the proteins linked to albumin. We hypothesized that the site-specific conjugation of albumin to a permissive site of a target protein will expand the utilities of albumin as a therapeutic activity extender to proteins with a complex structure. We show here the genetic incorporation of a non-natural amino acid (NNAA) followed by chemoselective albumin conjugation to prolong therapeutic activity in vivo. Urate oxidase (Uox), a therapeutic enzyme for treatment of hyperuricemia, is a homotetramer with multiple surface lysines, limiting conventional approaches for albumination. Incorporation of p-azido-l-phenylalanine into two predetermined positions of Uox allowed site-specific linkage of dibenzocyclooctyne-derivatized human serum albumin (HSA) through strain-promoted azide-alkyne cycloaddition (SPAAC). The bio-orthogonality of SPAAC resulted in the production of a chemically well-defined conjugate, Uox-HSA, with a retained enzymatic activity. Uox-HSA had a half-life of 8.8 h in mice, while wild-type Uox had a half-life of 1.3 h. The AUC increased 5.5-fold (1657 vs. 303 mU/mL × h). These results clearly demonstrated that site-specific albumination led to the prolonged enzymatic activity of Uox in vivo. Site-specific albumination enabled by NNAA incorporation and orthogonal chemistry demonstrates its promise for the development of long-acting protein therapeutics with high potency and safety. PMID:25862515

  18. Site-specific albumination of a therapeutic protein with multi-subunit to prolong activity in vivo.

    PubMed

    Lim, Sung In; Hahn, Young S; Kwon, Inchan

    2015-06-10

    Albumin fusion/conjugation (albumination) has been an effective method to prolong in vivo half-life of therapeutic proteins. However, its broader application to proteins with complex folding pathway or multi-subunit is restricted by incorrect folding, poor expression, heterogeneity, and loss of native activity of the proteins linked to albumin. We hypothesized that the site-specific conjugation of albumin to a permissive site of a target protein will expand the utilities of albumin as a therapeutic activity extender to proteins with a complex structure. We show here the genetic incorporation of a non-natural amino acid (NNAA) followed by chemoselective albumin conjugation to prolong therapeutic activity in vivo. Urate oxidase (Uox), a therapeutic enzyme for treatment of hyperuricemia, is a homotetramer with multiple surface lysines, limiting conventional approaches for albumination. Incorporation of p-azido-l-phenylalanine into two predetermined positions of Uox allowed site-specific linkage of dibenzocyclooctyne-derivatized human serum albumin (HSA) through strain-promoted azide-alkyne cycloaddition (SPAAC). The bio-orthogonality of SPAAC resulted in the production of a chemically well-defined conjugate, Uox-HSA, with a retained enzymatic activity. Uox-HSA had a half-life of 8.8 h in mice, while wild-type Uox had a half-life of 1.3 h. The AUC increased 5.5-fold (1657 vs. 303 mU/mL x h). These results clearly demonstrated that site-specific albumination led to the prolonged enzymatic activity of Uox in vivo. Site-specific albumination enabled by NNAA incorporation and orthogonal chemistry demonstrates its promise for the development of long-acting protein therapeutics with high potency and safety.

  19. Light Harvesting and White-Light Generation in a Composite of Carbon Dots and Dye-Encapsulated BSA-Protein-Capped Gold Nanoclusters.

    PubMed

    Barman, Monoj Kumar; Paramanik, Bipattaran; Bain, Dipankar; Patra, Amitava

    2016-08-01

    Several strategies have been adopted to design an artificial light-harvesting system in which light energy is captured by peripheral chromophores and it is subsequently transferred to the core via energy transfer. A composite of carbon dots and dye-encapsulated BSA-protein-capped gold nanoclusters (AuNCs) has been developed for efficient light harvesting and white light generation. Carbon dots (C-dots) act as donor and AuNCs capped with BSA protein act as acceptor. Analysis reveals that energy transfer increases from 63 % to 83 % in presence of coumarin dye (C153), which enhances the cascade energy transfer from carbon dots to AuNCs. Bright white light emission with a quantum yield of 19 % under the 375 nm excitation wavelength is achieved by changing the ratio of components. Interesting findings reveal that the efficient energy transfer in carbon-dot-metal-cluster nanocomposites may open up new possibilities in designing artificial light harvesting systems for future applications. PMID:27383453

  20. Chlorpromazine interactions to sera albumins. A study by the quenching of fluorescence

    NASA Astrophysics Data System (ADS)

    Silva, Dilson; Cortez, Célia M.; Louro, Sônia R. W.

    2004-04-01

    Binding of chlorpromazine (CPZ) and hemin (Hmn) to human (HSA) and bovine (BSA) serum albumin was studied by fluorescence quenching technique. Intrinsic fluorescences of BSA and HSA were measured by selectively exciting their tryptophan residues. Gradual quenching was observed by titration of both proteins with CPZ and Hmn. CPZ is a widely used anti-psychosis drug that causes severe side effects and strongly interacts with biomembranes, both in its lipidic and proteic regions. CPZ also interacts with blood components, influences bioavailability, and affects the function of several biomolecules. Albumin plays an important role in the transport and storage of hormones, ions, fatty acids and others substances, including CPZ, affecting the regulation of their plasmatic concentration. Hmn is an important ferric residue of hemoglobin that binds within the hydrophobic region of albumin with great specificity. Hmn added to HSA and BSA solutions at a molar ratio of 1:1 quenched about half of their fluorescence. Stern-Volmer plots obtained from experiments carried out at 25 and 35 °C showed the quenching of fluorescence of HSA and BSA by CPZ to be a collisional phenomenon. Hmn quenches fluorescence by a static process, which specifically indicates the formation of a complex. Our results suggest the prime binding site for CPZ and Hmn on both HSA and BSA to be near tryptophan residues.

  1. Photoacids as a new fluorescence tool for tracking structural transitions of proteins: following the concentration-induced transition of bovine serum albumin.

    PubMed

    Amdursky, Nadav

    2015-12-21

    Spectroscopy-based techniques for assessing structural transitions of proteins follow either an intramolecular chromophore, as in absorption-based circular dichroism (CD) or fluorescence-based tryptophan emission, or an intermolecular chromophore such as fluorescent probes. Here a new fluorescent probe method to probe the structural transition of proteins by photoacids is presented, which has a fundamentally different photo-physical origin to that of common fluorescent probes. Photoacids are molecules that release a proton upon photo-excitation. By following the steady-state and time-resolved emission of the protonated and de-protonated species of the photoacid we probe the environment of its binding site in bovine serum albumin (BSA) in a wide range of weight concentrations (0.001-8%). We found a unique concentration-induced structural transition of BSA at pH2 and at concentrations of >0.75%, which involves the exposure of its hydrophobic core to the solution. We confirm our results with the common tryptophan emission method, and show that the use of photoacids can result in a much more sensitive tool. We also show that common fluorescent probes and the CD methodologies have fundamental restrictions that limit their use in a concentration-dependent study. The use of photoacids is facile and requires only a fluorospectrometer (and preferably, but not mandatorily, a time-resolution emission system). The photoacid can be either non-covalently (as in this study) or covalently attached to the molecule, and can be readily employed to follow the local environment of numerous (bio-)systems. PMID:26573990

  2. Recognition and binding of β-lactam antibiotics to bovine serum albumin by frontal affinity chromatography in combination with spectroscopy and molecular docking.

    PubMed

    Li, Qian; Zhang, Tianlong; Bian, Liujiao

    2016-03-01

    Serum albumins are the most abundant carrier proteins in blood plasma and participate in the binding and transportation of various exogenous and endogenous compounds in the body. This work was designed to investigate the recognition and binding of three typical β-lactam antibiotics including penicillin G (Pen G), penicillin V (Pen V) and cefalexin (Cef) with bovine serum albumin (BSA) by frontal affinity chromatography in combination with UV-vis absorption spectra, fluorescence emission spectra, binding site marker competitive experiment and molecular docking under simulated physiological conditions. The results showed that a BSA only bound with one antibiotic molecule in the binding process, and the binding constants for Pen G-BSA, Pen V-BSA and Cef-BSA complexes were 4.22×10(1), 4.86×10(2) and 3.32×10(3) (L/mol), respectively. All the three β-lactam antibiotics were mainly inserted into the subdomain IIA (binding site 1) of BSA by hydrogen bonds and Van der Waals forces. The binding capacity between the antibiotics and BSA was closely related to the functional groups and flexibility of side chains in antibiotics. This study provided an important insight into the molecular recognition and binding interaction of BSA with β-lactam antibiotics, which may be a useful guideline for the innovative clinical medications and new antibiotic designs with effective pharmacological properties. PMID:26882128

  3. Development of etoposide-loaded bovine serum albumin nanosuspensions for parenteral delivery.

    PubMed

    Wang, Zhonglan; Li, Zhongwen; Zhang, Dong; Miao, Lei; Huang, Guihua

    2015-01-01

    Nanosuspensions emerge as a promising strategy for delivery of poorly water-soluble drugs. Albumin is a versatile protein carrier for drug delivery and targeting. The purpose of this study was to develop a formulation of etoposide-loaded bovine serum albumin (BSA) nanosuspensions, to study in vitro characterization, and to estimate the in vivo safety and tissue distribution of etoposide-loaded BSA nanosuspensions for parenteral delivery. Etoposide-loaded BSA nanosuspensions were prepared by high-pressure homogenization-solvent precipitation method. The particle size, zeta potential, drug entrapment efficiency, and drug loading of the lyophilized formulation were 182.3 nm, -22.18 mV, 86.44%, and 8.49% respectively. In vitro release files of the formulation presented sustained release properties. Preliminary safety study was conducted to evaluate the delivery system, and results indicated that myelosuppression effect of the etoposide-loaded BSA nanosuspensions group was significantly lower than the Injection® group. Furthermore, results of tissue distribution studies showed that the concentration and AUC of etoposide were increased significantly in lung, liver, spleen while reduced in heart, kidney compared with the etoposide injection® group after i.v. administration of etoposide-loaded BSA nanosuspensions. The formulation played a role in targeting delivery to lung, reduce toxicity, and side effects of etoposide. In conclusion, etoposide-loaded BSA nanosuspensions were promising for parenteral delivery of etoposide.

  4. Spectroscopic and molecular modelling studies of binding mechanism of metformin with bovine serum albumin

    NASA Astrophysics Data System (ADS)

    Sharma, Deepti; Ojha, Himanshu; Pathak, Mallika; Singh, Bhawna; Sharma, Navneet; Singh, Anju; Kakkar, Rita; Sharma, Rakesh K.

    2016-08-01

    Metformin is a biguanide class of drug used for the treatment of diabetes mellitus. It is well known that serum protein-ligand binding interaction significantly influence the biodistribution of a drug. Current study was performed to characterize the binding mechanism of metformin with serum albumin. The binding interaction of the metformin with bovine serum albumin (BSA) was examined using UV-Vis absorption spectroscopy, fluorescence, circular dichroism, density functional theory and molecular docking studies. Absorption spectra and fluorescence emission spectra pointed out the weak binding of metformin with BSA as was apparent from the slight change in absorbance and fluorescence intensity of BSA in presence of metformin. Circular dichroism study implied the significant change in the conformation of BSA upon binding with metformin. Density functional theory calculations showed that metformin has non-planar geometry and has two energy states. The docking studies evidently signified that metformin could bind significantly to the three binding sites in BSA via hydrophobic, hydrogen bonding and electrostatic interactions. The data suggested the existence of non-covalent specific binding interaction in the complexation of metformin with BSA. The present study will certainly contribute to the development of metformin as a therapeutic molecule.

  5. Silver nanoclusters emitting weak NIR fluorescence biomineralized by BSA

    NASA Astrophysics Data System (ADS)

    Li, Baoshun; Li, Jianjun; Zhao, Junwu

    2015-01-01

    Noble metal (e.g., gold and silver) nanomaterials possess unique physical and chemical properties. In present work, silver nanoclusters (also known as silver quantum clusters or silver quantum dots) were synthesized by bovine serum albumin (BSA) biomineralization. The synthesized silver nanoclusters were characterized by UV-VIS absorption spectroscopy, fluorescence spectroscopy, upconversion emission spectroscopy, TEM, HRTEM and FTIR spectroscopy. TEM results showed that the average size of the silver nanoclusters was 2.23 nm. Fluorescence results showed that these silver nanoclusters could emit weak near-infrared (NIR) fluorescence (the central emission wavelength being about 765 nm). And the central excitation wavelength was about 395 nm, in the UV spectral region. These silver nanoclusters showed an extraordinarily large gap (about 370 nm) between the central excitation wavelength and central emission wavelength. In addition, it was found that these silver nanoclusters possess upconversion emission property. Upconversion emission results showed that the upconversion emission spectrum of the silver nanoclusters agreed well with their normal fluorescence emission spectrum. The synthesized silver nanoclusters showed high stability in aqueous solution and it was considered that they might be confined in BSA molecules. It was found that silver nanoclusters might enhance and broaden the absorption of proteins, and the protein absorption peak showed an obvious red shift (being 7 nm) after the formation of silver nanoclusters.

  6. The effect of nonenzymatic protein on lignocellulose enzymatic hydrolysis and simultaneous saccharification and fermentation.

    PubMed

    Wang, Hui; Kobayashi, Shinichi; Hiraide, Hatsue; Cui, Zongjun; Mochidzuki, Kazuhiro

    2015-01-01

    Nonenzymatic protein was added to cellulase hydrolysis and simultaneous saccharification and fermentation (SSF) of different biomass materials. Adding bovine serum albumin (BSA) and corn steep before cellulase enhanced enzyme activity in solution and increased cellulose and xylose conversion rates. The cellulose conversion rate of filter paper hydrolysis was increased by 32.5 % with BSA treatment. When BSA was added before cellulase, the remaining activity in the solution was higher than that in a control without BSA pretreatment. During SSF with pretreated rice straw as the substrate, adding 1.0 mg/mL BSA increased the ethanol yield by 13.6 % and final xylose yield by 42.6 %. The results indicated that lignin interaction is not the only mechanism responsible for the positive BSA effect. BSA had a stabilizing effect on cellulase and relieved cumulative sugar inhibition of enzymatic hydrolysis of biomass materials. Thus, nonenzymatic protein addition represents a promising strategy in the biorefining of lignocellulose materials.

  7. Endocytosis of albumin and thyroglobulin at the basolateral membrane of thyrocytes organized in follicles.

    PubMed

    Gire, V; Kostrouch, Z; Bernier-Valentin, F; Rabilloud, R; Munari-Silem, Y; Rousset, B

    1996-02-01

    Serum proteins such as albumin are present inside thyroid follicles in both normal and pathological situations. To analyze the mechanism of entry of these proteins, we investigated the ability of polarized thyrocytes to internalize soluble molecules at their basolateral pole. Experiments were conducted on in vitro reconstituted thyroid follicles using BSA and pig thyroglobulin (Tg) coupled to gold particles for electron microscopy, conjugated to fluorescein for conventional and confocal fluorescence microscopy, or radioiodinated for biochemical measurements. Incubations were carried out at 37 C. BSA and Tg coupled to gold particles were rapidly internalized from the culture medium and sequentially found in small vesicles and early endosomes and in late endosomes and lysosomes. Fluorescence microscope analyses revealed that the majority of cells forming reconstituted thyroid follicles are capable of internalizing BSA and Tg, but that Tg was more efficiently endocytosed than BSA. Using radioiodinated ligands, it was observed that the endocytosis of Tg was 10 times higher than that of BSA. The internalization of [125I]Tg was inhibited by increasing concentrations of unlabeled Tg. In contrast, endocytosis of 125I-labeled BSA was independent of the unlabeled BSA concentration. Experiments performed at 4 C indicated the presence of a basolateral membrane binding activity for [125I]Tg; the Tg concentration that reduced the binding of labeled Tg by 50% ranged from 4-6 microM. These data are evidence of a process of internalization of soluble molecules at the basolateral pole of thyrocytes, with BSA being internalized by fluid phase endocytosis and Tg by selective endocytosis. Our findings explain how serum albumin can enter thyroid follicles and disclose a new cellular handling and transport pathway of Tg. We propose that selective uptake of Tg operating on molecules secreted at the basolateral surface of thyrocytes could control the amount of Tg released in the

  8. Release and pharmacokinetics of near-infrared labeled albumin from monodisperse poly(d,l-lactic-co-hydroxymethyl glycolic acid) microspheres after subcapsular renal injection.

    PubMed

    Kazazi-Hyseni, F; van Vuuren, S H; van der Giezen, D M; Pieters, E H; Ramazani, F; Rodriguez, S; Veldhuis, G J; Goldschmeding, R; van Nostrum, C F; Hennink, W E; Kok, R J

    2015-08-01

    Subcapsular renal injection is a novel administration method for local delivery of therapeutics for the treatment of kidney related diseases. The aim of this study was to investigate the feasibility of polymeric microspheres for sustained release of protein therapeutics in the kidney and study the subsequent redistribution of the released protein. For this purpose, monodisperse poly(d,l-lactic-co-hydroxymethyl glycolic acid) (PLHMGA) microspheres (40 μm in diameter) loaded with near-infrared dye-labeled bovine serum albumin (NIR-BSA) were prepared by a membrane emulsification method. Rats were injected with either free NIR-BSA or with NIR-BSA loaded microspheres (NIR-BSA-ms) and the pharmacokinetics of the released NIR-BSA was studied for 3 weeks by ex vivo imaging of organs and blood. Quantitative release data were obtained from kidney homogenates and possible metabolism of the protein was investigated by SDS-PAGE analysis of the samples. The ex vivo images showed a rapid decrease of the NIR signal within 24h in kidneys injected with free NIR-BSA, while, importantly, the signal of the labeled protein was still visible at day 21 in kidneys injected with NIR-BSA-ms. SDS-PAGE analysis of the kidney homogenates showed that intact NIR-BSA was released from the microspheres. The locally released NIR-BSA drained to the systemic circulation and subsequently accumulated in the liver, where it was degraded and excreted renally. The in vivo release of NIR-BSA was calculated after extracting the protein from the remaining microspheres in kidney homogenates. The in vivo release rate was faster (89 ± 4% of the loading in 2 weeks) compared to the in vitro release of NIR-BSA (38 ± 1% in 2 weeks). In conclusion, PLHMGA microspheres injected under the kidney capsule provide a local depot from which a formulated protein is released over a prolonged time-period.

  9. Quenching of tryptophan fluorescence of bovine serum albumin under the effect of ions of heavy metals

    NASA Astrophysics Data System (ADS)

    Plotnikova, O. A.; Mel'nikov, A. G.; Mel'nikov, G. V.; Gubina, T. I.

    2016-01-01

    The interaction of heavy metals with bovine serum albumin (BSA) has been studied using data of quenching of intrinsic fluorescence of the protein by the ions of the heavy metals. Under the assumption of static quenching with formation of nonfluorescent complexes of fluorophores of BSA with heavy metals, conclusions have been drawn on the peculiarities of binding of the heavy metals to the protein. The values of the Stern-Volmer constants of association and those of the constants of BSA binding to the heavy metals decrease in the order Cu(II) > Pb(II) > Cd(II). It has been experimentally found that the copper ions have greater capacity to bind to the protein with the formation of the nonfluorescent complexes, which results in a significant decrease in the fluorescence intensity of the protein.

  10. Interactions and effects of BSA-functionalized single-walled carbon nanotubes on different cell lines

    NASA Astrophysics Data System (ADS)

    Muzi, Laura; Tardani, Franco; La Mesa, Camillo; Bonincontro, Adalberto; Bianco, Alberto; Risuleo, Gianfranco

    2016-04-01

    Functionalized carbon nanotubes (CNTs) have shown great promise in several biomedical contexts, spanning from drug delivery to tissue regeneration. Thanks to their unique size-related properties, single-walled CNTs (SWCNTs) are particularly interesting in these fields. However, their use in nanomedicine requires a clear demonstration of their safety in terms of tissue damage, toxicity and pro-inflammatory response. Thus, a better understanding of the cytotoxicity mechanisms, the cellular interactions and the effects that these materials have on cell survival and on biological membranes is an important first step for an appropriate assessment of their biocompatibility. In this study we show how bovine serum albumin (BSA) is able to generate homogeneous and stable dispersions of SWCNTs (BSA-CNTs), suggesting their possible use in the biomedical field. On the other hand, this study wishes to shed more light on the impact and the interactions of protein-stabilized SWCNTs with two different cell types exploiting multidisciplinary techniques. We show that BSA-CNTs are efficiently taken up by cells. We also attempt to describe the effect that the interaction with cells has on the dielectric characteristics of the plasma membrane and ion flux using electrorotation. We then focus on the BSA-CNTs’ acute toxicity using different cellular models. The novel aspect of this work is the evaluation of the membrane alterations that have been poorly investigated to date.

  11. Forced Desorption of Bovine Serum Albumin and Lysozyme from Graphite: Insights from Molecular Dynamics Simulation.

    PubMed

    Mücksch, Christian; Urbassek, Herbert M

    2016-08-18

    We use molecular dynamics (MD) simulation to study the adsorption and desorption of two widely different proteins, bovine serum albumin (BSA) and lysozyme, on a graphite surface. The adsorption is modeled using accelerated MD to allow the proteins to find optimum conformations on the surface. Our results demonstrate that the "hard protein" lysozyme retains much of its secondary structure during adsorption, whereas BSA loses it almost completely. BSA has a considerably larger adsorption energy compared to that of lysozyme, which does not scale with chain length. Desorption simulations are carried out using classical steered MD. The BSA molecule becomes fully unzipped during pull-off, whereas several helices survive this process in lysozyme. The unzipping process shows up in the force-distance curve of BSA as a series of peaks, whereas only a single or few, depending on protein orientation, force peaks occur for lysozyme. The maximum desorption force is larger for BSA than for lysozyme, but only by a factor of about 2.3.

  12. Heat-induced cross-linking and degradation of wheat gluten, serum albumin, and mixtures thereof.

    PubMed

    Rombouts, Ine; Lagrain, Bert; Delcour, Jan A

    2012-10-10

    Some wheat-based food systems, such as cakes, cookies, and egg noodles, contain mixtures of animal and plant (gluten) proteins and are processed under (mildly) alkaline conditions. Although changes in these proteins during processing can affect end product quality, they have seldom been studied. This study investigated protein cross-linking and degradation during heating (0-120 min, pH 8.0, 50-130 °C) of (mixtures of) wheat gluten and bovine serum albumin (BSA). The decrease in protein extractabilities in sodium dodecyl sulfate containing buffer under (non)reducing conditions and the levels of (cross-linked) amino acids were measured. No indications for polymerization at 50 °C were found. Below 100 °C, BSA polymerized more readily than wheat gluten. Above 100 °C, the opposite was observed. The kinetics of heat-induced polymerization of a 1:1 gluten-BSA mixture were similar to that of isolated gluten, implying that gluten decelerated BSA denaturation. Severe heating (130 °C, >15 min) induced degradation reactions in gluten but not in BSA. At all conditions used in this study, disulfide (SS) bonds contributed to the extractability loss. In addition, above 110 °C, β-elimination of cystine led to non-SS cross-links. Intramolecular SS bonds more often transformed in intermolecular non-SS bonds in BSA than in gluten. PMID:22950662

  13. Investigating the influence of effective parameters on molecular characteristics of bovine serum albumin nanoparticles

    NASA Astrophysics Data System (ADS)

    Rohiwal, S. S.; Satvekar, R. K.; Tiwari, A. P.; Raut, A. V.; Kumbhar, S. G.; Pawar, S. H.

    2015-04-01

    The protein nanoparticles formulation is a challenging task as they are prone to undergo conformational transitions while processing which may affect bioavailability for bioactive compounds. Herein, a modified desolvation method is employed to prepare Bovine Serum Albumin nanoparticles, with controllable particle size ranging from 100 to 300 nm and low polydispersity index. The factors influencing the size and structure of BSA NPs viz. protein concentration, pH and the conditions for purification are well investigated. The structure of BSA NPs is altered due to processing, and may affect the effective binding ability with drugs and bioactive compounds. With that aims, investigations of molecular characteristics of BSA NPs are carried out in detail by using spectroscopic techniques. UV-visible absorption and Fourier Transform Infrared demonstrate the alteration in protein structure of BSA NPs whereas the FT-Raman spectroscopy investigates changes in the secondary and tertiary structures of the protein. The conformational changes of BSA NPs are observed by change in fluorescence intensity and emission maximum wavelength of tryptophan residue by fluorescence spectroscopy. The field emission scanning electron and atomic force microscopy micrographs confirm the size and semi-spherical morphology of the BSA NPs. The effect of concentration and pH on particle size distribution is studied by particle size analyzer.

  14. High Mobility Group Box Protein 1 Boosts Endothelial Albumin Transcytosis through the RAGE/Src/Caveolin-1 Pathway

    PubMed Central

    Shang, Dan; Peng, Tao; Gou, Shanmiao; Li, Yiqing; Wu, Heshui; Wang, Chunyou; Yang, Zhiyong

    2016-01-01

    High-mobility group box protein 1 (HMGB1), an inflammatory mediator, has been reported to destroy cell-cell junctions, resulting in vascular endothelial hyperpermeability. Here, we report that HMGB1 increases the endothelial transcytosis of albumin. In mouse lung vascular endothelial cells (MLVECs), HMGB1 at a concentration of 500 ng/ml or less did not harm cell-cell junctions but rapidly induced endothelial hyperpermeability to 125I-albumin. HMGB1 induced an increase in 125I-albumin and AlexaFluor 488-labeled albumin internalization in endocytosis assays. Depletion of receptor for advanced glycation end products (RAGE), but not TLR2 or TLR4, suppressed HMGB1-induced albumin transcytosis and endocytosis. Genetic and pharmacological destruction of lipid rafts significantly inhibited HMGB1-induced albumin endocytosis and transcytosis. HMGB1 induced the rapid phosphorylation of caveolin (Cav)-1 and Src. Either RAGE gene silencing or soluble RAGE suppressed Cav-1 Tyr14 phosphorylation and Src Tyr418 phosphorylation. The Src inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl) pyrazolo[3,4-d] pyrimidine (PP2) blocked HMGB1-induced Cav-1 Tyr14 phosphorylation. PP2 and overexpression of Cav-1 with a T14F mutation significantly inhibited HMGB1-induced transcytosis and albumin endocytosis. Our findings suggest that HMGB1 induces the transcytosis of albumin via RAGE-dependent Src phosphorylation and Cav-1 phosphorylation. These studies revealed a new mechanism of HMGB1-induced endothelial hyperpermeability. PMID:27572515

  15. High Mobility Group Box Protein 1 Boosts Endothelial Albumin Transcytosis through the RAGE/Src/Caveolin-1 Pathway.

    PubMed

    Shang, Dan; Peng, Tao; Gou, Shanmiao; Li, Yiqing; Wu, Heshui; Wang, Chunyou; Yang, Zhiyong

    2016-01-01

    High-mobility group box protein 1 (HMGB1), an inflammatory mediator, has been reported to destroy cell-cell junctions, resulting in vascular endothelial hyperpermeability. Here, we report that HMGB1 increases the endothelial transcytosis of albumin. In mouse lung vascular endothelial cells (MLVECs), HMGB1 at a concentration of 500 ng/ml or less did not harm cell-cell junctions but rapidly induced endothelial hyperpermeability to (125)I-albumin. HMGB1 induced an increase in (125)I-albumin and AlexaFluor 488-labeled albumin internalization in endocytosis assays. Depletion of receptor for advanced glycation end products (RAGE), but not TLR2 or TLR4, suppressed HMGB1-induced albumin transcytosis and endocytosis. Genetic and pharmacological destruction of lipid rafts significantly inhibited HMGB1-induced albumin endocytosis and transcytosis. HMGB1 induced the rapid phosphorylation of caveolin (Cav)-1 and Src. Either RAGE gene silencing or soluble RAGE suppressed Cav-1 Tyr14 phosphorylation and Src Tyr418 phosphorylation. The Src inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl) pyrazolo[3,4-d] pyrimidine (PP2) blocked HMGB1-induced Cav-1 Tyr14 phosphorylation. PP2 and overexpression of Cav-1 with a T14F mutation significantly inhibited HMGB1-induced transcytosis and albumin endocytosis. Our findings suggest that HMGB1 induces the transcytosis of albumin via RAGE-dependent Src phosphorylation and Cav-1 phosphorylation. These studies revealed a new mechanism of HMGB1-induced endothelial hyperpermeability. PMID:27572515

  16. The Virucidal EB Peptide Protects Host Cells from Herpes Simplex Virus Type 1 Infection in the Presence of Serum Albumin and Aggregates Proteins in a Detergent-Like Manner▿

    PubMed Central

    Bultmann, Hermann; Girdaukas, Gary; Kwon, Glen S.; Brandt, Curtis R.

    2010-01-01

    The linear cationic amphiphilic EB peptide, derived from the FGF4 signal sequence, was previously shown to be virucidal and to block herpes simplex type I (HSV-1) entry (H. Bultmann, J. S. Busse, and C. R. Brandt, J. Virol. 75:2634–2645, 2001). Here we show that cells treated with EB (RRKKAAVALLPAVLLALLAP) for less than 5 min are also protected from infection with HSV-1. Though protection was lost over a period of 5 to 8 h, it was reinduced as rapidly as during the initial treatment. Below a 20 μM concentration of EB, cells gained protection in a serum-dependent manner, requiring bovine serum albumin (BSA) as a cofactor. Above 40 μM, EB coprecipitated with BSA under hypotonic conditions. Coprecipitates retained antiviral activity and released active peptide. NaCl (≥0.3 M) blocked coprecipitation without interfering with antiviral activity. As shown for β-galactosidase, EB below 20 μM acted as an enzyme inhibitor, whereas above 40 to 100 μM EB, β-galactosidase was precipitated as was BSA or other unrelated proteins. Pyrene fluorescence spectroscopy revealed that in the course of protein aggregation, EB acted like a cationic surfactant and self associated in a process resembling micelle formation. Both antiviral activity and protein aggregation did not depend on stereospecific EB interactions but depended strongly on the sequence of the peptide's hydrophobic tail. EB resembles natural antimicrobial peptides, such as melittin, but when acting in a nonspecific detergent-like manner, it primarily seems to target proteins. PMID:20643896

  17. Bovine serum albumin adsorption onto colloidal Al2O3 particles: a new model based on zeta potential and UV-vis measurements.

    PubMed

    Rezwan, Kurosch; Meier, Lorenz P; Rezwan, Mandana; Vörös, Janos; Textor, Marcus; Gauckler, Ludwig J

    2004-11-01

    We investigated the adsorption of bovine serum albumin (BSA) on colloidal Al2O3 particles in an aqueous environment. Changes in the zeta potential of the Al2O3 particles upon the adsorption of BSA were measured using an electro-acoustic technique. The mass of protein adsorbed was determined by using UV-vis spectroscopy. The change of the isoelectric point of the Al2O3 powder-protein suspension was found to be a function of adsorbed protein mass. It was shown that approximately one monolayer of BSA was needed to fully mask the surface and to compromise the charge of Al2O3. From titration experiments it follows that about 30-36% of the negatively charged groups of the protein form bonds with the protonated and charged Al2O3 surface. On the basis of our observations we introduced a new adsorption model for BSA on Al2O3 particles.

  18. Mechanism of denaturation of bovine serum albumin by dodecyl trimethylammonium bromide.

    PubMed

    Moosavi-Movahedi, A A; Bordbar, A K; Taleshi, A A; Naderimanesh, H M; Ghadam, P

    1996-09-01

    Bovine serum albumin (BSA) denaturation has been extensively studied by different anionic and cationic surfactant. Dodecyl trimethylammonium bromide (DTAB) is a cationic surfactant, and it is suggested that it binds to the C-terminal section of BSA. In the present study, the thermodynamical denaturation of BSA by dodecyl trimethylammonium bromide (DTAB) has been studied with various experimental techniques. Equilibrium dialysis, thermal denaturation, gel electrophoresis, titration microcalorimetry at pH 7, I = 0.005, and different temperatures were all performed. The enthalpy obtained from the van't Hoff relation and calorimetry method as well as electrophoresis results were utilized to explain the BSA tranistion state. Major findings included: the binding isotherm shifts at a low free concentrations of DTAB and at a higher temperature suggest endothermicity for enthalpy of interaction; the calorimetry enthalpy (delta Hcal) of interaction was smaller than the van't Hoff enthalpy (delta HvH) for BSA-DTAB interaction; and the aggregation of BSA increased with increasing DTAB concentration. This study suggests that BSA unfolding induced by DTAB follows a multistate transition model and does not follow the two-state mechanism assumed for most single subunit proteins.

  19. Conformational and adsorptive characteristics of albumin affect interfacial protein boundary lubrication: from experimental to molecular dynamics simulation approaches.

    PubMed

    Fang, Hsu-Wei; Hsieh, Man-Ching; Huang, Huei-Ting; Tsai, Cheng-Yen; Chang, Min-Hui

    2009-02-01

    The lifetime of artificial joints is mainly determined by their biotribological properties. Synovial fluid which consists of various biological molecules acts as the lubricant. Among the compositions of synovial fluid, albumin is the most abundant protein. Under high load and low sliding speed articulation of artificial joint, it is believed the lubricants form protective layers on the sliding surfaces under the boundary lubrication mechanism. The protective molecular layer keeps two surfaces from direct collision and thus decreases the possibility of wear damage. However, the lubricating ability of the molecular layer may vary due to the conformational change of albumin in the process. In this study, we investigated the influence of albumin conformation on the adsorption behaviors on the articulating surfaces and discuss the relationship between adsorbed albumin and its tribological behaviors. We performed the friction tests to study the effects of albumin unfolding on the frictional behaviors. The novelty of this research is to further carry out molecular dynamics simulation, and protein adsorption experiments to investigate the mechanisms of the albumin-mediated boundary lubrication of arthroplastic materials. It was observed that the thermal processes induce the loss of secondary structure of albumin. The compactness of the unfolded structure leads to a higher adsorption rate onto the articulating material surface and results in the increase of friction coefficient.

  20. Interaction of bovine serum albumin with starch nanoparticles prepared by TEMPO-mediated oxidation.

    PubMed

    Fan, Haoran; Ji, Na; Zhao, Mei; Xiong, Liu; Sun, Qingjie

    2015-01-01

    The objective of this study was to elucidate the interaction of starch nanoparticles prepared through TEMPO oxidation (TEMPO-SNPs) with protein (bovine serum albumin) by various spectroscopic techniques and transmission electron microscopy (TEM). The enhanced absorbance observed by UV spectra and the decrease in fluorescence spectroscopy of bovine serum albumin (BSA) induced by TEMPO-SNPs demonstrated the occurrence of an interaction between BSA and TEMPO-SNPs. The quenching constant was inversely correlated with temperature, showing that the quenching effect of TEMPO-SNPs was static quenching. Electrostatic force had a leading contribution to the binding roles of BSA on TEMPO-SNPs, which was confirmed by negative enthalpy change and positive entropy change. When interacting with TEMPO-SNPs at different concentrations, the content of the α-helix structure in BSA decreased and β-sheet and random coil structures increased, indicating that TEMPO-SNPs had an effect on the secondary conformation of BSA. Furthermore, TEM images suggested that nanoparticle-protein complexes were formed.

  1. Interaction of bovine serum albumin with starch nanoparticles prepared by TEMPO-mediated oxidation.

    PubMed

    Fan, Haoran; Ji, Na; Zhao, Mei; Xiong, Liu; Sun, Qingjie

    2015-01-01

    The objective of this study was to elucidate the interaction of starch nanoparticles prepared through TEMPO oxidation (TEMPO-SNPs) with protein (bovine serum albumin) by various spectroscopic techniques and transmission electron microscopy (TEM). The enhanced absorbance observed by UV spectra and the decrease in fluorescence spectroscopy of bovine serum albumin (BSA) induced by TEMPO-SNPs demonstrated the occurrence of an interaction between BSA and TEMPO-SNPs. The quenching constant was inversely correlated with temperature, showing that the quenching effect of TEMPO-SNPs was static quenching. Electrostatic force had a leading contribution to the binding roles of BSA on TEMPO-SNPs, which was confirmed by negative enthalpy change and positive entropy change. When interacting with TEMPO-SNPs at different concentrations, the content of the α-helix structure in BSA decreased and β-sheet and random coil structures increased, indicating that TEMPO-SNPs had an effect on the secondary conformation of BSA. Furthermore, TEM images suggested that nanoparticle-protein complexes were formed. PMID:25907012

  2. Tuning the interactions of PEG-coated gold nanorods with BSA and model proteins through insertion of amino or carboxylate groups.

    PubMed

    Scaletti, Federica; Feis, Alessandro; Centi, Sonia; Pini, Roberto; Rotello, Vincent M; Messori, Luigi

    2015-09-01

    Gold nanorods (GNRs) are important platforms for biosensing and drug delivery. As for most nanomaterials, appropriate coatings such as polyethylene glycol (PEG) are needed to stabilize GNRs within biological fluids. We show here that the interactions of GNRs with proteins can be finely modulated through surface modification using PEG-containing chains bearing charged headgroups. Interestingly, introduction of amino or carboxylate groups produces relevant and differential changes in GNR interactions with three representative proteins: lysozyme, cytochrome c, and bovine serum albumin. These effects were explored through the direct monitoring of plasmonic bands of the GNRs and are supported by independent dynamic light scattering (DLS) and circular dichroism (CD) determinations. Notably, GNR-protein interactions observed for these charged GNRs can be almost completely reversed by salt addition. These observations demonstrate the importance of electrostatic effects in governing GNR-protein interactions, and provide a basis for new sensing and delivery platforms.

  3. Protein stability induced by ligand binding correlates with changes in protein flexibility

    PubMed Central

    Celej, María Soledad; Montich, Guillermo G.; Fidelio, Gerardo D.

    2003-01-01

    The interaction between ligands and proteins usually induces changes in protein thermal stability with modifications in the midpoint denaturation temperature, enthalpy of unfolding, and heat capacity. These modifications are due to the coupling of unfolding with binding equilibrium. Furthermore, they can be attained by changes in protein structure and conformational flexibility induced by ligand interaction. To study these effects we have used bovine serum albumin (BSA) interacting with three different anilinonaphthalene sulfonate derivatives (ANS). These ligands have different effects on protein stability, conformation, and dynamics. Protein stability was studied by differential scanning calorimetry and fluorescence spectroscopy, whereas conformational changes were detected by circular dichroism and infrared spectroscopy including kinetics of hydrogen/deuterium exchange. The order of calorimetric midpoint of denaturation was: 1,8-ANS-BSA > 2,6-ANS-BSA > free BSA >> (nondetected) bis-ANS-BSA. Both 1,8-ANS and 2,6-ANS did not substantially modify the secondary structure of BSA, whereas bis-ANS induced a distorted α-helix conformation with an increase of disordered structure. Protein flexibility followed the order: 1,8-ANS-BSA < 2,6-ANS-BSA < free BSA << bis-ANS-BSA, indicating a clear correlation between stability and conformational flexibility. The structure induced by an excess of bis-ANS to BSA is compatible with a molten globule-like state. Within the context of the binding landscape model, we have distinguished five conformers (identified by subscript): BSA1,8-ANS, BSA2,6-ANS, BSAfree, BSAbis-ANS, and BSAunfolded among the large number of possible states of the conformational dynamic ensemble. The relative population of each distinguishable conformer depends on the type and concentration of ligand and the temperature of the system. PMID:12824495

  4. Locating the Binding Sites of Pb(II) Ion with Human and Bovine Serum Albumins

    PubMed Central

    Belatik, Ahmed; Hotchandani, Surat; Carpentier, Robert; Tajmir-Riahi, Heidar-Ali

    2012-01-01

    Lead is a potent environmental toxin that has accumulated above its natural level as a result of human activity. Pb cation shows major affinity towards protein complexation and it has been used as modulator of protein-membrane interactions. We located the binding sites of Pb(II) with human serum (HSA) and bovine serum albumins (BSA) at physiological conditions, using constant protein concentration and various Pb contents. FTIR, UV-visible, CD, fluorescence and X-ray photoelectron spectroscopic (XPS) methods were used to analyse Pb binding sites, the binding constant and the effect of metal ion complexation on HSA and BSA stability and conformations. Structural analysis showed that Pb binds strongly to HSA and BSA via hydrophilic contacts with overall binding constants of KPb-HSA = 8.2 (±0.8)×104 M−1 and KPb-BSA = 7.5 (±0.7)×104 M−1. The number of bound Pb cation per protein is 0.7 per HSA and BSA complexes. XPS located the binding sites of Pb cation with protein N and O atoms. Pb complexation alters protein conformation by a major reduction of α-helix from 57% (free HSA) to 48% (metal-complex) and 63% (free BSA) to 52% (metal-complex) inducing a partial protein destabilization. PMID:22574219

  5. The studies of density, apparent molar volume, and viscosity of bovine serum albumin, egg albumin, and lysozyme in aqueous and RbI, CsI, and DTAB aqueous solutions at 303.15 K.

    PubMed

    Singh, Man; Chand, Hema; Gupta, K C

    2005-06-01

    Density (rho), apparent molar volume (V(phi)), and viscosity (eta) of 0.0010 to 0.0018% (w/v) of bovine serum albumin (BSA), egg albumin, and lysozyme in 0.0002, 0.0004, and 0.0008 M aqueous RbI and CsI, and (dodecyl)(trimethyl)ammonium bromide (DTAB) solutions were obtained. The experimental data were regressed against composition, and constants are used to elucidate the conformational changes in protein molecules. With salt concentration, the density of proteins is found to decrease, and the order of the effect of additives on density is observed as CsI > RbI > DTAB. The trend of apparent molar volume of proteins is found as BSA > egg-albumin > lysozyme for three additives. In general, eta values of BSA remain higher for all compositions of RbI than that of egg-albumin for CsI and DTAB. These orders of the data indicate the strength of intermolecular forces between proteins and salts, and are helpful for understanding the denaturation of proteins.

  6. Ellipsometric studies of synthetic albumin-binding chitosan-derivatives and selected blood plasma proteins

    NASA Astrophysics Data System (ADS)

    Sarkar, Sabyasachi

    This dissertation summarizes work on the synthesis of chitosan-derivatives and the development of ellipsometric methods to characterize materials of biological origin. Albumin-binding chitosan-derivatives were synthesized via addition reactions that involve amine groups naturally present in chitosan. These surfaces were shown to have an affinity towards human serum albumin via ELISA, UV spectroscopy and SDS PAGE. Modified surfaces were characterized with IR ellipsometry at various stages of their synthesis using appropriate optical models. It was found that spin cast chitosan films were anisotropic in nature. All optical models used for characterizing chitosan-derivatives were thus anisotropic. Chemical signal dependence on molecular structure and composition was illustrated via IR spectroscopic ellipsometry (IRSE). An anisotropic optical model of an ensemble of Lorentz oscillators were used to approximate material behavior. The presence of acetic acid in spin-cast non-neutralized chitosan samples was thus shown. IRSE application to biomaterials was also demonstrated by performing a step-wise chemical characterizations during synthesis stages. Protein adsorbed from single protein solutions on these modified surfaces was monitored by visible in-situ variable wavelength ellipsometry. Based on adsorption profiles obtained from single protein adsorption onto silicon surfaces, lumped parameter kinetic models were developed. These models were used to fit experimental data of immunoglobulin-G of different concentrations and approximate conformational changes in fibrinogen adsorption. Biomaterial characterization by ellipsometry was further extended to include characterization of individual protein solutions in the IR range. Proteins in an aqueous environment were characterized by attenuated total internal reflection (ATR) IR ellipsometry using a ZnSe prism. Parameterized dielectric functions were created for individual proteins using Lorentz oscillators. These

  7. Comprehensive studies on the nature of interaction between carboxylated multi-walled carbon nanotubes and bovine serum albumin.

    PubMed

    Lou, Kai; Zhu, Zhaohua; Zhang, Hongmei; Wang, Yanqing; Wang, Xiaojiong; Cao, Jian

    2016-01-01

    Herein, the interaction between carboxylated multi-walled carbon nanotubes (MWCNTs-COOH) and bovine serum albumin has been investigated by using circular dichroism, UV-vis, and fluorescence spectroscopic methods and molecular modeling in order to better understand the basic behavior of carbon nanotubes in biological systems. The spectral results showed that MWCNTs-COOH bound to BSA and induced the relatively large changes in secondary structure of protein by mainly hydrophobic forces and π-π stacking interactions. Thermal denaturation of BSA in the presence of MWCNTs-COOH indicated that carbon nanotubes acted as a structure destabilizer for BSA. In addition, the putative binding site of MWCNTs-COOH on BSA was near to domain II. With regard to human health, the present study could provide a better understanding of the biological properties, cytotocicity of surface modified carbon nanotubes.

  8. Biomimetic synthesis of hollow calcium carbonate with the existence of the agar matrix and bovine serum albumin.

    PubMed

    Feng, Jianhua; Wu, Gang; Qing, Chengsong

    2016-01-01

    Proteins play important roles in the process of biomineralization. Vaterite and calcite have been synthesized by the reaction of Na2CO3 and CaCl2 in the bovine serum albumin (BSA) and agar system. The samples have been characterized by Fourier transform infrared spectroscopy (FT-IR) and X-ray diffraction (XRD). The shape of CaCO3 crystal has been analyzed by scanning electronic microscopy (SEM). The results show that calcite is a single product in the absence of BSA, but the product is a mixture of calcite and vaterite in the presence of BSA. The spheral shell of CaCO3 crystal was obtained when the concentration of BSA increased to 9.0mg/mL.

  9. Roles of bovine serum albumin and copper in the assay and stability of ammonia monooxygenase activity in vitro.

    PubMed Central

    Juliette, L Y; Hyman, M R; Arp, D J

    1995-01-01

    We investigated the effects of bovine serum albumin (BSA) on both the assay and the stability of ammonia-oxidizing activity in cell extracts of Nitrosomonas europaea. Ammonia-dependent O2 uptake activity of freshly prepared extracts did not require BSA. However, a dependence on BSA developed in extracts within a short time. The role of BSA in the assay of ammonia-oxidizing activity apparently is to absorb endogenous free fatty acids which are present in the extracts, because (i) only proteins which bind fatty acids, e.g., BSA or beta-lactoglobulin, supported ammonia-oxidizing activity; (ii) exogenous palmitoleic acid completely inhibited ammonia-dependent O2 uptake activity; (iii) the inhibition caused by palmitoleic acid was reversed only by proteins which bind fatty acids; and (iv) the concentration of endogenous free palmitoleic acid increased during aging of cell extracts. Additionally, the presence of BSA (10 mg/ml) or CuCl2 (500 microM) stabilized ammonia-dependent O2 uptake activity for 2 to 3 days at 4 degrees C. The stabilizing effect of BSA or CuCl2 was apparently due to an inhibition of lipolysis, because both additives inhibited the increase in concentrations of free palmitoleic acid in aging extracts. Other additives which are known to modify lipase activity were also found to stabilize ammonia-oxidizing activity. These additives included HgCl2, lecithin, and phenylmethylsulfonyl fluoride. PMID:7665467

  10. New insight into the stereoselective interactions of quinine and quinidine, with bovine serum albumin.

    PubMed

    Liu, Yan; Chen, Mingmao; Wang, Shuaihua; Lin, Jingjing; Cai, Lizhen; Song, Ling

    2014-05-01

    Quinine (QN) and quinidine (QD), the chief quinoline alkaloids of various species of cinchona bark, are stereoisomers to each other. In this study, a series of appropriate and efficient methods have been applied to compare the binding modes of QN and QD with bovine serum albumin (BSA). The isothermal titration calorimetry and room temperature phosphorescence results show that both QN and QD can interact with BSA at one binding site to form drug-protein complexes, mainly through enthalpic driving force with the binding affinity order: QN > QD. The fluorescence resonance energy transfer and time-resolved fluorescence spectroscopy exhibits that QN has a larger energy transfer and more intensified binding capacity for BSA than QD. Data of dynamic light scattering reveal that the aggregate state of BSA is changed during this binding process, and the particle size distribution of QN-BSA bioconjugate is larger than that of QD. Nuclear magnetic resonance analysis indicates that aromatic protons make more contribution during ligand-protein complexation than that of aliphatic protons. The circular dichroism spectra exhibit different degrees of changes in BSA secondary structures in the presence of QN and QD, respectively.

  11. Albumin and multiple sclerosis.

    PubMed

    LeVine, Steven M

    2016-04-12

    Leakage of the blood-brain barrier (BBB) is a common pathological feature in multiple sclerosis (MS). Following a breach of the BBB, albumin, the most abundant protein in plasma, gains access to CNS tissue where it is exposed to an inflammatory milieu and tissue damage, e.g., demyelination. Once in the CNS, albumin can participate in protective mechanisms. For example, due to its high concentration and molecular properties, albumin becomes a target for oxidation and nitration reactions. Furthermore, albumin binds metals and heme thereby limiting their ability to produce reactive oxygen and reactive nitrogen species. Albumin also has the potential to worsen disease. Similar to pathogenic processes that occur during epilepsy, extravasated albumin could induce the expression of proinflammatory cytokines and affect the ability of astrocytes to maintain potassium homeostasis thereby possibly making neurons more vulnerable to glutamate exicitotoxicity, which is thought to be a pathogenic mechanism in MS. The albumin quotient, albumin in cerebrospinal fluid (CSF)/albumin in serum, is used as a measure of blood-CSF barrier dysfunction in MS, but it may be inaccurate since albumin levels in the CSF can be influenced by multiple factors including: 1) albumin becomes proteolytically cleaved during disease, 2) extravasated albumin is taken up by macrophages, microglia, and astrocytes, and 3) the location of BBB damage affects the entry of extravasated albumin into ventricular CSF. A discussion of the roles that albumin performs during MS is put forth.

  12. Albumin and multiple sclerosis.

    PubMed

    LeVine, Steven M

    2016-01-01

    Leakage of the blood-brain barrier (BBB) is a common pathological feature in multiple sclerosis (MS). Following a breach of the BBB, albumin, the most abundant protein in plasma, gains access to CNS tissue where it is exposed to an inflammatory milieu and tissue damage, e.g., demyelination. Once in the CNS, albumin can participate in protective mechanisms. For example, due to its high concentration and molecular properties, albumin becomes a target for oxidation and nitration reactions. Furthermore, albumin binds metals and heme thereby limiting their ability to produce reactive oxygen and reactive nitrogen species. Albumin also has the potential to worsen disease. Similar to pathogenic processes that occur during epilepsy, extravasated albumin could induce the expression of proinflammatory cytokines and affect the ability of astrocytes to maintain potassium homeostasis thereby possibly making neurons more vulnerable to glutamate exicitotoxicity, which is thought to be a pathogenic mechanism in MS. The albumin quotient, albumin in cerebrospinal fluid (CSF)/albumin in serum, is used as a measure of blood-CSF barrier dysfunction in MS, but it may be inaccurate since albumin levels in the CSF can be influenced by multiple factors including: 1) albumin becomes proteolytically cleaved during disease, 2) extravasated albumin is taken up by macrophages, microglia, and astrocytes, and 3) the location of BBB damage affects the entry of extravasated albumin into ventricular CSF. A discussion of the roles that albumin performs during MS is put forth. PMID:27067000

  13. About the structural role of disulfide bridges in serum albumins: evidence from protein simulated unfolding.

    PubMed

    Paris, Guillaume; Kraszewski, Sebastian; Ramseyer, Christophe; Enescu, Mironel

    2012-11-01

    The role of the 17 disulfide (S-S) bridges in preserving the native conformation of human serum albumin (HSA) is investigated by performing classical molecular dynamics (MD) simulations on protein structures with intact and, respectively, reduced S-S bridges. The thermal unfolding simulations predict a clear destabilization of the protein secondary structure upon reduction of the S-S bridges as well as a significant distortion of the tertiary structure that is revealed by the changes in the protein native contacts fraction. The effect of the S-S bridges reduction on the protein compactness was tested by calculating Gibbs free energy profiles with respect to the protein gyration radius. The theoretical results obtained using the OPLS-AA and the AMBER ff03 force fields are in agreement with the available experimental data. Beyond the validation of the simulation method, the results here reported provide new insights into the mechanism of the protein reductive/oxidative unfolding/folding processes. It is predicted that in the native conformation of the protein, the thiol (-SH) groups belonging to the same reduced S-S bridge are located in potential wells that maintain them in contact. The -SH pairs can be dispatched by specific conformational transitions of the peptide chain located in the neighborhood of the cysteine residues.

  14. Characterization and optimization of heroin hapten-BSA conjugates: method development for the synthesis of reproducible hapten-based vaccines.

    PubMed

    Torres, Oscar B; Jalah, Rashmi; Rice, Kenner C; Li, Fuying; Antoline, Joshua F G; Iyer, Malliga R; Jacobson, Arthur E; Boutaghou, Mohamed Nazim; Alving, Carl R; Matyas, Gary R

    2014-09-01

    A potential new treatment for drug addiction is immunization with vaccines that induce antibodies that can abrogate the addictive effects of the drug of abuse. One of the challenges in the development of a vaccine against drugs of abuse is the availability of an optimum procedure that gives reproducible and high yielding hapten-protein conjugates. In this study, a heroin/morphine surrogate hapten (MorHap) was coupled to bovine serum albumin (BSA) using maleimide-thiol chemistry. MorHap-BSA conjugates with 3, 5, 10, 15, 22, 28, and 34 haptens were obtained using different linker and hapten ratios. Using this optimized procedure, MorHap-BSA conjugates were synthesized with highly reproducible results and in high yields. The number of haptens attached to BSA was compared by 2,4,6-trinitrobenzenesulfonic acid (TNBS) assay, modified Ellman's test and matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). Among the three methods, MALDI-TOF MS discriminated subtle differences in hapten density. The effect of hapten density on enzyme-linked immunosorbent assay (ELISA) performance was evaluated with seven MorHap-BSA conjugates of varying hapten densities, which were used as coating antigens. The highest antibody binding was obtained with MorHap-BSA conjugates containing 3-5 haptens. This is the first report that rigorously analyzes, optimizes and characterizes the conjugation of haptens to proteins that can be used for vaccines against drugs of abuse. The effect of hapten density on the ELISA detection of antibodies against haptens demonstrates the importance of careful characterization of the hapten density by the analytical techniques described.

  15. Interaction of drugs with bovine and human serum albumin

    NASA Astrophysics Data System (ADS)

    Sułkowska, Anna

    2002-09-01

    The study on the interaction of antithyroid drugs: 2-mercapto-1-methylimidazole (Methimazole, MMI) and 6 n-propyl-2-thiouracil (PTU) with two kinds of serum albumin: bovine (BSA) and human (HSA) has been undertaken. Fluorescence emission spectra of serum albumin in the presence of MMI or PTU, recorded at the excitation wavelengths 280 and 295 nm, clearly show that the studied drugs act as quenchers. A decrease in fluorescence intensity at 340 or 350 nm, when excited at 280 or 295 nm, respectively, is attributed to changes in the environment of the protein fluorophores caused to the presence of the ligand. The 295 nm lights excites tryptophan residues, while the 280 nm lights excites both tryptophan and tyrosine residues. A comparison of quenching effects, when protein is excited at 295 and 280 nm, reveals that the tryptophanyl group interacts with the ligand. The differences in interactions of pyrimidine derivatives with HSA and BSA were observed using spectrofluorimetry technique. As the HSA structure contains only one tryptophanyl residue (Trp 214), while BSA has two ones (Trp 135 and Trp 214), the similar decrease of fluorescence points at the subdomain IIA, where Trp 214 was located, as a binding site of the studied drugs.

  16. Quarter variation and correlations of colostrum albumin, immunoglobulin G1 and G2 in dairy cows.

    PubMed

    Samarütel, Jaak; Baumrucker, Craig R; Gross, Josef J; Dechow, Chad D; Bruckmaier, Rupert M

    2016-05-01

    A high variation in immunoglobulin G1 (IgG1) concentration in first milked quarter colostrum has been reported, but BSA quarter colostrum variation is not known. The occurrence of serum albumin in milk has been attributed to increased blood-milk barrier penetration. Reports of serum albumin binding to the Fc Receptor of the neonate, the receptor thought to be responsible for IgG1 transcytosis, suggested that a correlation with the appearance of IgG1 in colostrum of dairy cows was likely. The objective of the study was to establish the quarter colostrum concentration and mass of immunoglobulins and serum albumin. First colostrum was quarter collected within 4 h of parturition from healthy udders of 31 multiparous dairy cows. Individual quarter colostrum weight was determined and a sample of each was frozen for subsequent analysis. Concentrations of immunoglobulin G1, G2, and BSA were measured by ELISA and total mass of components was calculated. In addition, colostrum was also analysed for L-lactate dehydrogenase activity. Analysis of concentration and mass of BSA, immunoglobulin G1, G2 established that the quarter variations were different by cow, quarter and quarter within cow. Partial correlations corrected for colostrum weight indicated that BSA and IgG2 concentration and mass are closely correlated while that of BSA and IgG1 concentration and mass exhibited no correlation suggesting that BSA and IgG1 may have different transport mechanisms. Interestingly, immunoglobulin G1 and G2 concentration and mass exhibited strong correlations suggesting that also some unknown mechanism of immunoglobulin G2 appearance in colostrum is occurring. Finally, no measured protein exhibited any correlation with the activity of lactate dehydrogenase in colostrum.

  17. Unraveling the binding mechanism of polyoxyethylene sorbitan esters with bovine serum albumin: a novel theoretical model based on molecular dynamic simulations.

    PubMed

    Delgado-Magnero, Karelia H; Valiente, Pedro A; Ruiz-Peña, Miriam; Pérez-Gramatges, Aurora; Pons, Tirso

    2014-04-01

    To gain a better understanding of the interactions governing the binding mechanism of proteins with non-ionic surfactants, the association processes of Tween 20 and Tween 80 with the bovine serum albumin (BSA) protein were investigated using molecular dynamics (MD) simulations. Protein:surfactant molar ratios were chosen according to the critical micelle concentration (CMC) of each surfactant in the presence of BSA. It was found that both the hydrophilic and the hydrophobic groups of the BSA equally contribute to the surface area of interaction with the non-ionic surfactants. A novel theoretical model for the interactions between BSA and these surfactants at the atomic level is proposed, where both surfactants bind to non-specific domains of the BSA three-dimensional structure mainly through their polyoxyethylene groups, by hydrogen bonds and van der Waals interactions. This is well supported by the strong electrostatic and van der Waals interaction energies obtained in the calculations involving surfactant polyoxyethylene groups and different protein regions. The results obtained from the MD simulations suggest that the formation of surfactant clusters over the BSA structure, due to further cooperative self-assembly of Tween molecules, could increase the protein conformational stability. These results extend the current knowledge on molecular interactions between globular proteins and non-ionic surfactants, and contribute to the fine-tuning design of protein formulations using polysorbates as excipients for minimizing the undesirable effects of protein adsorption and aggregation.

  18. Fluorescence spectroscopic study of serum albumin-bromadiolone interaction: fluorimetric determination of bromadiolone.

    PubMed

    Deepa, Subbiah; Mishra, Ashok K

    2005-07-01

    Bromadiolone (BRD), a substituted 4-hydroxycoumarin derivative, is known to possess anti-coagulant activity with acute toxicity. In this paper, we report a study on the interaction of bromadiolone with the plasma proteins bovine serum albumin (BSA) and human serum albumin (HSA), using the intrinsic fluorescence emission properties of bromadiolone. Bromadiolone is weakly fluorescent in aqueous buffer medium, with an emission at 397 nm. Binding of bromadiolone with serum albumins (SA) leads to a marked enhancement in the fluorescence emission intensity and steady state fluorescence anisotropy (r(ss)), accompanied by a blueshift of 10 nm. In the serum albumin-bromadiolone complex, selective excitation of tryptophan (Trp) residue results in emission from bromadiolone, thereby indicating a Förster type energy transfer from Trp to BRD. This quenching of Trp fluorescence by BRD was used to estimate the binding constant of the SA-BRD complex. The binding constants for BRD with BSA and HSA were 7.5 x 10(4) and 3.7 x 10(5)L mol(-1), respectively. Based on this, a new method involving SA as fluorescence-enhancing reagent for estimation of BRD in aqueous samples has been suggested. The detection limits of bromadiolone under the optimum conditions were 0.77 and 0.19 microg mL(-1) in presence of BSA and HSA, respectively. PMID:15925260

  19. Albumin adsorption on CoCrMo alloy surfaces

    NASA Astrophysics Data System (ADS)

    Yan, Yu; Yang, Hongjuan; Su, Yanjing; Qiao, Lijie

    2015-12-01

    Proteins can adsorb on the surface of artificial joints immediately after being implanted. Although research studying protein adsorption on medical material surfaces has been carried out, the mechanism of the proteins’ adsorption which affects the corrosion behaviour of such materials still lacks in situ observation at the micro level. The adsorption of bovine serum albumin (BSA) on CoCrMo alloy surfaces was studied in situ by AFM and SKPFM as a function of pH and the charge of CoCrMo alloy surfaces. Results showed that when the specimens were uncharged, hydrophobic interaction could govern the process of the adsorption rather than electrostatic interaction, and BSA molecules tended to adsorb on the surfaces forming a monolayer in the side-on model. Results also showed that adsorbed BSA molecules could promote the corrosion process for CoCrMo alloys. When the surface was positively charged, the electrostatic interaction played a leading role in the adsorption process. The maximum adsorption occurred at the isoelectric point (pH 4.7) of BSA.

  20. Albumin adsorption on CoCrMo alloy surfaces

    PubMed Central

    Yan, Yu; Yang, Hongjuan; Su, Yanjing; Qiao, Lijie

    2015-01-01

    Proteins can adsorb on the surface of artificial joints immediately after being implanted. Although research studying protein adsorption on medical material surfaces has been carried out, the mechanism of the proteins’ adsorption which affects the corrosion behaviour of such materials still lacks in situ observation at the micro level. The adsorption of bovine serum albumin (BSA) on CoCrMo alloy surfaces was studied in situ by AFM and SKPFM as a function of pH and the charge of CoCrMo alloy surfaces. Results showed that when the specimens were uncharged, hydrophobic interaction could govern the process of the adsorption rather than electrostatic interaction, and BSA molecules tended to adsorb on the surfaces forming a monolayer in the side-on model. Results also showed that adsorbed BSA molecules could promote the corrosion process for CoCrMo alloys. When the surface was positively charged, the electrostatic interaction played a leading role in the adsorption process. The maximum adsorption occurred at the isoelectric point (pH 4.7) of BSA. PMID:26673525

  1. Adhesion of Staphylococcus epidermidis to biomaterials is inhibited by fibronectin and albumin.

    PubMed

    Linnes, J C; Mikhova, K; Bryers, J D

    2012-08-01

    Decades of contradictory results have obscured the exact role of adsorbed fibronectin in the adhesion of the bacterium, Staphylococcus epidermidis, to biomaterials. Here, the ability of adsorbed fibronectin (FN) or bovine serum albumin (BSA) to modulate S. epidermidis adhesion to various biomaterials is reported. FN or BSA was adsorbed in increasing surface densities up to saturated monolayer coverage onto various common biomaterials, including poly(ethylene terephthalate), fluorinated ethylene propylene, poly(ether urethane), silicone, and borosilicate glass. Despite the wide range of surface characteristics represented, adsorption isotherms varied only subtly between materials for the two proteins considered. S. epidermidis adhesion to the various protein-coated biomaterials was quantified in a static-fluid batch adhesion assay. Although slight differences in overall adherent cell numbers were observed between the various protein-coated substrata, all materials exhibited significant dose-dependent decreases in S. epidermidis adhesion with increasing adsorption of either protein (FN, BSA) to all surfaces. Results here indicate that S. epidermidis adhesion to FN-coated surfaces is not a specific adhesion (i.e., receptor: ligand) mediated process, as no significant difference in adhesion was found between FN- and BSA-coated materials. Rather, results indicate that increasing surface density of either FN or BSA actually inhibited S. epidermidis adhesion to all biomaterials examined.

  2. Interaction of copper(II) complex of compartmental Schiff base ligand N, N'-bis(3-hydroxysalicylidene)ethylenediamine with bovine serum albumin

    NASA Astrophysics Data System (ADS)

    Boghaei, Davar M.; Farvid, Shokouh S.; Gharagozlou, Mehrnaz

    2007-03-01

    Circular dichroism (CD) spectroscopy, cyclic voltammetry (CV) and differential pulse voltammetry (DPV) were used to investigate the interaction between copper(II) complex of compartmental Schiff base ligand (L), N, N'-bis(3-hydroxysalicylidene)ethylenediamine, and bovine serum albumin (BSA) in 0.1 mol dm -3 phosphate buffer solution adjusted to physiological pH 7.0 containing 20% (w/w) dimethylsulfoxide at room temperature. CD spectra show that the interaction of the copper(II) complex with BSA leads to changes in the α-helical content of BSA and therefore changes in secondary structure of the protein with the slight red shift (2 nm) in CD spectra. From the voltammetric data, i.e. changes in limiting current with addition of BSA, the binding constant ( K) of the interaction of copper(II) complex with BSA was found to be 1.96 × 10 4 dm 3 mol -1. From the shifts in potential with the addition of BSA, the equilibrium constant ratio ( K2/ K1) for the binding of the oxidized Cu IIL ( K1) and reduced Cu IL ( K2) species to BSA was found to be 3.77, which shows that the reduced form Cu IL is bound more strongly to BSA than the oxidized form Cu IIL.

  3. The response of type three secretion system needle proteins MxiHDelta5, BsaLDelta5, and PrgIDelta5 to temperature and pH.

    PubMed

    Barrett, Brooke S; Picking, Wendy L; Picking, William D; Middaugh, C Russell

    2008-11-15

    The type III secretion system (TTSS) is a specialized supramolecular injectisome composed of 25 or more proteins which form basal and extracellular domains and share gross architectural similarities with bacterial flagella. The extracellular component of the "needle complex" is primarily composed of a single monomeric subunit organized in a helical array surrounding a hollow pore and protrudes from the bacterial membrane. It is through this surface appendage that virulence factors are translocated to the host cell cytoplasm and thereby subvert normal host cell functions. We present here a comprehensive biophysical analysis of the dynamic conformational behavior of the truncated monomeric needle subunit proteins MxiH(Delta5) (Shigella flexneri), BsaL(Delta5) (Burkholderia pseudomallei), and PrgI(Delta5) (Salmonella typhimurium) as well as their thermal stability over a pH range of 3-8. Circular dichroism spectroscopy indicates the secondary structure is largely alpha helical in all three proteins, and surprisingly thermally labile with transition midpoints in the range of 35-50 degrees C over the pH range of 3-8. Additionally, at the concentrations examined, the very broad thermal transitions were >90% reversible. Second derivative UV absorbance spectroscopy data indicates some disruption of the protein's tertiary structure occurs at temperatures in the range of 29-46 degrees C. The difference in the pH of maximal stability for each of the proteins and the variation for each protein with respect to both secondary and tertiary structural elements is striking. It appears, that at physiological temperatures all three proteins experience intermediate non-native molten globule like states in which they display significant secondary structure in the absence of extensive tertiary interactions. Because of the size difference between the inner pore of the needle and the fully folded needle proteins, it seems clear that the needle subunits must be secreted in a partially or

  4. Investigations of acetaminophen binding to bovine serum albumin in the presence of fatty acid: Fluorescence and 1H NMR studies

    NASA Astrophysics Data System (ADS)

    Bojko, B.; Sułkowska, A.; Maciążek-Jurczyk, M.; Równicka, J.; Sułkowski, W. W.

    2009-04-01

    The binding of acetaminophen to bovine serum albumin (BSA) was studied by the quenching fluorescence method and the proton nuclear magnetic resonance technique ( 1H NMR). For fluorescence measurements 1-anilino-9-naphthalene sulfonate (ANS) hydrophobic probe was used to verify subdomain IIIA as acetaminophen's likely binding site. Three binding sites of acetaminophen in subdomain IIA of bovine serum albumin were found. Quenching constants calculated by the Stern-Volmer modified method were used to estimate the influence of myristic acid (MYR) on the drug binding to the albumin. The influence of [fatty acid]/[albumin] molar ratios on the affinity of the protein towards acetaminophen was described. Changes of chemical shifts and relaxation times of the drug indicated that the presence of MYR inhibits interaction in the AA-albumin complex. It is suggested that the elevated level of fatty acids does not significantly influence the pharmacokinetics of acetaminophen.

  5. Arginine and lysine reduce the high viscosity of serum albumin solutions for pharmaceutical injection.

    PubMed

    Inoue, Naoto; Takai, Eisuke; Arakawa, Tsutomu; Shiraki, Kentaro

    2014-05-01

    Therapeutic protein solutions for subcutaneous injection must be very highly concentrated, which increases their viscosity through protein-protein interactions. However, maintaining a solution viscosity below 50 cP is important for the preparation and injection of therapeutic protein solutions. In this study, we examined the effect of various amino acids on the solution viscosity of very highly concentrated bovine serum albumin (BSA) and human serum albumin (HSA) at a physiological pH. Among the amino acids tested, l-arginine hydrochloride (ArgHCl) and l-lysine hydrochloride (LysHCl) (50-200 mM) successfully reduced the viscosity of both BSA and HSA solutions; guanidine hydrochloride (GdnHCl), NaCl, and other sodium salts were equally as effective, indicating the electrostatic shielding effect of these additives. Fourier transform infrared spectroscopy showed that BSA is in its native state even in the presence of ArgHCl, LysHCl, and NaCl at high protein concentrations. These results indicate that weakened protein-protein interactions play a key role in reducing solution viscosity. ArgHCl and LysHCl, which are also non-toxic compounds, will be used as additives to reduce the solution viscosity of concentrated therapeutic proteins.

  6. The role of colloid particles in the albumin-lanthanides interaction: The study of aggregation mechanisms.

    PubMed

    Tikhonova, Tatiana N; Shirshin, Evgeny A; Romanchuk, Anna Yu; Fadeev, Victor V

    2016-10-01

    We studied the interaction between bovine serum albumin (BSA) and lanthanide ions in aqueous solution in the 4.0÷9.5pH range. A strong increase of the solution turbidity was observed at pH values exceeding 6, which corresponds to the formation of Ln(OH)3 nanoparticles, while no changes were observed near the isoelectric point of BSA (pH 4.7). The results of the dynamic light scattering and protein adsorption measurements clearly demonstrated that the observed turbidity enhancement was caused by albumin sorption on the surface of Ln(OH)3 and colloid particles bridging via adsorbed protein molecules. Upon pH increase from 4.5 to 6.5, albumin adsorption on lanthanide colloids was observed, while the following increase of pH from 6.5 to 9.5 led to protein desorption. The predominant role of the electrostatic interactions in the adsorption and desorption processes were revealed in the zeta-potential measurements. No reversibility was observed upon decreasing pH from 9.5 to 4.5 that was suggested to be due to the other interaction mechanisms present in the system. It was shown that while for all lanthanide ions the interaction mechanism with BSA was similar, its manifestation in the optical properties of the system was significantly different. This was interpreted as a consequence of the differences in lanthanides hydrolysis constants. PMID:27419645

  7. Effects of PEG size on structure, function and stability of PEGylated BSA.

    PubMed

    Plesner, Bitten; Fee, Conan J; Westh, Peter; Nielsen, Anders D

    2011-10-01

    The effects of PEGylation on the structural, thermal and functional stability of bovine serum albumin (BSA) were investigated using BSA and 6 linear mono-PEGylated BSA compounds. The secondary and tertiary structure of BSA measured by circular dichroism (CD) was independent of PEGylation. In contrast, the thermal stability of BSA was affected by PEGylation. The apparent unfolding temperature T(max) measured by differential scanning calorimetry (DSC) decreased with PEGylation, whereas the temperature of aggregation, T(agg), measured by dynamic light scattering (DLS) increased with PEGylation. The unfolding temperature and the temperature of aggregation were both independent of the molecular weight of the PEG chain. Possible functional changes of BSA after PEGylation were measured by Isothermal Titration Calorimetry (ITC), where the binding of sodium dodecyl sulphate (SDS) to BSA and PEGylated BSA was analysed. At 25°C, two distinct classes of binding sites (high affinity and low affinity) for BSA and one class of binding site (low affinity) for PEGylated BSA were identified. The binding isotherm was modelled assuming independence and thermodynamic equivalence of the sites within each class. From the present biophysical characterisation, it is concluded that after PEGylation BSA appears to be unaffected structurally (secondary and tertiary structure), slightly destabilised thermally (unfolding temperature), stabilised kinetically (temperature of aggregation) and has an altered functionality (binding profile). These biophysical characteristics are all independent of the molecular weight of the attached polymer chain.

  8. Adsorption of proteins at physiological concentrations on pegylated surfaces and the compatibilizing role of adsorbed albumin with respect to other proteins according to optical waveguide lightmode spectroscopy (OWLS).

    PubMed

    Leclercq, Laurent; Modena, Enrico; Vert, Michel

    2013-01-01

    In literature, contacts between pegylated compounds and blood proteins are generally discussed in terms of excluded volume-related repulsions although adsorption and compatibility have been reported for some of these proteins occasionally. The major problem to investigate the behavior of blood in contact with pegylated surfaces is the complexity of the medium and especially the presence of albumin in large excess. In a model approach, optical waveguide lightmode spectroscopy (OWLS) was used to monitor the fate of albumin, fibrinogen, and γ-globulins at physiological concentrations in pH = 7.4 isotonic HEPES buffer after contact with SiTiO2 chips coated with diblock poly(DL-lactic acid)-block-poly(ethylene oxide)s and triblock poly(DL-lactic acid)-block-poly(ethylene oxide)-block-poly(DL-lactic acid) copolymers. Corresponding homopolymers were used as controls. The three protein systems were investigated separately, as a mixture and when added successively according to different orders of addition. OWLS gave access to the mass and the thickness of adhering protein layers that resist washing with HEPES buffer. Protein depositions were detected regardless of the presence of poly(ethylene glycol) segments on surfaces. Adsorption depended on the protein, on the surface and also on the presence of the other proteins. Unexpectedly any surface coated with a layer of adsorbed albumin prevented deposition of other proteins, including albumin itself. This outstanding finding suggests that it was the presence of albumin adsorbed on a surface, pegylated or not, that made that surface compatible with other proteins. As a consequence, dipping a device to be in contact with the blood of a patient in a solution of albumin could be a very simple means to avoid further protein deposition and maybe platelets adhesion after in vivo implantation.

  9. Effect of zeta potentials on bovine serum albumin adsorption to hydroxyapatite surfaces.

    PubMed

    Miyake, Nahoko; Sato, Toru; Maki, Yoshinobu

    2013-01-01

    The aim of the present study was to examine the adsorption of bovine serum albumin (BSA) to hydroxyapatite surfaces by means of zeta potential. The electrophoretic mobility of both hydroxyapatite and BSA were negative, with BSA itself less negative than hydroxyapatite. The zeta potential of the surface of BSA-adsorbed hydroxyapatite was significantly more negative than that of hydroxyapatite alone (p<0.0001). The BSA histogram indicated two negative peaks, and the zeta potential of BSA-adsorbed hydroxyapatite also showed two similar negative peaks. These results suggest that BSA adsorption to hydroxyapatite surfaces is related to electrostatic interaction. PMID:23903580

  10. 17 beta-estradiol-BSA conjugates and 17 beta-estradiol regulate growth plate chondrocytes by common membrane associated mechanisms involving PKC dependent and independent signal transduction.

    PubMed

    Sylvia, V L; Walton, J; Lopez, D; Dean, D D; Boyan, B D; Schwartz, Z

    2001-01-01

    Nuclear receptors for 17 beta-estradiol (E(2)) are present in growth plate chondrocytes from both male and female rats and regulation of chondrocytes through these receptors has been studied for many years; however, recent studies indicate that an alternative pathway involving a membrane receptor may also be involved in the cell response. E(2) was found to directly affect the fluidity of chondrocyte membranes derived from female, but not male, rats. In addition, E(2) activates protein kinase C (PKC) in a nongenomic manner in female cells, and chelerythrine, a specific inhibitor of PKC, inhibits E(2)-dependent alkaline phosphatase activity and proteoglycan sulfation in these cells, indicating PKC is involved in the signal transduction mechanism. The aims of the present study were: (1) to examine the effect of a cell membrane-impermeable 17 beta-estradiol-bovine serum albumin conjugate (E(2)-BSA) on chondrocyte proliferation, differentiation, and matrix synthesis; (2) to determine the pathway that mediates the membrane effect of E(2)-BSA on PKC; and (3) to compare the action of E(2)-BSA to that of E(2). Confluent, fourth passage resting zone (RC) and growth zone (GC) chondrocytes from female rat costochondral cartilage were treated with 10(-9) to 10(-7) M E(2) or E(2)-BSA and changes in alkaline phosphatase specific activity, proteoglycan sulfation, and [(3)H]-thymidine incorporation measured. To examine the pathway of PKC activation, chondrocyte cultures were treated with E(2)-BSA in the presence or absence of GDP beta S (inhibitor of G-proteins), GTP gamma S (activator of G-proteins), U73122 or D609 (inhibitors of phospholipase C [PLC]), wortmannin (inhibitor of phospholipase D [PLD]) or LY294002 (inhibitor of phosphatidylinositol 3-kinase). E(2)-BSA mimicked the effects of E(2) on alkaline phosphatase specific activity and proteoglycan sulfation, causing dose-dependent increases in both RC and GC cell cultures. Both forms of estradiol inhibited [(3)H

  11. Effect of bovine serum albumin on the structure and properties of Langmuir Blodgett films based phosphocholine and cholesterol

    NASA Astrophysics Data System (ADS)

    Dubatovka, K. I.; Zhavnerko, G. K.; Agabekov, V. E.

    2014-02-01

    Mono- and bilayer Langmuir-Blodgett films based on phosphocholine and cholesterol and prepared by horizontal and vertical deposition are investigated by atomic force microscopy. It was found that bovine serum albumin (BSA) included at the stage of film formation. At the same time, isolation has a considerable effect on their structure. It was shown that the globular formation of nanostructures with heights of 4-7 nm occurs as a result of transferring lipids to a hydrophobic surface from a subphase containing BSA, indicating the reorganization of monolayers during protein isolation and inclusion in its composition.

  12. Role of Serum Interleukin 6, Albumin and C-Reactive Protein in COPD Patients

    PubMed Central

    Emami Ardestani, Mohammad

    2015-01-01

    Background: Chronic obstructive pulmonary disease (COPD) is a non-specific inflammation, which involves the airways, lung parenchyma and pulmonary vessels. The inflammation causes the activation of inflammatory cells and the release of various inflammatory mediators such as interleukin-8 (IL-8), IL-6 and tumor necoris factor alpha (TNF-a). The purpose of the present study was to measure serum IL-6, C-reactive protein (CRP) (as a positive phase reactant) and albumin level (as a negative phase reactant) in COPD patients (only due to cigarette smoking not bio-mass), non COPD smokers and healthy subjects using enzyme-linked immunosorbent assay (ELISA); we compared the differences in inflammatory factors among groups. Materials and Methods: A total of 180 males were enrolled in this study and divided into three equal groups. The first group was 60 smokers who had COPD. The second group included 60 smokers without COPD and the third group consisted of people who were not smokers and did not have COPD; 5 mL of venous blood was taken from all participants and it was collected in a test tube containing anticoagulant and then centrifuged at 3000 rpm for 10 minutes. Serum was separated and used to measure the amount of IL-6, CRP and albumin. Spirometry was performed according to the criteria set by the American Thoracic Society. Results: The mean serum level of IL-6 was 83.2±7.5 pg/mL in group I, 54.9±24.3 pg/mL in group II and 46.9±10.4 pg/mL in group III. There was a significant difference among the three groups (P<0.001). The mean serum level of CRP was 28.9±14.9 mg/dL in the first group, 19.9±8.5 mg/dL in the second group and 4.2±2.3 mg/dL in the third group (P=0.02). But by controlling the confounding effects of age, this difference was not significant (P=0.49). The mean serum level of albumin was I 4.1±0.57 mg/dL in group I, 4.3±0.56 mg/dL in group II and 4.1±0.53 mg/dL in group III. There was no significant difference among the three groups in this regard (P=0

  13. Comparing encapsulation efficiency and ultrasound-triggered release for protein between phospholipid-based microbubbles and liposomes.

    PubMed

    Lu, Cui-Tao; Zhao, Ying-Zheng; Gao, Hui-Sheng; Tian, Ji-Lai; Zhou, Zhi-Cai; Zhao, Gang-Tao; Tang, Qin-Qin; Jin, Zhuo; Xu, Yan-Yan; Huang, Pin-Tong; Han, Jing; Wang, Liang; Li, Xiao-Kun

    2010-01-01

    This work was to compare the encapsulation efficiency and ultrasound-triggered release for protein between microbubbles and liposomes. Bovine serum albumin (BSA) was used as a model. Final ratios between BSA and HPC in microbubbles and liposomes were 1:5, 1:7 and 1:10, respectively. Morphologic characteristics and contrast enhancement of loaded microbubbles and liposomes were measured. Encapsulation efficiency and ultrasound-stimulated release profile were detected. The mean size of loaded microbubbles and liposomes was 3.4 microm and 1.7 microm, respectively. Encapsulation efficiency of microbubbles had an inverse relationship with the ratio between BSA and HPC, while loaded liposomes remained nearly unchanged in the designed range of the ratio between BSA and HPC. Microbubbles resulted in significant enhancement of CnTi images. After ultrasound, more than 90% of the entrapped BSA was released from microbubbles, but less than 5% of BSA released from liposomes. Microbubbles are a promising delivery system for proteins.

  14. Spectral characterization of the binding and conformational changes of serum albumins upon interaction with an anticancer drug, anastrozole

    NASA Astrophysics Data System (ADS)

    Punith, Reeta; Seetharamappa, J.

    2012-06-01

    The present study employed different optical spectroscopic techniques viz., fluorescence, FTIR, circular dichroism (CD) and UV-vis absorption spectroscopy to investigate the mechanism of interaction of an anticancer drug, anastrozole (AZ) with transport proteins viz., bovine serum albumin (BSA) and human serum albumin (HSA). The drug, AZ quenched the intrinsic fluorescence of protein and the analysis of results revealed the presence of dynamic quenching mechanism. The binding characteristics of drug-protein were computed. The thermodynamic parameters, enthalpy change (ΔH°) and entropy change (ΔS°) were calculated to be +92.99 kJ/mol and +159.18 J/mol/K for AZ-BSA and, +99.43 kJ/mol and +159.19 J/mol/K for AZ-HSA, respectively. These results indicated that the hydrophobic forces stabilized the interaction between the drug and protein. CD, FTIR, absorption, synchronous and 3D fluorescence results indicated that the binding of AZ to protein induced structural perturbation in both serum albumins. The distance, r between the drug and protein was calculated based on the theory of Förster's resonance energy transfer and found to be 5.9 and 6.24 nm, respectively for AZ-BSA and AZ-HSA.

  15. Association of a high normalized protein catabolic rate and low serum albumin level with carpal tunnel syndrome in hemodialysis patients

    PubMed Central

    Huang, Wen-Hung; Hsu, Ching-Wei; Weng, Cheng-Hao; Yen, Tzung-Hai; Lin, Jui-Hsiang; Lee, Meng

    2016-01-01

    Abstract Carpal tunnel syndrome (CTS) is the most common mononeuropathy in patients with end-stage renal disease (ESRD). The association between chronic inflammation and CTS in hemodialysis (HD) patients has rarely been investigated. HD patients with a high normalized protein catabolic rate (nPCR) and low serum albumin level likely have adequate nutrition and inflammation. In this study, we assume that a low serum albumin level and high nPCR is associated with CTS in HD patients. We recruited 866 maintenance hemodialysis (MHD) patients and divided them into 4 groups according to their nPCR and serum albumin levels: (1) nPCR <1.2 g/kg/d and serum albumin level <4 g/dL; (2) nPCR ≥1.2 g/kg/d and serum albumin level <4 g/dL; (3) nPCR <1.2 g/kg/d and serum albumin level ≥4 g/dL; and (4) nPCR ≥1.2 g/kg/d and serum albumin level ≥4 g/dL. After adjustment for related variables, HD duration and nPCR ≥1.2 g/kg/d and serum albumin level <4 g/dL were positively correlated with CTS. By calculating the area under the receiver-operating characteristic curve, we calculated that the nPCR and HD duration cut-off points for obtaining the most favorable Youden index were 1.29 g/kg/d and 7.5 years, respectively. Advance multivariate logistic regression analysis revealed that in MHD patients, nPCR ≥1.29 g/kg/d and serum albumin <4 g/dL, and also HD duration >7.5 years were associated with CTS. A high nPCR and low serum albumin level, which likely reflect adequate nutrition and inflammation, were associated with CTS in MHD patients. PMID:27368039

  16. Synthesis, purification and mass spectrometric characterisation of a fluorescent Au9@BSA nanocluster and its enzymatic digestion by trypsin

    NASA Astrophysics Data System (ADS)

    Fernández-Iglesias, Nerea; Bettmer, Jörg

    2013-12-01

    Nanoclusters of noble metals like Ag and Au have attracted great attention as they form a missing link between isolated metal atoms and nanoparticles. Their particular properties like luminescence in the visible range and nontoxicity make them attractive for bioimaging and biolabelling purposes, especially with use of proteins as stabilising agents. In this context, this study intends the synthesis of a specific Au nanocluster covered by bovine serum albumin (BSA). It is shown that size-exclusion chromatography is feasible for the purification and isolation of the nanocluster. A mass spectrometric characterisation, preferably by ESI-MS, indicates the presence of an Au9@BSA nanocluster. Enzymatic digestion of the nanocluster with trypsin results in a significant increase of the fluorescence intensity at 650 and 710 nm, whereas complementary MALDI-MS studies are presented for the identification of generated peptides and show a distinctive pattern in comparison to the pure protein. It can be concluded that Au9@BSA might be, in future, an interesting candidate for in vitro studies of protease activities.Nanoclusters of noble metals like Ag and Au have attracted great attention as they form a missing link between isolated metal atoms and nanoparticles. Their particular properties like luminescence in the visible range and nontoxicity make them attractive for bioimaging and biolabelling purposes, especially with use of proteins as stabilising agents. In this context, this study intends the synthesis of a specific Au nanocluster covered by bovine serum albumin (BSA). It is shown that size-exclusion chromatography is feasible for the purification and isolation of the nanocluster. A mass spectrometric characterisation, preferably by ESI-MS, indicates the presence of an Au9@BSA nanocluster. Enzymatic digestion of the nanocluster with trypsin results in a significant increase of the fluorescence intensity at 650 and 710 nm, whereas complementary MALDI-MS studies are presented

  17. Investigating the BSA protein adsorption and bacterial adhesion of Al-alloy surfaces after creating a hierarchical (micro/nano) superhydrophobic structure.

    PubMed

    Moazzam, Parisa; Razmjou, Amir; Golabi, Mohsen; Shokri, Dariush; Landarani-Isfahani, Amir

    2016-09-01

    Bacterial adhesion and subsequent biofilm formation on metals such as aluminum (Al) alloys lead to serious issues in biomedical and industrial fields from both an economical and health perspective. Here, we showed that a careful manipulation of Al surface characteristics via a facile two-steps superhydrophobic modification can provide not only biocompatibility and an ability to control protein adsorption and bacterial adhesion, but also address the issue of apparent long-term toxicity of Al-alloys. To find out the roles of surface characteristics, surface modification and protein adsorption on microbial adhesion and biofilm formation, the surfaces were systematically characterized by SEM, EDX, XPS, AFM, FTIR, water contact angle (WCA) goniometry, surface free energy (SFE) measurement, MTT, Bradford, Lowry and microtiter plate assays and also flow-cytometry and potentiostat analyses. Results showed that WCA and SFE changed from 70° to 163° and 36.3 to 0.13 mN m(-1) , respectively. The stable and durable modification led to a substantial reduction in static/dynamic BSA adsorption. The effect of such a treatment on the biofilm formation was analyzed by using three different bacteria of Pseudomonas aeruginosa, Staphylococcus epidermidis, and Staphylococcus aureus. The microtiter plate assay and flow cytometry analysis showed that the modification not only could substantially reduce the bacterial adhesion but this biofouling resistance is independent of bacterium type. An excellent cell viability after exposure of HeLa cells to waters incubated with the modified samples was observed. Finally, the corrosion rate reduced sharply from 856.6 to 0.119 MPY after superhydrophobic modifications, which is an excellent stable corrosion inhibition property. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 2220-2233, 2016. PMID:27104583

  18. Investigating the BSA protein adsorption and bacterial adhesion of Al-alloy surfaces after creating a hierarchical (micro/nano) superhydrophobic structure.

    PubMed

    Moazzam, Parisa; Razmjou, Amir; Golabi, Mohsen; Shokri, Dariush; Landarani-Isfahani, Amir

    2016-09-01

    Bacterial adhesion and subsequent biofilm formation on metals such as aluminum (Al) alloys lead to serious issues in biomedical and industrial fields from both an economical and health perspective. Here, we showed that a careful manipulation of Al surface characteristics via a facile two-steps superhydrophobic modification can provide not only biocompatibility and an ability to control protein adsorption and bacterial adhesion, but also address the issue of apparent long-term toxicity of Al-alloys. To find out the roles of surface characteristics, surface modification and protein adsorption on microbial adhesion and biofilm formation, the surfaces were systematically characterized by SEM, EDX, XPS, AFM, FTIR, water contact angle (WCA) goniometry, surface free energy (SFE) measurement, MTT, Bradford, Lowry and microtiter plate assays and also flow-cytometry and potentiostat analyses. Results showed that WCA and SFE changed from 70° to 163° and 36.3 to 0.13 mN m(-1) , respectively. The stable and durable modification led to a substantial reduction in static/dynamic BSA adsorption. The effect of such a treatment on the biofilm formation was analyzed by using three different bacteria of Pseudomonas aeruginosa, Staphylococcus epidermidis, and Staphylococcus aureus. The microtiter plate assay and flow cytometry analysis showed that the modification not only could substantially reduce the bacterial adhesion but this biofouling resistance is independent of bacterium type. An excellent cell viability after exposure of HeLa cells to waters incubated with the modified samples was observed. Finally, the corrosion rate reduced sharply from 856.6 to 0.119 MPY after superhydrophobic modifications, which is an excellent stable corrosion inhibition property. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 2220-2233, 2016.

  19. Electrochemical characterization of albumin protein on Ti-6AL-4V alloy immersed in a simulated plasma solution.

    PubMed

    Padilla, Norma; Bronson, Arturo

    2007-06-01

    The effect of oxygen and albumin on the electrochemical behavior of a Ti-6Al-4V alloy immersed in a simulated inorganic plasma (SIP) solution was studied with a rotating-cylindrical electrode configuration to focus on the surface/electrolyte reactions. Potentiokinetic scans and electrochemical impedance spectroscopy have been used to characterize the interface by determining the passive current density and capacitance. For the polarization scans, an albumin addition of 37.7 mg/cm(3) to the SIP solution (oxygenated and unoxygenated) decreased the passive current density, indicating a lowering of the corrosive rate. The surface capacitance for the Ti-6Al-4V alloy immersed in a SIP solution averaged 13 microF/cm(2), which transformed after albumin addition (37.7 mg/cm(3)) from a potential independent behavior to the capacitance ranging from 23 to 6 microF/cm(2) with increasing potentials from -800 to 1500 mV(SCE), respectively, indicative of albumin adsorption. Within the same potential range and albumin addition to oxygenated solutions, the capacitances expanded slightly with a similar decreasing trend from 31 to 6 microF/cm(2), although the capacitance depicts an interaction between the hydrated passive film and the adsorbed albumin from -550 to 500 mV(SCE) in which the capacitance plateaued at 15 microF/cm(2). The hydrated porous oxide film results from the porous rutile layer reacting with H(2)O(2) formed as an intermediary component of oxygen reduction at the Ti-6Al-4V surface. The passive film-albumin interaction would affect the processing of titanium alloys in their surface preparation for biocompatibility, as well as determining the reactivity of titanium alloys to proteins.

  20. A Comprehensive Spectroscopic and Computational Investigation to Probe the Interaction of Antineoplastic Drug Nordihydroguaiaretic Acid with Serum Albumins.

    PubMed

    Nusrat, Saima; Siddiqi, Mohammad Khursheed; Zaman, Masihuz; Zaidi, Nida; Ajmal, Mohammad Rehan; Alam, Parvez; Qadeer, Atiyatul; Abdelhameed, Ali Saber; Khan, Rizwan Hasan

    2016-01-01

    Exogenous drugs that are used as antidote against chemotheray, inflammation or viral infection, gets absorbed and interacts reversibly to the major serum transport protein i.e. albumins, upon entering the circulatory system. To have a structural guideline in the rational drug designing and in the synthesis of drugs with greater efficacy, the binding mechanism of an antineoplastic and anti-inflammatory drug Nordihydroguaiaretic acid (NDGA) with human and bovine serum albumins (HSA & BSA) were examined by spectroscopic and computational methods. NDGA binds to site II of HSA with binding constant (Kb) ~105 M-1 and free energy (ΔG) ~ -7.5 kcal.mol-1. It also binds at site II of BSA but with lesser binding affinity (Kb) ~105 M-1 and ΔG ~ -6.5 kcal.mol-1. The negative value of ΔG, ΔH and ΔS for both the albumins at three different temperatures confirmed that the complex formation process between albumins and NDGA is spontaneous and exothermic. Furthermore, hydrogen bonds and hydrophobic interactions are the main forces involved in complex formation of NDGA with both the albumins as evaluated from fluorescence and molecular docking results. Binding of NDGA to both the albumins alter the conformation and causes minor change in the secondary structure of proteins as indicated by the CD spectra. PMID:27391941

  1. A Comprehensive Spectroscopic and Computational Investigation to Probe the Interaction of Antineoplastic Drug Nordihydroguaiaretic Acid with Serum Albumins

    PubMed Central

    Nusrat, Saima; Siddiqi, Mohammad Khursheed; Zaman, Masihuz; Zaidi, Nida; Ajmal, Mohammad Rehan; Alam, Parvez; Qadeer, Atiyatul; Abdelhameed, Ali Saber

    2016-01-01

    Exogenous drugs that are used as antidote against chemotheray, inflammation or viral infection, gets absorbed and interacts reversibly to the major serum transport protein i.e. albumins, upon entering the circulatory system. To have a structural guideline in the rational drug designing and in the synthesis of drugs with greater efficacy, the binding mechanism of an antineoplastic and anti-inflammatory drug Nordihydroguaiaretic acid (NDGA) with human and bovine serum albumins (HSA & BSA) were examined by spectroscopic and computational methods. NDGA binds to site II of HSA with binding constant (Kb) ~105 M-1 and free energy (ΔG) ~ -7.5 kcal.mol-1. It also binds at site II of BSA but with lesser binding affinity (Kb) ~105 M-1 and ΔG ~ -6.5 kcal.mol-1. The negative value of ΔG, ΔH and ΔS for both the albumins at three different temperatures confirmed that the complex formation process between albumins and NDGA is spontaneous and exothermic. Furthermore, hydrogen bonds and hydrophobic interactions are the main forces involved in complex formation of NDGA with both the albumins as evaluated from fluorescence and molecular docking results. Binding of NDGA to both the albumins alter the conformation and causes minor change in the secondary structure of proteins as indicated by the CD spectra. PMID:27391941

  2. Differential sensing using proteins: exploiting the cross-reactivity of serum albumin to pattern individual terpenes and terpenes in perfume.

    PubMed

    Adams, Michelle M; Anslyn, Eric V

    2009-12-01

    There has been a growing interest in the use of differential sensing for analyte classification. In an effort to mimic the mammalian senses of taste and smell, which utilize protein-based receptors, we have introduced serum albumins as nonselective receptors for recognition of small hydrophobic molecules. Herein, we employ a sensing ensemble consisting of serum albumins, a hydrophobic fluorescent indicator (PRODAN), and a hydrophobic additive (deoxycholate) to detect terpenes. With the aid of linear discriminant analysis, we successfully applied our system to differentiate five terpenes. We then extended our terpene analysis and utilized our sensing ensemble for terpene discrimination within the complex mixtures found in perfume.

  3. Fluorescence resonance energy transfer between ZnSe ZnS quantum dots and bovine serum albumin in bioaffinity assays of anticancer drugs

    NASA Astrophysics Data System (ADS)

    Shu, Chang; Ding, Li; Zhong, Wenying

    2014-10-01

    In the current work, using ZnSe ZnS quantum dots (QDs) as representative nanoparticles, the affinities of seven anticancer drugs for bovine serum albumin (BSA) were studied using fluorescence resonance energy transfer (FRET). The FRET efficiency of BSA-QD conjugates can reach as high as 24.87% by electrostatic interaction. The higher binding constant (3.63 × 107 L mol-1) and number of binding sites (1.75) between ZnSe ZnS QDs and BSA demonstrated that the QDs could easily associate to plasma proteins and enhance the transport efficacy of drugs. The magnitude of binding constants (103-106 L mol-1), in the presence of QDs, was between drugs-BSA and drugs-QDs in agreement with common affinities of drugs for serum albumins (104-106 L mol-1) in vivo. ZnSe ZnS QDs significantly increased the affinities for BSA of Vorinostat (SAHA), Docetaxel (DOC), Carmustine (BCNU), Doxorubicin (Dox) and 10-Hydroxycamptothecin (HCPT). However, they slightly reduced the affinities of Vincristine (VCR) and Methotrexate (MTX) for BSA. The recent work will not only provide useful information for appropriately understanding the binding affinity and binding mechanism at the molecular level, but also illustrate the ZnSe ZnS QDs are perfect candidates for nanoscal drug delivery system (DDS).

  4. Binding of [3H]palmitate to BSA.

    PubMed

    Elmadhoun, B M; Wang, G Q; Templeton, J F; Burczynski, F J

    1998-10-01

    Determination of the BSA-palmitate high-affinity binding constant (Ka) traditionally relied on the heptane-water partitioning technique. We used this technique to calculate Ka for the BSA-[3H]palmitate complex, to determine if Ka was independent of protein concentration, and to determine if the unbound [3H]palmitate concentration is constant at different BSA concentrations using constant BSA-to-palmitate molar ratios (range 1:1 to 1:4). After extensive extraction of non-[3H]palmitate radiolabeled substances, the heptane-to-buffer partition ratio, in the absence of BSA, was 702 +/- 19 (mean +/- SD, n = 6). This value was much lower than the predicted value of 1,376 and was highly dependent on which phase (organic or aqueous) initially contained the [3H]palmitic acid. The data were consistent with the notion of self-association of [3H]palmitate in the aqueous phase. Ka for the BSA-[3H]palmitate complex was determined to be similar (2.2 +/- 0.1) x 10(8) M-1 (mean +/- SD, P > 0.05) at all BSA concentrations studied. At each BSA-to-palmitate molar ratio, the equilibrium unbound ligand concentration was constant only at low BSA concentrations (<10 microM) and at low BSA-to-palmitate molar ratios (i.e., 1:1 and 1:2). At higher BSA concentrations and molar ratios, the unbound ligand concentration increased with an increase in protein concentration. Hepatocyte uptake using the manufacturer-supplied radiolabeled product was significantly higher than with the purified product, suggesting that a non-[3H]palmitate radiolabel is also a substrate for the uptake process.

  5. Grafting of bovine serum albumin proteins on plasma-modified polymers for potential application in tissue engineering

    NASA Astrophysics Data System (ADS)

    Kasálková, Nikola Slepičková; Slepička, Petr; Kolská, Zdeňka; Hodačová, Petra; Kučková, Štěpánka; Švorčík, Václav

    2014-04-01

    In this work, an influence of bovine serum albumin proteins grafting on the surface properties of plasma-treated polyethylene and poly- l-lactic acid was studied. The interaction of the vascular smooth muscle cells with the modified polymer surface was determined. The surface properties were characterized by X-ray photoelectron spectroscopy, atomic force microscopy, nano-LC-ESI-Q-TOF mass spectrometry, electrokinetic analysis, and goniometry. One of the motivations for this work is the idea that by the interaction of the cell with substrate surface, the proteins will form an interlayer between the cell and the substrate. It was proven that when interacting with the plasma-treated high-density polyethylene and poly- l-lactic acid, the bovine serum albumin protein is grafted on the polymer surface. Since the proteins are bonded to the substrate surface, they can stimulate cell adhesion and proliferation.

  6. Regulation of inflammation-primed activation of macrophages by two serum factors, vitamin D3-binding protein and albumin.

    PubMed Central

    Yamamoto, N; Kumashiro, R; Yamamoto, M; Willett, N P; Lindsay, D D

    1993-01-01

    A very small amount (0.0005 to 0.001%) of an ammonium sulfate [50% saturated (NH4)2SO4]-precipitable protein fraction of alpha 2-globulin efficiently supported inflammation-primed activation of macrophages. This fraction contains vitamin D3-binding protein essential for macrophage activation. Comparative macrophage activation studies with fetal calf serum, alpha 2-globulin fraction, 50% (NH4)2SO4 precipitate, and purified bovine vitamin D3-binding protein revealed that fetal calf serum and alpha 2-globulin fraction appear to contain an inhibitor for macrophage activation while ammonium sulfate precipitate contains no inhibitor. This inhibitor was found to be serum albumin. When bovine serum albumin (25 micrograms/ml) was added to a medium supplemented with 0.0005 to 0.05% (NH4)2SO4 precipitate or 1 to 10 ng of vitamin D3-binding protein per ml, activation of macrophages was inhibited. PMID:8225612

  7. Grafting of bovine serum albumin proteins on plasma-modified polymers for potential application in tissue engineering

    PubMed Central

    2014-01-01

    In this work, an influence of bovine serum albumin proteins grafting on the surface properties of plasma-treated polyethylene and poly-l-lactic acid was studied. The interaction of the vascular smooth muscle cells with the modified polymer surface was determined. The surface properties were characterized by X-ray photoelectron spectroscopy, atomic force microscopy, nano-LC-ESI-Q-TOF mass spectrometry, electrokinetic analysis, and goniometry. One of the motivations for this work is the idea that by the interaction of the cell with substrate surface, the proteins will form an interlayer between the cell and the substrate. It was proven that when interacting with the plasma-treated high-density polyethylene and poly-l-lactic acid, the bovine serum albumin protein is grafted on the polymer surface. Since the proteins are bonded to the substrate surface, they can stimulate cell adhesion and proliferation. PMID:24708858

  8. Effects of N-acetyl-L-cysteine-capped CdTe quantum dots on bovine serum albumin and bovine hemoglobin: isothermal titration calorimetry and spectroscopic investigations.

    PubMed

    Sun, Haoyu; Cui, Erqian; Tan, Zhigang; Liu, Rutao

    2014-12-01

    The interactions of N-acetyl-L-cysteine-capped CdTe quantum dots (QDs) with bovine serum albumin (BSA) and bovine hemoglobin (BHb) were investigated by isothermal titration calorimetry (ITC), fluorescence, synchronous fluorescence, fluorescence lifetime, ultraviolet-visible absorption, and circular dichroism techniques. Fluorescence data of BSA-QDs and BHb-QDs revealed that the quenching was static in every system. While CdTe QDs changed the microenvironment of tryptophan in BHb, the microenvironment of BSA kept unchanged. Adding CdTe QDs affected the skeleton and secondary structure of the protein (BSA and BHb). The ITC results indicated that the interaction between the protein (BSA and BHb) and QDs-612 was spontaneous and the predominant force was hydrophobic interaction. In addition, the binding constants were determined to be 1.19 × 10(5) L mol(-1) (BSA-QDs) and 2.19 × 10(5) L mol(-1) (BHb-QDs) at 298 K. From these results, we conclude that CdTe QDs have a larger impact on the structure of BHb than BSA.

  9. EPR studies of intermolecular interactions and competitive binding of drugs in a drug-BSA binding model.

    PubMed

    Akdogan, Y; Emrullahoglu, M; Tatlidil, D; Ucuncu, M; Cakan-Akdogan, G

    2016-08-10

    Understanding intermolecular interactions between drugs and proteins is very important in drug delivery studies. Here, we studied different binding interactions between salicylic acid and bovine serum albumin (BSA) using electron paramagnetic resonance (EPR) spectroscopy. Salicylic acid was labeled with a stable radical (spin label) in order to monitor its mobilized (free) or immobilized (bound to BSA) states. In addition to spin labeled salicylic acid (SL-salicylic acid), its derivatives including SL-benzoic acid, SL-phenol, SL-benzene, SL-cyclohexane and SL-hexane were synthesized to reveal the effects of various drug binding interactions. EPR results of these SL-molecules showed that hydrophobic interaction is the main driving force. Whereas each of the two functional groups (-COOH and -OH) on the benzene ring has a minute but detectable effect on the drug-protein complex formation. In order to investigate the effect of electrostatic interaction on drug binding, cationic BSA (cBSA) was synthesized, altering the negative net charge of BSA to positive. The salicylic acid loading capacity of cBSA is significantly higher compared to that of BSA, indicating the importance of electrostatic interaction in drug binding. Moreover, the competitive binding properties of salicylic acid, ibuprofen and aspirin to BSA were studied. The combined EPR results of SL-salicylic acid/ibuprofen and SL-ibuprofen/salicylic acid showed that ibuprofen is able to replace up to ∼83% of bound SL-salicylic acid, and salicylic acid can replace only ∼14% of the bound SL-ibuprofen. This indicates that ∼97% of all salicylic acid and ibuprofen binding sites are shared. On the other hand, aspirin replaces only ∼23% of bound SL-salicylic acid, and salicylic acid replaces ∼50% of bound SL-aspirin, indicating that ∼73% of all salicylic acid and aspirin binding sites are shared. These results show that EPR spectroscopy in combination with the spin labeling technique is a very powerful

  10. EPR studies of intermolecular interactions and competitive binding of drugs in a drug-BSA binding model.

    PubMed

    Akdogan, Y; Emrullahoglu, M; Tatlidil, D; Ucuncu, M; Cakan-Akdogan, G

    2016-08-10

    Understanding intermolecular interactions between drugs and proteins is very important in drug delivery studies. Here, we studied different binding interactions between salicylic acid and bovine serum albumin (BSA) using electron paramagnetic resonance (EPR) spectroscopy. Salicylic acid was labeled with a stable radical (spin label) in order to monitor its mobilized (free) or immobilized (bound to BSA) states. In addition to spin labeled salicylic acid (SL-salicylic acid), its derivatives including SL-benzoic acid, SL-phenol, SL-benzene, SL-cyclohexane and SL-hexane were synthesized to reveal the effects of various drug binding interactions. EPR results of these SL-molecules showed that hydrophobic interaction is the main driving force. Whereas each of the two functional groups (-COOH and -OH) on the benzene ring has a minute but detectable effect on the drug-protein complex formation. In order to investigate the effect of electrostatic interaction on drug binding, cationic BSA (cBSA) was synthesized, altering the negative net charge of BSA to positive. The salicylic acid loading capacity of cBSA is significantly higher compared to that of BSA, indicating the importance of electrostatic interaction in drug binding. Moreover, the competitive binding properties of salicylic acid, ibuprofen and aspirin to BSA were studied. The combined EPR results of SL-salicylic acid/ibuprofen and SL-ibuprofen/salicylic acid showed that ibuprofen is able to replace up to ∼83% of bound SL-salicylic acid, and salicylic acid can replace only ∼14% of the bound SL-ibuprofen. This indicates that ∼97% of all salicylic acid and ibuprofen binding sites are shared. On the other hand, aspirin replaces only ∼23% of bound SL-salicylic acid, and salicylic acid replaces ∼50% of bound SL-aspirin, indicating that ∼73% of all salicylic acid and aspirin binding sites are shared. These results show that EPR spectroscopy in combination with the spin labeling technique is a very powerful

  11. Experimental, computational and chemometrics studies of BSA-vitamin B6 interaction by UV-Vis, FT-IR, fluorescence spectroscopy, molecular dynamics simulation and hard-soft modeling methods.

    PubMed

    Manouchehri, Firouzeh; Izadmanesh, Yahya; Aghaee, Elham; Ghasemi, Jahan B

    2016-10-01

    The interaction of pyridoxine (Vitamin B6) with bovine serum albumin (BSA) is investigated under pseudo-physiological conditions by UV-Vis, fluorescence and FTIR spectroscopy. The intrinsic fluorescence of BSA was quenched by VB6, which was rationalized in terms of the static quenching mechanism. According to fluorescence quenching calculations, the bimolecular quenching constant (kq), dynamic quenching (KSV) and static quenching (KLB) at 310K were obtained. The efficiency of energy transfer and the distance between the donor (BSA) and the acceptor (VB6) were calculated by Foster's non-radiative energy transfer theory and were equal to 41.1% and 2.11nm. The collected UV-Vis and fluorescence spectra were combined into a row-and column-wise augmented matrix and resolved by multivariate curve resolution-alternating least squares (MCR-ALS). MCR-ALS helped to estimate the stoichiometry of interactions, concentration profiles and pure spectra for three species (BSA, VB6 and VB6-BSA complex) existed in the interaction procedure. Based on the MCR-ALS results, using mass balance equations, a model was developed and binding constant of complex was calculated using non-linear least squares curve fitting. FT-IR spectra showed that the conformation of proteins was altered in presence of VB6. Finally, the combined docking and molecular dynamics (MD) simulations were used to estimate the binding affinity of VB6 to BSA. Five-nanosecond MD simulations were performed on bovine serum albumin (BSA) to study the conformational features of its ligand binding site. From MD results, eleven BSA snapshots were extracted, at every 0.5ns, to explore the binding affinity (GOLD score) of VB6 using a docking procedure. MD simulations indicated that there is a considerable flexibility in the structure of protein that affected ligand recognition. Structural analyses and docking simulations indicated that VB6 binds to site I and GOLD score values depend on the conformations of both BSA and ligand

  12. Experimental, computational and chemometrics studies of BSA-vitamin B6 interaction by UV-Vis, FT-IR, fluorescence spectroscopy, molecular dynamics simulation and hard-soft modeling methods.

    PubMed

    Manouchehri, Firouzeh; Izadmanesh, Yahya; Aghaee, Elham; Ghasemi, Jahan B

    2016-10-01

    The interaction of pyridoxine (Vitamin B6) with bovine serum albumin (BSA) is investigated under pseudo-physiological conditions by UV-Vis, fluorescence and FTIR spectroscopy. The intrinsic fluorescence of BSA was quenched by VB6, which was rationalized in terms of the static quenching mechanism. According to fluorescence quenching calculations, the bimolecular quenching constant (kq), dynamic quenching (KSV) and static quenching (KLB) at 310K were obtained. The efficiency of energy transfer and the distance between the donor (BSA) and the acceptor (VB6) were calculated by Foster's non-radiative energy transfer theory and were equal to 41.1% and 2.11nm. The collected UV-Vis and fluorescence spectra were combined into a row-and column-wise augmented matrix and resolved by multivariate curve resolution-alternating least squares (MCR-ALS). MCR-ALS helped to estimate the stoichiometry of interactions, concentration profiles and pure spectra for three species (BSA, VB6 and VB6-BSA complex) existed in the interaction procedure. Based on the MCR-ALS results, using mass balance equations, a model was developed and binding constant of complex was calculated using non-linear least squares curve fitting. FT-IR spectra showed that the conformation of proteins was altered in presence of VB6. Finally, the combined docking and molecular dynamics (MD) simulations were used to estimate the binding affinity of VB6 to BSA. Five-nanosecond MD simulations were performed on bovine serum albumin (BSA) to study the conformational features of its ligand binding site. From MD results, eleven BSA snapshots were extracted, at every 0.5ns, to explore the binding affinity (GOLD score) of VB6 using a docking procedure. MD simulations indicated that there is a considerable flexibility in the structure of protein that affected ligand recognition. Structural analyses and docking simulations indicated that VB6 binds to site I and GOLD score values depend on the conformations of both BSA and ligand

  13. In vivo genome editing of the albumin locus as a platform for protein replacement therapy

    PubMed Central

    Sharma, Rajiv; Anguela, Xavier M.; Doyon, Yannick; Wechsler, Thomas; DeKelver, Russell C.; Sproul, Scott; Paschon, David E.; Miller, Jeffrey C.; Davidson, Robert J.; Shivak, David; Zhou, Shangzhen; Rieders, Julianne; Gregory, Philip D.; Holmes, Michael C.; Rebar, Edward J.

    2015-01-01

    Site-specific genome editing provides a promising approach for achieving long-term, stable therapeutic gene expression. Genome editing has been successfully applied in a variety of preclinical models, generally focused on targeting the diseased locus itself; however, limited targeting efficiency or insufficient expression from the endogenous promoter may impede the translation of these approaches, particularly if the desired editing event does not confer a selective growth advantage. Here we report a general strategy for liver-directed protein replacement therapies that addresses these issues: zinc finger nuclease (ZFN) –mediated site-specific integration of therapeutic transgenes within the albumin gene. By using adeno-associated viral (AAV) vector delivery in vivo, we achieved long-term expression of human factors VIII and IX (hFVIII and hFIX) in mouse models of hemophilia A and B at therapeutic levels. By using the same targeting reagents in wild-type mice, lysosomal enzymes were expressed that are deficient in Fabry and Gaucher diseases and in Hurler and Hunter syndromes. The establishment of a universal nuclease-based platform for secreted protein production would represent a critical advance in the development of safe, permanent, and functional cures for diverse genetic and nongenetic diseases. PMID:26297739

  14. Application of MoS2 modified screen-printed electrodes for highly sensitive detection of bovine serum albumin.

    PubMed

    Kukkar, Manil; Sharma, Ashish; Kumar, Parveen; Kim, Ki-Hyun; Deep, Akash

    2016-10-01

    The present work reports the application of a new molybdenum disulphide (MoS2)-based electrochemical platform for highly sensitive quantitation of an iron-binding protein, bovine serum albumin (BSA). The gold screen-printed electrodes were modified with MoS2 nanoflakes, followed by bioconjugation with anti-BSA antibodies. Using the above bioelectrode, cyclic voltammetric analysis was carried out in the presence of a Fe(3+)/Fe(2+) redox probe which exhibited a linear response of peak current with varying concentrations of BSA up to 10 ng/mL (with a detection limit of 0.006 ng/mL). This study is novel in that it shows a considerable enhancement of signal during electrochemical sensing of a protein. PMID:27639148

  15. Spectroscopic studies of interaction between CuO nanoparticles and bovine serum albumin.

    PubMed

    Esfandfar, Paniz; Falahati, Mojtaba; Saboury, AliAkbar

    2016-09-01

    Recently, the great interests in manufacturing and application of metal oxide nanoparticles in commercial and industrial products have led to focus on the potential impact of these particles on biomacromolecules. In the present study, the interaction of copper oxide (CuO) nanoparticles with bovine serum albumin (BSA) was studied by spectroscopic techniques. The zeta potential value for BSA and CuO nanoparticles with average diameter of around 50 nm at concentration of 10 μM in the deionized (DI) water were -5.8 and -22.5 mV, respectively. Circular dichroism studies did not show any changes in the content of secondary structure of the protein after CuO nanoparticles interaction. Fluorescence data revealed that the fluorescence quenching of BSA by CuO nanoparticles was the result of the formed complex of CuO nanoparticles - BSA. Binding constants and other thermodynamic parameters were determined at three different temperatures. The hydrogen bond interactions are the predominant intermolecular forces to stabilize the CuO nanoparticle - BSA complex. This study provides important insight into the interaction of CuO nanoparticles with proteins, which may be of importance for further application of these nanoparticles in biomedical applications. PMID:26555383

  16. Quercetin Influence on Thermal Denaturation of Bovine Serum Albumin.

    PubMed

    Precupas, Aurica; Sandu, Romica; Popa, Vlad T

    2016-09-01

    The effect of quercetin (QUER) binding on bovine serum albumin (BSA) thermal denaturation was systematically investigated by means of differential scanning calorimetry (DSC). Additional information concerning thermodynamic and structural binding parameters was provided by isothermal titration calorimetry (ITC) and molecular docking. The most relevant effect of QUER is manifested in the modification of the two-step thermal fingerprint of protein denaturation. Higher QUER concentrations result in a single-step denaturation thermogram, ascribed to the interplay between specific and nonspecific binding and enhancement of the solvent unfolding action. Analysis of ITC data indicate sequential binding of two molecules of QUER occurring spontaneously at different binding sites of BSA involving hydrophobic, electrostatic and hydrogen binding forces. Identification of QUER binding sites was possible through corroboration of DSC runs in the presence of site markers and molecular docking. Modeling of ligand-protein interaction confirmed the experimental data. On one hand, a neutral form of QUER binds in a nonplanar conformation to Sudlow's site I, a large hydrophobic cavity of subdomain IIA of BSA and decreases its thermal stability. On the other hand, a second molecule of QUER, the anionic form, is bound in planar conformation to Sudlow's site II, situated in the subdomain IIIA of the folded protein, and increases the thermal stability of the corresponding structural domain of the protein. PMID:27505141

  17. Interaction of water-soluble amino acid Schiff base complexes with bovine serum albumin: Fluorescence and circular dichroism studies

    NASA Astrophysics Data System (ADS)

    Gharagozlou, Mehrnaz; Boghaei, Davar M.

    2008-12-01

    Fluorescence spectroscopy in combination with circular dichroism (CD) spectroscopy were used to investigate the interaction of water-soluble amino acid Schiff base complexes, [Zn(L 1,2)(phen)] where phen is 1,10-phenanthroline and H 2L 1,2 is amino acid Schiff base ligands, with bovine serum albumin (BSA) under the physiological conditions in phosphate buffer solution adjusted to pH 7.0. The quenching mechanism of fluorescence was suggested as static quenching according to the Stern-Volmer equation. Quenching constants were determined using the Stern-Volmer equation to provide a measure of the binding affinity between amino acid Schiff base complexes and BSA. The thermodynamic parameters Δ G, Δ H and Δ S at different temperatures (298, 310 and 318 K) were calculated. The results indicate that the hydrophobic and hydrogen bonding interactions play a major role in [Zn(L 1)(phen)]-BSA association, whereas hydrophobic and electrostatic interactions participate a main role in [Zn(L 2)(phen)]-BSA binding process. Binding studies concerning the number of binding sites and apparent binding constant Kb were performed by fluorescence quenching method. The distance R between the donor (BSA) and acceptor (amino acid Schiff base complexes) has been obtained utilizing fluorescence resonant energy transfer (FRET). Furthermore, CD spectra were used to investigate the structural changes of the BSA molecule with the addition of amino acid Schiff base complexes. The results indicate that the interaction of amino acid Schiff base complexes with BSA leads to changes in the secondary structure of the protein. Fractional contents of the secondary structure of BSA ( fα, fβ, fturn and frandom) were calculated with and without amino acid Schiff base complexes utilizing circular dichroism spectroscopy. Our results clarified that amino acid Schiff base complexes could bind to BSA and be effectively transported and eliminated in the body, which could be a useful guideline for further drug

  18. (Na+ + K+)-ATPase Is a Target for Phosphoinositide 3-Kinase/Protein Kinase B and Protein Kinase C Pathways Triggered by Albumin*

    PubMed Central

    Peruchetti, Diogo B.; Pinheiro, Ana Acacia S.; Landgraf, Sharon S.; Wengert, Mira; Takiya, Christina M.; Guggino, William B.; Caruso-Neves, Celso

    2011-01-01

    In recent decades, evidence has confirmed the crucial role of albumin in the progression of renal disease. However, the possible role of signaling pathways triggered by physiologic concentrations of albumin in the modulation of proximal tubule (PT) sodium reabsorption has not been considered. In the present work, we have shown that a physiologic concentration of albumin increases the expression of the α1 subunit of (Na+ + K+)-ATPase in LLC-PK1 cells leading to an increase in enzyme activity. This process involves the sequential activation of PI3K/protein kinase B and protein kinase C pathways promoting inhibition of protein kinase A. This integrative network is inhibited when albumin concentration is increased, similar to renal disease, leading to a decrease in the α1 subunit of (Na+ + K+)-ATPase expression. Together, the results indicate that variation in albumin concentration in PT cells has an important effect on PT sodium reabsorption and, consequently, on renal sodium excretion. PMID:22057272

  19. Blood serum and BSA, but neither red blood cells nor hemoglobin can support vitellogenesis and egg production in the dengue vector Aedes aegypti

    PubMed Central

    Gonzales, Kristina K.; Tsujimoto, Hitoshi

    2015-01-01

    Aedes aegypti is the major vector of dengue, yellow fever and chikungunya viruses that put millions of people in endemic countries at risk. Mass rearing of this mosquito is crucial for strategies that use modified insects to reduce vector populations and transmission of pathogens, such as sterile insect technique or population replacement. A major problem for vector mosquito mass rearing is the requirement of vertebrate blood for egg production since it poses significant costs as well as potential health hazards. Also, regulations for human and animal use as blood source can pose a significant obstacle. A completely artificial diet that supports egg production in vector mosquitoes can solve this problem. In this study, we compared different blood fractions, serum and red blood cells, as dietary protein sources for mosquito egg production. We also tested artificial diets made from commercially available blood proteins (bovine serum albumin (BSA) and hemoglobin). We found that Ae. aegypti performed vitellogenesis and produced eggs when given whole bovine blood, serum, or an artificial diet containing BSA. Conversely, egg production was impaired after feeding of the red blood cell fraction or an artificial diet containing only hemoglobin. We also found that egg viability of serum-fed mosquitoes were comparable to that of whole blood and an iron supplemented BSA meal produced more viable eggs than a meal containing BSA alone. Our results indicate that serum proteins, not hemoglobin, may replace vertebrate blood in artificial diets for mass mosquito rearing. PMID:26020000

  20. Blood serum and BSA, but neither red blood cells nor hemoglobin can support vitellogenesis and egg production in the dengue vector Aedes aegypti.

    PubMed

    Gonzales, Kristina K; Tsujimoto, Hitoshi; Hansen, Immo A

    2015-01-01

    Aedes aegypti is the major vector of dengue, yellow fever and chikungunya viruses that put millions of people in endemic countries at risk. Mass rearing of this mosquito is crucial for strategies that use modified insects to reduce vector populations and transmission of pathogens, such as sterile insect technique or population replacement. A major problem for vector mosquito mass rearing is the requirement of vertebrate blood for egg production since it poses significant costs as well as potential health hazards. Also, regulations for human and animal use as blood source can pose a significant obstacle. A completely artificial diet that supports egg production in vector mosquitoes can solve this problem. In this study, we compared different blood fractions, serum and red blood cells, as dietary protein sources for mosquito egg production. We also tested artificial diets made from commercially available blood proteins (bovine serum albumin (BSA) and hemoglobin). We found that Ae. aegypti performed vitellogenesis and produced eggs when given whole bovine blood, serum, or an artificial diet containing BSA. Conversely, egg production was impaired after feeding of the red blood cell fraction or an artificial diet containing only hemoglobin. We also found that egg viability of serum-fed mosquitoes were comparable to that of whole blood and an iron supplemented BSA meal produced more viable eggs than a meal containing BSA alone. Our results indicate that serum proteins, not hemoglobin, may replace vertebrate blood in artificial diets for mass mosquito rearing.

  1. Binding of several benzodiazepines to bovine serum albumin: Fluorescence study

    NASA Astrophysics Data System (ADS)

    Machicote, Roberta G.; Pacheco, María E.; Bruzzone, Liliana

    2010-10-01

    The interactions of lorazepam, oxazepam and bromazepam with bovine serum albumin (BSA) were studied by fluorescence spectrometry. The Stern-Volmer quenching constants and corresponding thermodynamic parameters Δ H, Δ G and Δ S were calculated. The binding constants and the number of binding sites were also investigated. The distances between the donor (BSA) and the acceptors (benzodiazepines) were obtained according to fluorescence resonance energy transfer and conformational changes of BSA were observed from synchronous fluorescence spectra.

  2. The complexity of condensed tannin binding to bovine serum albumin--An isothermal titration calorimetry study.

    PubMed

    Kilmister, Rachel L; Faulkner, Peta; Downey, Mark O; Darby, Samuel J; Falconer, Robert J

    2016-01-01

    Isothermal titration calorimetry was applied to study the binding of purified proanthocyanidin oligomers to bovine serum albumin (BSA). The molecular weight of the proanthocyanidin oligomer had a major impact on its binding to BSA. The calculated change in enthalpy (ΔH) and association constant (Ka) became greater as the oligomer size increased then plateaued at the heptameric oligomer. These results support a model for precipitation of proteins by proanthocyanidin where increased oligomer size enhanced the opportunity for cross linkages between proteins ultimately forming sediment-able complexes. The authors suggest tannin binding to proteins is opportunistic and involves multiple sites, each with a different Ka and ΔH of binding. The ΔH of binding comprises both an endothermic hydrophobic interaction and exothermic hydrogen bond component. This suggests the calculated entropy value (ΔS) for tannin-protein interactions is subject to a systematic error and should be interpreted with caution.

  3. Spectroscopic and molecular docking studies on the charge transfer complex of bovine serum albumin with quinone in aqueous medium and its influence on the ligand binding property of the protein

    NASA Astrophysics Data System (ADS)

    Satheshkumar, Angupillai; Elango, Kuppanagounder P.

    2014-09-01

    The spectral techniques such as UV-Vis, 1H NMR and fluorescence and electrochemical experiments have been employed to investigate the interaction between 2-methoxy-3,5,6-trichloro-1,4-benzoquinone (MQ; a water soluble quinone) and bovine serum albumin (BSA) in aqueous medium. The fluorescence of BSA was quenched by MQ via formation of a 1:1 BSA-MQ charge transfer adduct with a formation constant of 3.3 × 108 L mol-1. Based on the Forster’s theory the binding distance between them is calculated as 2.65 nm indicating high probability of binding. For the first time, influence of quinone on the binding property of various types of ligands such as aspirin, ascorbic acid, nicotinimide and sodium stearate has also been investigated. The results indicated that the strong and spontaneous binding existing between BSA and MQ, decreased the intensity of binding of these ligands with BSA. Since Tryptophan (Trp) is the basic residue present in BSA, a comparison between binding property of Trp-MQ adduct with that of BSA-MQ with these ligands has also been attempted. 1H NMR titration study indicated that the Trp forms a charge transfer complex with MQ, which reduces the interaction of Trp with the ligands. Molecular docking study supported the fact that the quinone interacts with the Trp212 unit of the BSA and the free energy change of binding (ΔG) for the BSA-MQ complex was found to be -46 kJ mol-1, which is comparable to our experimental free energy of binding (-49 kJ mol-1) obtained from fluorescence study.

  4. Single Particle Dynamic Imaging and Fe3+ Sensing with Bright Carbon Dots Derived from Bovine Serum Albumin Proteins

    NASA Astrophysics Data System (ADS)

    Yang, Qingxiu; Wei, Lin; Zheng, Xuanfang; Xiao, Lehui

    2015-12-01

    In this work, we demonstrated a convenient and green strategy for the synthesis of highly luminescent and water-soluble carbon dots (Cdots) by carbonizing carbon precursors, i.e., Bovine serum albumin (BSA) nanoparticles, in water solution. Without post surface modification, the as-synthesized Cdots exhibit fluorescence quantum yield (Q.Y.) as high as 34.8% and display superior colloidal stability not only in concentrated salt solutions (e.g. 2 M KCl) but also in a wide range of pH solutions. According to the FT-IR measurements, the Cdots contain many carboxyl groups, providing a versatile route for further chemical and biological functionalization. Through conjugation of Cdots with the transacting activator of transcription (TAT) peptide (a kind of cell penetration peptide (CPP)) derived from human immunodeficiency virus (HIV), it is possible to directly monitor the dynamic interactions of CPP with living cell membrane at single particle level. Furthermore, these Cdots also exhibit a dosage-dependent selectivity toward Fe3+ among other metal ions, including K+, Na+, Mg2+, Hg2+, Co2+, Cu2+, Pb2+ and Al3+. We believed that the Cdots prepared by this strategy would display promising applications in various areas, including analytical chemistry, nanomedicine, biochemistry and so on.

  5. Single Particle Dynamic Imaging and Fe3+ Sensing with Bright Carbon Dots Derived from Bovine Serum Albumin Proteins

    PubMed Central

    Yang, Qingxiu; Wei, Lin; Zheng, Xuanfang; Xiao, Lehui

    2015-01-01

    In this work, we demonstrated a convenient and green strategy for the synthesis of highly luminescent and water-soluble carbon dots (Cdots) by carbonizing carbon precursors, i.e., Bovine serum albumin (BSA) nanoparticles, in water solution. Without post surface modification, the as-synthesized Cdots exhibit fluorescence quantum yield (Q.Y.) as high as 34.8% and display superior colloidal stability not only in concentrated salt solutions (e.g. 2 M KCl) but also in a wide range of pH solutions. According to the FT-IR measurements, the Cdots contain many carboxyl groups, providing a versatile route for further chemical and biological functionalization. Through conjugation of Cdots with the transacting activator of transcription (TAT) peptide (a kind of cell penetration peptide (CPP)) derived from human immunodeficiency virus (HIV), it is possible to directly monitor the dynamic interactions of CPP with living cell membrane at single particle level. Furthermore, these Cdots also exhibit a dosage-dependent selectivity toward Fe3+ among other metal ions, including K+, Na+, Mg2+, Hg2+, Co2+, Cu2+, Pb2+ and Al3+. We believed that the Cdots prepared by this strategy would display promising applications in various areas, including analytical chemistry, nanomedicine, biochemistry and so on. PMID:26634992

  6. ANALYSIS OF DRUG-PROTEIN BINDING BY ULTRAFAST AFFINITY CHROMATOGRAPHY USING IMMOBILIZED HUMAN SERUM ALBUMIN

    PubMed Central

    Mallik, Rangan; Yoo, Michelle J.; Briscoe, Chad J.; Hage, David S.

    2010-01-01

    Human serum albumin (HSA) was explored for use as a stationary phase and ligand in affinity microcolumns for the ultrafast extraction of free drug fractions and the use of this information for the analysis of drug-protein binding. Warfarin, imipramine, and ibuprofen were used as model analytes in this study. It was found that greater than 95% extraction of all these drugs could be achieved in as little as 250 ms on HSA microcolumns. The retained drug fraction was then eluted from the same column under isocratic conditions, giving elution in less than 40 s when working at 4.5 mL/min. The chromatographic behavior of this system gave a good fit with that predicted by computer simulations based on a reversible, saturable model for the binding of an injected drug with immobilized HSA. The free fractions measured by this method were found to be comparable to those determined by ultrafiltration, and equilibrium constants estimated by this approach gave good agreement with literature values. Advantages of this method include its speed and the relatively low cost of microcolumns that contain HSA. The ability of HSA to bind many types of drugs also creates the possibility of using the same affinity microcolumn to study and measure the free fractions for a variety of pharmaceutical agents. These properties make this technique appealing for use in drug binding studies and in the high-throughput screening of new drug candidates. PMID:20227701

  7. Ratio of C-Reactive Protein to Albumin Predicts Muscle Mass in Adult Patients Undergoing Hemodialysis

    PubMed Central

    Chen, Yu-Tong; Wu, Pei-Yu; Chen, Hsi-Hsien; Chen, Tso-Hsiao; Hsu, Yung-Ho

    2016-01-01

    Recent studies have indicated that the ratio of C-reactive protein to albumin (CRP–Alb ratio) is associated with clinical outcomes in patients with disease. We examined the predictive value of this ratio in patients undergoing hemodialysis (HD). In this cross-sectional study, 91 eligible adult HD patients were analyzed, and the correlation between the CRP–Alb ratio and skeletal muscle mass normalized for body weight (SMM/wt; estimated using a bioelectrical impedance analyzer) was investigated. The mean age of the study participants was 54.9 ± 6.6 years (ranging from 27 to 64 years); 43 (47.2%) were men. The mean values for the SMM/wt were 39.1% ± 5.4%. The CRP–Alb ratio was found to be negatively correlated with SMM/wt (r = −0.33, P = 0.002) and creatinine (r = −0.20, P = 0.056). All the univariate significant and nonsignificant relevant covariates were selected for multivariable stepwise regression analysis. We determined that the homeostasis model assessment-estimated insulin resistance and CRP–Alb ratio were independent risk determinants for SMM/wt (βHOMA-IR = −0.18 and βCRP–Alb ratio = −3.84, adjusted R2 = 0.32). This study indicated that the CRP–Alb ratio may help clinicians in predicting muscle mass in adult patients undergoing HD. PMID:27768746

  8. A spectroscopic study on interaction between bovine serum albumin and titanium dioxide nanoparticle synthesized from microwave-assisted hybrid chemical approach.

    PubMed

    Ranjan, Shivendu; Dasgupta, Nandita; Srivastava, Priyanka; Ramalingam, Chidambaram

    2016-08-01

    The use of nanoparticles in food or pharma requires a molecular-level perceptive of how NPs interact with protein corona once exposed to a physiological environment. In this study, the conformational changes of bovine serum albumin (BSA) were investigated in detail when exposed to different concentration of titanium dioxide nanoparticle by various techniques. To analyze the effects of NPs on proteins, the interaction between bovine serum albumin and titanium dioxide nanoparticles at different concentrations were investigated. The interaction, BSA conformations, kinetics, and adsorption were analyzed by dynamic light scattering, Fourier transform infrared spectroscopy and fluorescence quenching. Dynamic light scattering analysis confirms the interaction with major changes in the size of the protein. Fluorescence quenching analysis confirms the side-on or end-on interaction of 1.1 molecules of serum albumin to titanium dioxide nanoparticles. Further, pseudo-second order kinetics was determined with equilibrium contact time of 20min. The spectroscopic analysis suggests that there is a conformational change both at secondary and tertiary structure levels. A distortion in both α-helix and β-sheets was observed by Fourier transform infrared (FTIR) spectroscopy. Fluorescence quenching analysis confirms the interaction of a molecule of bovine serum albumin to the single TiO2 nanoparticle. Further, pseudo-second order kinetics was determined with equilibrium contact time of 20min. The data of the present study determines the detailed evaluation of BSA adsorption on TiO2 nanoparticle along with mechanism and adsorption kinetics.

  9. A spectroscopic study on interaction between bovine serum albumin and titanium dioxide nanoparticle synthesized from microwave-assisted hybrid chemical approach.

    PubMed

    Ranjan, Shivendu; Dasgupta, Nandita; Srivastava, Priyanka; Ramalingam, Chidambaram

    2016-08-01

    The use of nanoparticles in food or pharma requires a molecular-level perceptive of how NPs interact with protein corona once exposed to a physiological environment. In this study, the conformational changes of bovine serum albumin (BSA) were investigated in detail when exposed to different concentration of titanium dioxide nanoparticle by various techniques. To analyze the effects of NPs on proteins, the interaction between bovine serum albumin and titanium dioxide nanoparticles at different concentrations were investigated. The interaction, BSA conformations, kinetics, and adsorption were analyzed by dynamic light scattering, Fourier transform infrared spectroscopy and fluorescence quenching. Dynamic light scattering analysis confirms the interaction with major changes in the size of the protein. Fluorescence quenching analysis confirms the side-on or end-on interaction of 1.1 molecules of serum albumin to titanium dioxide nanoparticles. Further, pseudo-second order kinetics was determined with equilibrium contact time of 20min. The spectroscopic analysis suggests that there is a conformational change both at secondary and tertiary structure levels. A distortion in both α-helix and β-sheets was observed by Fourier transform infrared (FTIR) spectroscopy. Fluorescence quenching analysis confirms the interaction of a molecule of bovine serum albumin to the single TiO2 nanoparticle. Further, pseudo-second order kinetics was determined with equilibrium contact time of 20min. The data of the present study determines the detailed evaluation of BSA adsorption on TiO2 nanoparticle along with mechanism and adsorption kinetics. PMID:27318604

  10. Proteolytically-induced changes of secondary structural protein conformation of bovine serum albumin monitored by Fourier transform infrared (FT-IR) and UV-circular dichroism spectroscopy

    NASA Astrophysics Data System (ADS)

    Güler, Günnur; Vorob'ev, Mikhail M.; Vogel, Vitali; Mäntele, Werner

    2016-05-01

    Enzymatically-induced degradation of bovine serum albumin (BSA) by serine proteases (trypsin and α-chymotrypsin) in various concentrations was monitored by means of Fourier transform infrared (FT-IR) and ultraviolet circular dichroism (UV-CD) spectroscopy. In this study, the applicability of both spectroscopies to monitor the proteolysis process in real time has been proven, by tracking the spectral changes together with secondary structure analysis of BSA as proteolysis proceeds. On the basis of the FTIR spectra and the changes in the amide I band region, we suggest the progression of proteolysis process via conversion of α-helices (1654 cm- 1) into unordered structures and an increase in the concentration of free carboxylates (absorption of 1593 and 1402 cm- 1). For the first time, the correlation between the degree of hydrolysis and the concentration of carboxylic groups measured by FTIR spectroscopy was revealed as well. The far UV-CD spectra together with their secondary structure analysis suggest that the α-helical content decreases concomitant with an increase in the unordered structure. Both spectroscopic techniques also demonstrate that there are similar but less spectral changes of BSA for the trypsin attack than for α-chymotrypsin although the substrate/enzyme ratio is taken the same.

  11. Fatty acid-binding site environments of serum vitamin D-binding protein and albumin are different

    PubMed Central

    Swamy, Narasimha; Ray, Rahul

    2008-01-01

    Vitamin D-binding protein (DBP) and albumin (ALB) are abundant serum proteins and both possess high-affinity binding for saturated and unsaturated fatty acids. However, certain differences exist. We surmised that in cases where serum albumin level is low, DBP presumably can act as a transporter of fatty acids. To explore this possibility we synthesized several alkylating derivatives of 14C-palmitic acid to probe the fatty acid binding pockets of DBP and ALB. We observed that N-ethyl-5-phenylisooxazolium-3′-sulfonate-ester (WRK ester) of 14C-palmitic acid specifically labeled DBP; but p-nitrophenyl- and N-hydroxysuccinimidyl-esters failed to do so. However, p-nitrophenyl ester of 14C-palmitic acid specifically labeled bovine ALB, indicating that the micro-environment of the fatty acid-binding domains of DBP and ALB may be different; and DBP may not replace ALB as a transporter of fatty acids. PMID:18374965

  12. Structural Studies of Protein-Surfactant Complexes

    SciTech Connect

    Chodankar, S. N.; Aswal, V. K.; Wagh, A. G.

    2008-03-17

    The structure of protein-surfactant complexes of two proteins bovine serum albumin (BSA) and lysozyme in presence of anionic surfactant sodium dodecyl sulfate (SDS) has been studied using small-angle neutron scattering (SANS). It is observed that these two proteins form different complex structures with the surfactant. While BSA protein undergoes unfolding on addition of surfactant, lysozyme does not show any unfolding even up to very high surfactant concentrations. The unfolding of BSA protein is caused by micelle-like aggregation of surfactant molecules in the complex. On the other hand, for lysozyme protein there is only binding of individual surfactant molecules to protein. Lysozyme in presence of higher surfactant concentrations has protein-surfactant complex structure coexisting with pure surfactant micelles.

  13. Enhanced extraction of bovine serum albumin with aqueous biphasic systems of phosphonium- and ammonium-based ionic liquids

    PubMed Central

    Pereira, Matheus M.; Pedro, Sónia N.; Quental, Maria V.; Lima, Álvaro S.; Coutinho, João A. P.; Freire, Mara G.

    2016-01-01

    Novel aqueous biphasic systems (ABS) composed of phosphonium- or ammonium-based ionic liquids (ILs), combined with a buffered aqueous solution of potassium citrate/citric acid (pH=7.0), were investigated for the extraction of proteins. For that purpose, the phase diagrams, tie-lines and tie-line lengths were determined at 25ºC, and the performance of these ABS for the extraction of bovine serum albumin (BSA) was then evaluated. The obtained results reveal that, with the exception of the more hydrophobic ILs, most of the systems investigated allow the complete extraction of BSA for the IL-rich phase in a single-step. These remarkable extraction efficiencies are far superior to those afforded by more conventional extraction systems previously reported. The composition of the biphasic systems, i.e., the amount of phase-forming components, was also investigated aiming at reducing the overall costs of the process without losing efficiency on the protein extraction. It is shown that the extraction efficiencies of BSA are maintained at 100% up to high protein concentrations (at least up to 10 g.L-1). The recovery of the BSA from the IL-rich phase by dialysis is also shown in addition to the demonstration of the IL recyclability and reusability, at least for 3 times. In the sequential three-step extractions (BSA recovery/IL reusability), the extraction efficiencies of BSA for the IL-rich phase were maintained at 100%. For the improved ABS, the preservation of the protein native conformation was confirmed by Size Exclusion High-Performance Liquid Chromatography (used also as the quantification method) and by Fourier Transform Infra-Red spectroscopy. According to the results herein reported, ABS composed of phosphonium- or ammonium-based ILs and a biodegradable organic salt represent an alternative and remarkable platform for the extraction of BSA and may be extended to other proteins of interest. PMID:25865275

  14. Enhanced extraction of bovine serum albumin with aqueous biphasic systems of phosphonium- and ammonium-based ionic liquids.

    PubMed

    Pereira, Matheus M; Pedro, Sónia N; Quental, Maria V; Lima, Álvaro S; Coutinho, João A P; Freire, Mara G

    2015-07-20

    Novel aqueous biphasic systems (ABS) composed of phosphonium- or ammonium-based ionic liquids (ILs), combined with a buffered aqueous solution of potassium citrate/citric acid (pH=7.0), were investigated for the extraction of proteins. For that purpose, the phase diagrams, tie-lines and tie-line lengths were determined at 25 °C, and the performance of these ABS for the extraction of bovine serum albumin (BSA) was then evaluated. The obtained results reveal that, with the exception of the more hydrophobic ILs, most of the systems investigated allow the complete extraction of BSA for the IL-rich phase in a single-step. These remarkable extraction efficiencies are far superior to those afforded by more conventional extraction systems previously reported. The composition of the biphasic systems, i.e., the amount of phase-forming components, was also investigated aiming at reducing the overall costs of the process without losing efficiency on the protein extraction. It is shown that the extraction efficiencies of BSA are maintained at 100% up to high protein concentrations (at least up to 10 g L(-1)). The recovery of the BSA from the IL-rich phase by dialysis is also shown in addition to the demonstration of the IL recyclability and reusability, at least for 3 times. In the sequential three-step extractions (BSA recovery/IL reusability), the extraction efficiencies of BSA for the IL-rich phase were maintained at 100%. For the improved ABS, the preservation of the protein native conformation was confirmed by Size Exclusion High-Performance Liquid Chromatography (used also as the quantification method) and by Fourier Transform Infra-Red spectroscopy. According to the results herein reported, ABS composed of phosphonium- or ammonium-based ILs and a biodegradable organic salt represent an alternative and remarkable platform for the extraction of BSA and may be extended to other proteins of interest. PMID:25865275

  15. Exploring the interaction between picoplatin and human serum albumin: The effects on protein structure and activity.

    PubMed

    Wang, Yanqing; Wu, Peirong; Zhou, Xinchun; Zhang, Hongmei; Qiu, Ligan; Cao, Jian

    2016-09-01

    For the first time, the effects of picoplatin on the structure and esterase-like catalytic activity of human serum albumin (HSA) have been investigated by spectroscopic approaches and molecular modeling. The circular dichroism (CD) spectral examinations indicated that the binding of picoplatin with HSA induced a slight decrease of a-helix content of protein and unfolded the constituent polypeptides of the protein. The synchronous fluorescence and three-dimensional fluorescence spectral methods were used to estimate the effect of picoplatin on the micro-environmental changes of the Trp and Tyr residues of HSA, indicating that the micro-environment around the Tyr and Trp residue is partly disturbed by picoplatin. UV-vis absorption spectral result indicated the formation of the ground state complex between picoplatin with HSA. The ANS binding assay indicated the existence of competitive combination of picoplatin and ANS with HSA. The studies on the effects of picoplatin on the binding of HSA with bilirubin and heme showed that picoplatin binding caused a change of angle between two chromophores of bound bilirubin and the binding site of picoplatin does not locate in subdomain IB in HSA that bound with heme. The molecular modeling results showed that picoplatin binds to the connection between domain I and domain II by hydrophobic, hydrogen bonds, and van der Waals forces. In addition, HSA maintains most of its esterase activity in the presence of picoplatin. The investigations on how picoplatin interacts with HSA are important for the understanding of the anticancer mechanism and toxicity of platinum-based anticancer drug. PMID:27484966

  16. Tandem mass spectral libraries of peptides in digests of individual proteins: Human Serum Albumin (HSA).

    PubMed

    Dong, Qian; Yan, Xinjian; Kilpatrick, Lisa E; Liang, Yuxue; Mirokhin, Yuri A; Roth, Jeri S; Rudnick, Paul A; Stein, Stephen E

    2014-09-01

    This work presents a method for creating a mass spectral library containing tandem spectra of identifiable peptide ions in the tryptic digestion of a single protein. Human serum albumin (HSA(1)) was selected for this purpose owing to its ubiquity, high level of characterization and availability of digest data. The underlying experimental data consisted of ∼3000 one-dimensional LC-ESI-MS/MS runs with ion-trap fragmentation. In order to generate a wide range of peptides, studies covered a broad set of instrument and digestion conditions using multiple sources of HSA and trypsin. Computer methods were developed to enable the reliable identification and reference spectrum extraction of all peptide ions identifiable by current sequence search methods. This process made use of both MS2 (tandem) spectra and MS1 (electrospray) data. Identified spectra were generated for 2918 different peptide ions, using a variety of manually-validated filters to ensure spectrum quality and identification reliability. The resulting library was composed of 10% conventional tryptic and 29% semitryptic peptide ions, along with 42% tryptic peptide ions with known or unknown modifications, which included both analytical artifacts and post-translational modifications (PTMs) present in the original HSA. The remaining 19% contained unexpected missed-cleavages or were under/over alkylated. The methods described can be extended to create equivalent spectral libraries for any target protein. Such libraries have a number of applications in addition to their known advantages of speed and sensitivity, including the ready re-identification of known PTMs, rejection of artifact spectra and a means of assessing sample and digestion quality.

  17. Safety concerns towards the biomedical application of PbS nanoparticles: An approach through protein-PbS interaction and corona formation

    SciTech Connect

    Kumar Bhunia, Amit; Saha, Satyajit; Kanti Samanta, Pijus; Kamilya, Tapanendu

    2014-03-24

    Semiconductor nanoparticles (NPs) with near-infrared (NIR) fluorescence has achieved great interest for early detection of colon tumors/cancer. We have synthesized lead sulphide (PbS) NPs (5–7 nm) having emission in NIR region and investigated its interaction with bovine serum albumin (BSA) to determine the bio-safety of PbS NPs. The interaction of PbS NPs with BSA occurs through formation of “hard” and “soft” protein NPs corona and follows exponential association. The hard corona represents that the core PbS NPs are fully covered by BSA with shell thickness of ∼8 nm, i.e., the dimension of BSA monomer. A large number of PbS NPs with hard corona of BSA forms “colony” with diameters in the range of 200–400 nm. The soft corona grows surrounding this colony. The quenching of fluorescence BSA in the presence of PbS NPs follows dynamic quenching process with tryptophan as major binding sites. Nearest to human body temperature, positive cooperative association between PbS NPs and BSA are found, and affinity of BSA to the PbS NPs gradually increases in superlinear fashion. The electrostatic interaction is the key force in binding of PbS NPs with BSA, and hydrophobic interaction between PbS NPs and BSA is responsible for conformational change of BSA.

  18. The investigation of the interaction between piracetam and bovine serum albumin by spectroscopic methods

    NASA Astrophysics Data System (ADS)

    Guo, Xingjia; Han, Xiaowei; Tong, Jian; Guo, Chuang; Yang, Wenfeng; Zhu, Jifen; Fu, Bing

    2010-03-01

    The interaction between piracetam (OPA) with bovine serum albumin (BSA) has been thoroughly studied by fluorescence quenching technique in combination with UV-vis absorption, Fourier transform infrared (FT-IR), and circular dichroism (CD) spectroscopies under the simulative physiological conditions. The quenching of BSA fluorescence by OPA was found to be a static quenching process. The binding constants ( K a) are 3.014, 2.926, and 2.503 × 10 3 M -1 at 292, 298, and 309 K, respectively. According to the van't Hoff equation, the thermodynamic functions standard enthalpy (Δ H) and standard entropy (Δ S) for the reaction were calculated to be -74.560 kJ mol -1 and -159.380 J mol -1 K -1, which indicated that OPA binds to BSA mainly by hydrogen bonds and van der Waals interactions. The binding distance between BSA and OPA was calculated to be 4.10 nm according to the theory of FÖrster's non-radiation energy transfer. The displacement experiments confirmed that OPA could bind to the site I of BSA. Furthermore, the effects of pH and some common ions on the binding constant were also examined. And the alterations of protein secondary structure in the presence of OPA were observed by the CD and FT-IR spectra.

  19. Binding interaction of quinclorac with bovine serum albumin: A biophysical study

    NASA Astrophysics Data System (ADS)

    Han, Xiao-Le; Mei, Ping; Liu, Yi; Xiao, Qi; Jiang, Feng-Lei; Li, Ran

    2009-10-01

    Quinclorac (QUC) is a new class of highly selective auxin herbicides. The interaction between QUC and bovine serum albumin (BSA) was investigated by fluorescence spectroscopy, synchronous fluorescence, three-dimensional fluorescence, CD spectroscopy and UV-vis absorption spectroscopy under simulative physiological condition. It was proved that the probable quenching mechanism of BSA by quinclorac was dynamic quenching. The Stern-Volmer quenching model has been successfully applied and the activation energy of the interaction as much as 8.03 kJ mol -1, corresponding thermodynamic parameters Δ Hθ, Δ Sθ and Δ Gθ were calculated. The results indicated that the acting forces between QUC and BSA were mainly hydrogen bonding and van der Waals forces. According to the Förster non-radiation energy transfer theory, the average binding distance between donor (BSA) and acceptor (QUC) was obtained ( r = 3.12 nm). The alterations of protein secondary structure in the presence of QUC were confirmed by the evidences from three-dimensional fluorescence, synchronous fluorescence and CD spectroscopy. Furthermore, the site marker competitive experiments indicated that the binding of QUC to BSA primarily took place in Sudlow site I.

  20. Synthesis, purification and mass spectrometric characterisation of a fluorescent Au9@BSA nanocluster and its enzymatic digestion by trypsin.

    PubMed

    Fernández-Iglesias, Nerea; Bettmer, Jörg

    2014-01-21

    Nanoclusters of noble metals like Ag and Au have attracted great attention as they form a missing link between isolated metal atoms and nanoparticles. Their particular properties like luminescence in the visible range and nontoxicity make them attractive for bioimaging and biolabelling purposes, especially with use of proteins as stabilising agents. In this context, this study intends the synthesis of a specific Au nanocluster covered by bovine serum albumin (BSA). It is shown that size-exclusion chromatography is feasible for the purification and isolation of the nanocluster. A mass spectrometric characterisation, preferably by ESI-MS, indicates the presence of an Au9@BSA nanocluster. Enzymatic digestion of the nanocluster with trypsin results in a significant increase of the fluorescence intensity at 650 and 710 nm, whereas complementary MALDI-MS studies are presented for the identification of generated peptides and show a distinctive pattern in comparison to the pure protein. It can be concluded that Au9@BSA might be, in future, an interesting candidate for in vitro studies of protease activities.

  1. Preparation of bovine serum albumin nanospheres as drug targeting carriers.

    PubMed

    Nakagawa, Y; Takayama, K; Ueda, H; Machida, Y; Nagai, T

    1987-12-01

    Bovine serum albumin nanospheres (BSA-NS) of mean diameter about 170 nm were prepared by means of the tanning method with glutaraldehyde, and their efficacy as drug targeting carriers was evaluated. To gain insight of biodegradability, BSA microspheres (BSA-MS) were first administered to rats and their distributions in the lungs and liver were observed by a scanning electron microscope. A large amount of BSA-MS was found in the lungs and their surface was slightly degraded at 1 week after the administration. For investigating biocompatibility, the weight increase of the spleen and liver was measured after the administration of the BSA-NS to mice. The spleen weight of the group receiving BSA-NS was equivalent to that of the control group, though the liver weight was significantly increased. It was observed that conjugates of BSA-NS with antibody selectively concentrated on the surface of Sepharose beads which were coated with antigen.

  2. Adsorption of bovine serum albumin on amorphous carbon surfaces studied with dip pen nanolithography

    NASA Astrophysics Data System (ADS)

    Yadav, Pradeep K.; McKavanagh, Fiona; Maguire, Paul D.; Lemoine, Patrick

    2011-10-01

    This article reports the use of dip pen nanolithography (DPN) for the study of adsorption of bovine serum albumin (BSA) proteins on amorphous carbon surfaces; tetrahedral amorphous carbon (t-aC) and silicon doped hydrogenated amorphous carbon (a-C:H:Si). Contact angle study shows that the BSA proteins reduce the contact angle on both carbon materials. We also noticed that the drop volume dependence is consistent with a negative line tension, i.e. due to an attractive protein/surface interaction. The DPN technique was used to write short-spaced (100 nm) BSA line patterns on both samples. We found a line merging effect, stronger in the case of the a-C:H:Si material. We discuss possible contributions from tip blunting, scratching, cross-talk between lever torsion and bending and nano-shaving of the patterns. We conclude that the observed effect is caused in large measure by the diffusion of BSA proteins on the amorphous carbon surfaces. This interpretation of the result is consistent with the contact angle data and AFM force curve analysis indicating larger tip/surface adhesion and spreading for the a-C:H:Si material. We conclude by discussing the advantages and limitations of DPN lithography to study biomolecular adsorption in nanoscale wetting environments.

  3. The nature of the apolar phase influences the structure of the protein emulsifier in oil-in-water emulsions stabilized by bovine serum albumin. A front-surface fluorescence study.

    PubMed

    Rampon, Vincent; Brossard, Chantal; Mouhous-Riou, Nadine; Bousseau, Benoît; Llamas, Geneviève; Genot, Claude

    2004-05-20

    Proteins are widely used as emulsifiers in food emulsions. Model emulsions, designed to study emulsifying properties of proteins and their conformation at the interfaces often contain a hydrocarbon as apolar phase instead of natural triglycerides as found in food products. Yet, some results indicate that the protein conformation at the interface depends on the nature of the apolar phase. Front-surface fluorescence spectroscopy was used to evidence differences in the structure of bovine serum albumin (BSA) adsorbed at the interface of emulsions prepared with different apolar phases: an hydrocarbon (n-dodecane), a synthetic medium-chain triglyceride (miglyol) and a natural vegetable oil (sunflower oil). Emulsions had similar size distributions of oil droplets. Front-surface fluorescence emission spectra of tryptophanyl residues of the protein (Trp) in emulsions, creams and serums varied as a function of the nature of hydrophobic phase. In emulsions and creams, wavelength of the maximum fluorescence intensities was blue-shifted as compared to the BSA solution. The shift was larger in creams than in emulsions and in samples containing dodecane than with the other apolar phases. Fourth derivative spectra of emulsions and creams exhibited two peaks assigned, respectively, to Trp located in hydrophilic and hydrophobic environments. The peaks were slightly red-shifted in the presence of sunflower oil as compared to miglyol and dodecane and the relative intensity of the "hydrophobic peak" was higher in dodecane. The effects were greater in creams than in emulsions. Fluorescence intensity of Trp was the highest in the serums of emulsions prepared with dodecane as compared to serums issued from sunflower oil and miglyol emulsions. Thus, proportion of adsorbed protein was lower in dodecane emulsions than with the other apolar phases. These results evidence that the mean environment of Trp was more hydrophobic in emulsions and creams than in solutions due to a displacement of

  4. Protein-binding and antioxidant potential of phenolics of mangosteen fruit (Garcinia mangostana).

    PubMed

    Naczk, M; Towsend, M; Zadernowski, R; Shahidi, F

    2011-09-15

    Phenolics were extracted from mangosteen fruit parts with 70% (v/v) aqueous acetone. The yield of crude extracts of phenolics (CP) ranged from 5.8% to 7.9%. The total phenolics (TPH) content ranged from 9.3mg to over 250mg of gallic acid equivalents per g of extract in the edible aril and skin, respectively. The formation of phenolic-protein complexes was assayed by both the dye-labelled bovine serum albumin (BSA) and the fluorescence quenching methods. Phenolics from peel and rind displayed a strong protein-precipitating potential. On the other hand, phenolics from edible aril exhibited greater affinity for BSA, as determined by the fluorescence quenching assay. The static quenching was the dominant mode of quenching of BSA fluorescence by mangosteen fruit phenolics. Mangosteen phenolics occupied two binding sites on BSA molecules located most likely in or near both tryptophan residues in the BSA molecule acting as an intrinsic fluorescence probe.

  5. Exploring the binding mechanism of 5-hydroxy-3‧,4‧,7-trimethoxyflavone with bovine serum albumin: Spectroscopic and computational approach

    NASA Astrophysics Data System (ADS)

    Sudha, A.; Srinivasan, P.; Thamilarasan, V.; Sengottuvelan, N.

    2016-03-01

    The current study was carried out to investigate the binding mechanism of a potential flavonoid compound 5-hydroxy-3‧,4‧,7-trimethoxyflavone (HTMF) with bovine serum albumin (BSA) using ultraviolet-visible, fluorescence, circular dichroism (CD) spectral measurements along with molecular docking and molecular dynamics (MD) simulation. It was confirmed from fluorescence spectra that the intrinsic fluorescence of BSA was robustly quenched by HTMF through a static quenching mechanism. The number of binding sites (n) for HTMF binding on BSA was found to be about one. The thermodynamic parameters estimated from the van't Hoff plot specified that hydrophobic force was the predominant force in the HTMF-BSA complex and there also exist hydrogen bonds and electrostatic interactions. The effect of HTMF on the BSA conformation examined using CD studies revealed that there is a decrease in the helical content of BSA upon HTMF interaction. The results of molecular docking study shed light on the binding mode which exposed that HTMF bind within the hydrophobic pocket of the subdomain IIIA of BSA. The stability of HTMF-BSA complex with respect to free protein was analyzed from the molecular dynamic studies. The electronic structure analysis of HTMF was achieved by using density functional theory (DFT) calculations at B3LYP/6-31G** level to support its antioxidant role. The results of computational analysis are in good consistence with the experimental data and the present findings suggested that HTMF exhibits a good binding propensity to BSA protein which will be helpful for the drug design.

  6. Preparation of protein imprinted materials by hierarchical imprinting techniques and application in selective depletion of albumin from human serum

    NASA Astrophysics Data System (ADS)

    Liu, Jinxiang; Deng, Qiliang; Tao, Dingyin; Yang, Kaiguang; Zhang, Lihua; Liang, Zhen; Zhang, Yukui

    2014-06-01

    Hierarchical imprinting was developed to prepare the protein imprinted materials, as the artificial antibody, for the selective depletion of HSA from the human serum proteome. Porcine serum albumin (PSA) was employed as the dummy template for the fabrication of the recognition sites. To demonstrate the advantages of the hierarchical imprinting, molecularly imprinted polymers prepared by hierarchical imprinting technique (h-MIPs) were compared with those obtained by bulk imprinting (b-MIPs), in terms of the binding capacity, adsorption kinetics, selectivity and synthesis reproducibility. The binding capacity of h-MIPs could reach 12 mg g-1. And saturation binding could be reached in less than 20 min for the h-MIPs. In the protein mixture, h-MIPs exhibit excellent selectivity for PSA, with imprinting factors as about 3.6, much higher than those for non-template proteins. For the proteomic application, the identified protein group number in serum treated by h-MIPs was increased to 422, which is 21% higher than that obtained from the original serum, meanwhile the identified protein group number for the Albumin Removal kit was only 376. The results demonstrate that protein imprinted polymers prepared by hierarchical imprinting technique, might become the artificial antibodies for the selective depletion of high abundance proteins in proteome study.

  7. Studying the denaturation of bovine serum albumin by a novel approach of difference-UV analysis.

    PubMed

    Nikolaidis, Athanasios; Moschakis, Thomas

    2017-01-15

    A novel approach in the analysis of difference-UV spectrophotometric data for determining the heat denaturation degree of bovine serum albumin (BSA) was assessed. Five different parameters of difference-UV spectra were obtained by subtracting spectra of unheated and denatured protein solutions at different temperature-time combinations. BSA was found to exhibit a maximum degree of heat denaturation of about 17% compared to the complete unfolding caused by 6M guanidine hydrochloride. This low degree of heat denaturation is probably caused by the aggregation of the initially unfolded protein molecules. The kinetic analysis exhibited discontinuities in the Arrhenius plots, distinguishing the unfolding and aggregation phases of the denaturation process, whereas such a discrimination could not be obtained by differential scanning calorimetry analyses. The proposed method is accurate, fast, simple and sensitive enough to detect changes in the protein heat denaturation even at short temperature-time intervals. PMID:27542472

  8. Interaction of weakly bound antibiotics neomycin and lincomycin with bovine and human serum albumin: biophysical approach.

    PubMed

    Keswani, Neelam; Choudhary, Sinjan; Kishore, Nand

    2010-07-01

    The thermodynamics of interaction of neomycin and lincomycin with bovine serum albumin (BSA) and human serum albumin (HSA) has been studied using isothermal titration calorimetry (ITC), in combination with UV-visible, steady state and time resolved fluorescence spectroscopic measurements. Neomycin is observed to bind weakly to BSA and HSA whereas lincomycin did not show any evidence for binding with the native state of these proteins, rather it interacts in the presence of surfactants. The ITC results suggest 1 : 1 binding stoichiometry for neomycin in the studied temperature range. The values of the van't Hoff enthalpy do not agree with the calorimetric enthalpy in the case of neomycin, suggesting conformational changes in the protein upon ligand binding, as well as with the rise in the temperature. Experiments at different ionic strengths, and in the presence of tetrabutyl ammonium bromide and surfactants suggest the predominant involvement of electrostatic interactions in the complexation process of neomycin with BSA and HSA, and non-specific interaction behaviour of lincomycin with these proteins.

  9. Grading of severity of the condition in burn patients by serum protein and albumin/globulin studies.

    PubMed

    Kumar, Pramod

    2010-07-01

    Capillary permeability increases after inflammation with consequent leak of fluid, electrolytes, and proteins. The albumin molecule size being smaller (69 kDa) than the globulin molecule (90-156 kDa) will leak relatively at an early stage of the disease (with moderate increase in capillary pore size) than globulin leading to albumin/globulin reversal. In cases with severe permeability changes with rapid progression to larger pore size with simultaneous leak of both albumin and globulin, albumin/globulin reversal will not occur. In this study estimation the serum protein and albumin/globulin (A/G) ratio at frequent intervals was done to grade the severity of the condition of burn patients by assessing the severity of capillary leak.A total of 61 admitted patients (from March 2002 to December 2004) based on the protein values were divided into 3 groups (group 1: 6-8 g/dL, group 2: 5.1-5.9 g/dL, group 3: < or =5.0 g/dL), and all the patients who showed change in their protein levels during the study were shifted to appropriate group and were classified as group shifters. The mean survival time and mortality of various groups were compared, and A/G ratio of all the expired cases was analyzed.Group 3 patients showed higher mortality (95%) as compared to that in other groups (group 1 and 2: 0% each and group shifters: 30.2%). Median survival time of group 3 was significantly low as compared to that of group 1 (P < 0.0026), group 2 (P < 0.0006), and group shifters (P < 0.0000). In group shifters the mean time (days) required for shifting from one group to other just before death or discharge in survivors was significantly higher than that in expired cases. Of 26 cases expired during the study, initial A/G ratio at the time of first assigning the group was not reversed in 22 cases (84.6%).The study concluded that the severity (indicated by lower serum protein values) and speed (judged by A/G ratio changes and median survival time analysis) of capillary permeability changes

  10. Binding to Bovine Serum Albumin Protects β-Carotene against Oxidative Degradation.

    PubMed

    Chang, Hui-Ting; Cheng, Hong; Han, Rui-Min; Zhang, Jian-Ping; Skibsted, Leif H

    2016-07-27

    Binding to bovine serum albumin (BSA) was found to protect β-carotene (β-Car) dissolved in air-saturated phosphate buffer solution/tetrahydrofuran (9:1, v/v) efficiently against photobleaching resulting from laser flash excitation at 532 nm. From dependence of the relative photobleaching yield upon the BSA concentration, an association constant of Ka = 4.67 × 10(5) L mol(-1) for β-Car binding to BSA was determined at 25 °C. Transient absorption spectroscopy confirmed less bleaching of β-Car on the microsecond time scale in the presence of BSA, while kinetics of triplet-state β-Car was unaffected by the presence of oxygen. The protection of β-Car against this type of reaction seems accordingly to depend upon dissipation of excitation energy from an excited state into the protein matrix. Static quenching of BSA fluorescence by β-Car had a Stern-Volmer constant of Ksv = 2.67 × 10(4) L mol(-1), with ΔH = 17 kJ mol(-1) and ΔS = 142 J mol(-1) K(-1) at 25 °C. Quenching of tryptophan (Trp) fluorescence by β-Car suggests involvement of Trp in binding of β-Car to BSA through hydrophobic interaction, while the lower value for the Stern-Volmer constant Ksv compared to the binding constant, Ka, may indicate involvement of β-Car aggregates. Bound β-Car increased the random coil fraction of BSA at the expense of α-helix, as shown by circular dichroism, affecting the β-Car configuration, as shown by Raman spectroscopy. PMID:27399620

  11. Analysis of protein transport in the Brassica oleracea vasculature reveals protein-specific destinations.

    PubMed

    Niu, Chenxing; Anstead, James; Verchot, Jeanmarie

    2012-03-01

    We investigated the vascular transport properties of exogenously applied proteins to Brassica oleracea plants and compared their delivery to various aerial parts of the plant with carboxy fluorescein (CF) dye. We identified unique properties for each protein. Alexafluor-tagged bovine serum albumin (Alexa-BSA) and Alexafluor-tagged Histone H1 (Alexa-Histone) moved slower than CF dye throughout the plant. Interestingly, Alexa-Histone was retained in the phloem and phloem parenchyma while Alexa-BSA moved into the apoplast. One possibility is that Alexa-Histone sufficiently resembles plant endogenous proteins and is retained in the vascular stream, while Alexa-BSA is exported from the cell as a foreign protein. Both proteins diffuse from the leaf veins into the leaf lamina. Alexa-BSA accumulated in the leaf epidermis while Alexa-Histone accumulated mainly in the mesophyll layers. Fluorescein-tagged hepatitis C virus core protein (fluorescein-HCV) was also delivered to B. oleracea plants and is larger than Alexa-BSA. This protein moves more rapidly than BSA through the plant and was restricted to the leaf veins. Fluorescein-HCV failed to unload to the leaf lamina. These combined data suggest that there is not a single default pathway for the vascular transfer of exogenous proteins in B. oleracea plants. Specific protein properties appear to determine their destination and transport properties within the phloem. PMID:22476467

  12. The effect of albumin fusion patterns on the production and bioactivity of the somatostatin-14 fusion protein in Pichia pastoris.

    PubMed

    Ding, Yuedi; Fan, Jun; Li, Wenxin; Yang, Runlin; Peng, Ying; Deng, Lili; Wu, Yu; Fu, Qiang

    2013-08-01

    Somatostatin is a natural inhibitor of growth hormone, and its analogues are clinically used for the therapy of acromegaly, gigantism, thyrotropinoma, and other carcinoid syndrome. However, natural somatostatin is limited for clinical usage because of its short half-life in vivo. Albumin fusion technology was used to construct long-acting fusion proteins, and Pichia pastoris was used as an expression system. Three fusion proteins, (somatostatin (SS)14)2-human serum albumin (HSA), (SS14)3-HSA, and HSA-(SS14)3, were constructed with different fusion copies of somatostatin-14 and fusion orientations. The expression level of (SS14)3-HSA and HSA-(SS14)3 was much lower than (SS14)2-HSA due to the additional fusion of the somatostatin-14 molecule. Matrix-assisted laser desorption ionization-time-of-flight mass spectrometry revealed that severe degradation occurred in the fermentation process. Similar to the standard of somatostatin-14, all three fusion proteins were able to inhibit growth hormone secretion in the blood, with (SS14)2-HSA being the most effective one. On the whole, (SS14)2-HSA was the most effective protein in both production level and bioactivity, and increasing the number of small protein copies fused to HSA may not be a suitable method to improve the protein bioactivity. PMID:23712794

  13. Simultaneous photometric determination of albumin and total protein in animal blood plasma employing a multicommutated flow system to carried out on line dilution and reagents solutions handling

    NASA Astrophysics Data System (ADS)

    Luca, Gilmara C.; Reis, Boaventura F.

    2004-02-01

    An automatic flow procedure for the simultaneous determination of albumin and total protein in blood plasma samples is proposed. The flow network comprised a set of three-way solenoid valves assembled to implement the multicommutation. The flow set up was controlled by means of a computer equipped with an electronic interface card which running a software wrote in QUICKBASIC 4.5 performed on line programmed dilution to allow the determination of both albumin and total protein in blood plasma. The photometric methods based on Bromocresol Green and Biuret reagents were selected for determination of albumin and total protein, respectively. Two LEDs based photometers coupled together the flow cells were employed as detector. After the adjustment of the operational parameters the proposed system presented the following features: an analytical throughput of 45 sample processing per hour for two analytes; relative standard deviations of 1.5 and 0.8% ( n=10) for a typical sample presenting 34 g l -1 albumin and 90 g l -1 total protein, respectively; linear responses ranging from 0 to 15 g l -1 albumin ( r=0.998) and total protein ( r=0.999); sample and reagents consumption, 140 μl serum solution, 0.015 mg VBC and 0.432 mg CuSO 4 per determination, respectively. Applying the paired t-test between results obtained using the proposed system and reference methods no significant difference at 95 and 90% confidence level for albumin and total protein, respectively, were observed.

  14. Simultaneous photometric determination of albumin and total protein in animal blood plasma employing a multicommutated flow system to carried out on line dilution and reagents solutions handling.

    PubMed

    Luca, Gilmara C; Reis, Boaventura F

    2004-02-01

    An automatic flow procedure for the simultaneous determination of albumin and total protein in blood plasma samples is proposed. The flow network comprised a set of three-way solenoid valves assembled to implement the multicommutation. The flow set up was controlled by means of a computer equipped with an electronic interface card which running a software wrote in QUICKBASIC 4.5 performed on line programmed dilution to allow the determination of both albumin and total protein in blood plasma. The photometric methods based on Bromocresol Green and Biuret reagents were selected for determination of albumin and total protein, respectively. Two LEDs based photometers coupled together the flow cells were employed as detector. After the adjustment of the operational parameters the proposed system presented the following features: an analytical throughput of 45 sample processing per hour for two analytes; relative standard deviations of 1.5 and 0.8% (n=10) for a typical sample presenting 34 g l(-1) albumin and 90 g l(-1) total protein, respectively; linear responses ranging from 0 to 15 g l(-1) albumin (r=0.998) and total protein (r=0.999); sample and reagents consumption, 140 microl serum solution, 0.015 mg VBC and 0.432 mg CuSO4 per determination, respectively. Applying the paired t-test between results obtained using the proposed system and reference methods no significant difference at 95 and 90% confidence level for albumin and total protein, respectively, were observed.

  15. Choline-induced selective fluorescence quenching of acetylcholinesterase conjugated Au@BSA clusters.

    PubMed

    Mathew, Meegle S; Baksi, Ananya; Pradeep, T; Joseph, Kuruvilla

    2016-07-15

    We have developed a highly selective sensitive fluorescent detection of acetylcholine (ACh) using bovine serum albumin (BSA) protected atomically precise clusters of gold. The gold quantum clusters (AuQC@BSA) synthesized using bovine serum albumin and conjugated with acetylcholinesterase (AChE), an enzyme specific for acetylcholine, resulting in AuQC@BSA-AChE. The enzyme, AChE hydrolyzes acetylcholine (ACh) to choline (Ch) which in turn interacts with AuQC@BSA-AChE and quenches its fluorescence, enabling sensing. We have carried out the real time monitoring of the hydrolysis of ACh using electrospray ionization mass spectrometry (ESI MS) to find out the mechanism of fluorescent quenching. The validity of present method for determination of concentration of acetylcholine in real system such as blood was demonstrated. Further, the sensor, AuQC@BSA-AChE can be easily coated on paper and an efficient and cheap sensor can be developed and detection limit for ACh is found to be 10nM. The fluorescent intensity of AuQC@BSA-AChE is sensitive towards acetylcholine in range of 10nM to 6.4µM. This suggests that AuQC@BSA-AChE has an excellent potential to be used for diagnosis of various neuropsychological and neuropsychiatric disorders.

  16. Resonance energy transfer between fluorescent BSA protected Au nanoclusters and organic fluorophores.

    PubMed

    Raut, Sangram; Rich, Ryan; Fudala, Rafal; Butler, Susan; Kokate, Rutika; Gryczynski, Zygmunt; Luchowski, Rafal; Gryczynski, Ignacy

    2014-01-01

    Bovine serum albumin (BSA) protected nanoclusters (Au and Ag) represent a group of nanomaterials that holds great promise in biophysical applications due to their unique fluorescence properties and lack of toxicity. These metal nanoclusters have utility in a variety of disciplines including catalysis, biosensing, photonics, imaging and molecular electronics. However, they suffer from several disadvantages such as low fluorescence quantum efficiency (typically near 6%) and broad emission spectrum (540 nm to 800 nm). We describe an approach to enhance the apparent brightness of BSA Au clusters by linking them with a high extinction donor organic dye pacific blue (PB). In this conjugate PB acts as a donor to BSA Au clusters and enhances its brightness by resonance energy transfer (RET). We found that the emission of BSA Au clusters can be enhanced by a magnitude of two-fold by resonance energy transfer (RET) from the high extinction donor PB, and BSA Au clusters can act as an acceptor to nanosecond lifetime organic dyes. By pumping the BSA Au clusters using a high extinction donor, one can increase the effective brightness of less bright fluorophores like BSA Au clusters. Moreover, we prepared another conjugate of BSA Au clusters with the near infrared (NIR) dye Dylight 750 (Dy750), where BSA Au clusters act as a donor to Dy750. We observed that BSA Au clusters can function as a donor, showing 46% transfer efficiency to the NIR dye Dy750 with a long lifetime component in the acceptor decay through RET. Such RET-based probes can be used to prevent the problems of a broad emission spectrum associated with the BSA Au clusters. Moreover, transferring energy from BSA Au clusters to Dy750 will result in a RET probe with a narrow emission spectrum and long lifetime component which can be utilized in imaging applications.

  17. Concurrent zero-dimensional and one-dimensional biomineralization of gold from a solution of Au3+ and bovine serum albumin

    NASA Astrophysics Data System (ADS)

    Hartings, Matthew R.; Benjamin, Noah; Briere, Floriene; Briscione, Maria; Choudary, Omar; Fisher, Tamra L.; Flynn, Laura; Ghias, Elizabeth; Harper, Michaela; Khamis, Nader; Koenigsknecht, Catherine; Lazor, Klare; Moss, Steven; Robbins, Elaine; Schultz, Susan; Yaman, Samiye; Haverhals, Luke M.; Trulove, Paul C.; De Long, Hugh C.; Miller, Abigail E.; Fox, Douglas M.

    2013-12-01

    A technique was developed for preparing a novel material that consists of gold nanoparticles trapped within a fiber of unfolded proteins. These fibers are made in an aqueous solution that contains HAuCl4 and the protein, bovine serum albumin (BSA). By changing the ratio of gold to BSA in solution, two different types of outcomes are observed. At lower gold to BSA ratios (30-120), a purple solution results after heating the mixture at 80 °C for 4 h. At higher gold to BSA ratios (130-170), a clear solution containing purple fibers results after heating the mixture at 80 °C for 4 h. UV-Vis spectroscopy and light scattering techniques show growth in nanocolloid size as gold to BSA ratio rises above 100. Data indicate that, for the higher gold to BSA ratios, the gold is sequestered within the solid material. The material mass, visible by eye, appears to be an aggregation of smaller individual fibers. Scanning electron microscopy and transmission electron microscopy indicate that these fibers are primarily one-dimensional aggregates, which can display some branching, and can be as narrow as 400 nm in size. The likely mechanism for the synthesis of the novel material is discussed.

  18. Comparative studies of the effects of copper sulfate and zinc sulfate on serum albumins

    NASA Astrophysics Data System (ADS)

    Plotnikova, O. A.; Melnikov, G. V.; Melnikov, A. G.; Kovalenko, A. V.

    2016-04-01

    The work is devoted to the study of the interaction of heavy metals with bovine serum albumin (BSA) and human serum albumin (HSA), by quenching of the intrinsic fluorescence of proteins and fluorescent probe pyrene by heavy metal ions. Sulfates of copper and zinc (CuSO4, ZnSO4) were taken as the metal salts. The value of the Stern-Volmer constants of quenching of intrinsic fluorescence of proteins and fluorescence probe pyrene reduced from Cu (II) to the Zn (II). It was experimentally found that the copper ions have a greater ability to fluorescence quenching, which is probably associated with the greater availability of protein chromophore groups to copper ions and with adsorbed fluorescent probe pyrene in the protein globule.

  19. Biofield-effect protein-sensor: Plasma functionalization of polyaniline, protein immobilization, and sensing mechanism

    NASA Astrophysics Data System (ADS)

    Cho, Chae-Ryong; Lee, Hyun-Uk; Ahn, Kyun; Jeong, Se-Young; Choi, Jun-Hee; Kim, Jinwoo; Cho, Jiung

    2014-06-01

    We report the fabrication of a biofield-effect protein-sensor (BioFEP) based on atmospheric-pressure plasma (AP) treatment of a conducting polyaniline (PANI) film. Successive H2 and O2 AP (OHAP) treatment generated dominant hydrophilic -OH and O=CO- functional groups on the PANI film surface, which served as strong binding sites to immobilize bovine serum albumin (BSA) protein molecules. The output current changes of the BioFEP as a function of BSA concentration were obtained. The resistance of the OHAP surface could be sensitively increased from 2.5 × 108 Ω to 2.0 × 1012 Ω with increasing BSA concentrations in the range of 0.025-4 μg/ml. The results suggest that the method is a simple and cost-effective tool to determine the concentration of BSA by measuring electrical resistance.

  20. Formation and reshuffling of disulfide bonds in bovine serum albumin demonstrated using tandem mass spectrometry with collision-induced and electron-transfer dissociation.

    PubMed

    Rombouts, Ine; Lagrain, Bert; Scherf, Katharina A; Lambrecht, Marlies A; Koehler, Peter; Delcour, Jan A

    2015-01-01

    Thermolysin hydrolyzates of freshly isolated, extensively stored (6 years, 6 °C, dry) and heated (60 min, 90 °C, in excess water) bovine serum albumin (BSA) samples were analyzed with liquid chromatography (LC) electrospray ionization (ESI) tandem mass spectrometry (MS/MS) using alternating electron-transfer dissociation (ETD) and collision-induced dissociation (CID). The positions of disulfide bonds and free thiol groups in the different samples were compared to those deduced from the crystal structure of native BSA. Results revealed non-enzymatic posttranslational modifications of cysteine during isolation, extensive dry storage, and heating. Heat-induced extractability loss of BSA was linked to the impact of protein unfolding on the involvement of specific cysteine residues in intermolecular and intramolecular thiol-disulfide interchange and thiol oxidation reactions. The here developed approach holds promise for exploring disulfide bond formation and reshuffling in various proteins under conditions relevant for chemical, biochemical, pharmaceutical and food processing. PMID:26193081

  1. Formation and reshuffling of disulfide bonds in bovine serum albumin demonstrated using tandem mass spectrometry with collision-induced and electron-transfer dissociation.

    PubMed

    Rombouts, Ine; Lagrain, Bert; Scherf, Katharina A; Lambrecht, Marlies A; Koehler, Peter; Delcour, Jan A

    2015-07-20

    Thermolysin hydrolyzates of freshly isolated, extensively stored (6 years, 6 °C, dry) and heated (60 min, 90 °C, in excess water) bovine serum albumin (BSA) samples were analyzed with liquid chromatography (LC) electrospray ionization (ESI) tandem mass spectrometry (MS/MS) using alternating electron-transfer dissociation (ETD) and collision-induced dissociation (CID). The positions of disulfide bonds and free thiol groups in the different samples were compared to those deduced from the crystal structure of native BSA. Results revealed non-enzymatic posttranslational modifications of cysteine during isolation, extensive dry storage, and heating. Heat-induced extractability loss of BSA was linked to the impact of protein unfolding on the involvement of specific cysteine residues in intermolecular and intramolecular thiol-disulfide interchange and thiol oxidation reactions. The here developed approach holds promise for exploring disulfide bond formation and reshuffling in various proteins under conditions relevant for chemical, biochemical, pharmaceutical and food processing.

  2. BSA modification to reduce CTAB induced nonspecificity and cytotoxicity of aptamer-conjugated gold nanorods

    NASA Astrophysics Data System (ADS)

    Yasun, Emir; Li, Chunmei; Barut, Inci; Janvier, Denisse; Qiu, Liping; Cui, Cheng; Tan, Weihong

    2015-05-01

    Aptamer-conjugated gold nanorods (AuNRs) are excellent candidates for targeted hyperthermia therapy of cancer cells. However, in high concentrations of AuNRs, aptamer conjugation alone fails to result in highly cell-specific AuNRs due to the presence of positively charged cetyltrimethylammonium bromide (CTAB) as a templating surfactant. Besides causing nonspecific electrostatic interactions with the cell surfaces, CTAB can also be cytotoxic, leading to uncontrolled cell death. To avoid the nonspecific interactions and cytotoxicity triggered by CTAB, we report the further biologically inspired modification of aptamer-conjugated AuNRs with bovine serum albumin (BSA) protein. Following this modification, interaction between CTAB and the cell surface was efficiently blocked, thereby dramatically reducing the side effects of CTAB. This approach may provide a general and simple method to avoid one of the most serious issues in biomedical applications of nanomaterials: nonspecific binding of the nanomaterials with biological cells.Aptamer-conjugated gold nanorods (AuNRs) are excellent candidates for targeted hyperthermia therapy of cancer cells. However, in high concentrations of AuNRs, aptamer conjugation alone fails to result in highly cell-specific AuNRs due to the presence of positively charged cetyltrimethylammonium bromide (CTAB) as a templating surfactant. Besides causing nonspecific electrostatic interactions with the cell surfaces, CTAB can also be cytotoxic, leading to uncontrolled cell death. To avoid the nonspecific interactions and cytotoxicity triggered by CTAB, we report the further biologically inspired modification of aptamer-conjugated AuNRs with bovine serum albumin (BSA) protein. Following this modification, interaction between CTAB and the cell surface was efficiently blocked, thereby dramatically reducing the side effects of CTAB. This approach may provide a general and simple method to avoid one of the most serious issues in biomedical applications

  3. Inhibition of neointimal proliferation in rabbits after vascular injury by a single treatment with a protein adduct of nitric oxide.

    PubMed Central

    Marks, D S; Vita, J A; Folts, J D; Keaney, J F; Welch, G N; Loscalzo, J

    1995-01-01

    Endothelium-derived relaxing factor is important for vascular homeostasis and possesses qualities that may modulate vascular injury, including vasodilation, platelet inhibition, and inhibition of smooth muscle proliferation. S-nitrososerum albumin is a naturally occurring adduct of nitric oxide (NO) with a prolonged biologic half-life and is a potent vasodilator and platelet inhibitor. Given the avidity of serum albumin for subendothelial matrix and the antiproliferative effects of NO, we investigated the effects of locally delivered S-nitroso-bovine serum albumin (S-NO-BSA) and a polythiolated form of bovine serum albumin (pS-BSA) modified to carry several S-nitrosothiol groups (pS-NO-BSA) on neointimal responses in an animal model of vascular injury. Locally delivered S-NO-BSA bound preferentially to denuded rabbit femoral vessels producing a 26-fold increase in local concentration compared with uninjured vessels (P = 0.029). pS-NO-BSA significantly reduced the intimal/medial ratio (P = 0.038) and did so in conjunction with elevations in platelet (P < 0.001) and vascular cGMP content (P < or = 0.001). pS-NO-BSA treatment also inhibited platelet deposition (P = 0.031) after denuding injury. Comparison of BSA, S-NO-BSA, pS-NO-BSA, and control revealed a dose-response relationship between the amount of displaceable NO delivered and the extent of inhibition of neointimal proliferation at 2 wk (P < or = 0.001). Local administration of a stable protein S-nitrosothiol inhibits intimal proliferation and platelet deposition after vascular arterial balloon injury. This strategy for the local delivery of a long-lived NO adduct has potential for preventing restenosis after angioplasty. Images PMID:8675628

  4. CdSe/ZnS quantum dots based electrochemical immunoassay for the detection of phosphorylated bovine serum albumin

    SciTech Connect

    Pinwattana, Kulwadee; Wang, Jun; Lin, Chiann Tso; Wu, Hong; Du, Dan; Lin, Yuehe; Chailapakul, Orawon

    2010-11-15

    A CdSe/ZnS quantum dot (QD) based electrochemical immunoassay of phosphorylated bovine serum albumin as a protein biomarker is presented. The QDs were used as labels and were conjugated with the secondary anti-phosphoserine antibody in a heterogeneous sandwich immunoassay. First, the primary BSA antibody was immobilized on polystyrene microwells, followed by the addition of BSA-OP. After that, the QD-labeled anti-phosphoserine antibody was added into microwells for immunorecognition. Finally, the bound QD was dissolved in an acid-dissolution step and was detected by electrochemical stripping analysis. The measured current responses were proportional to the concentration of BSA-OP. Under optimal conditions, the voltammetric response was linear over the range of 0.5 - 500 ng mL-1 of BSA-OP, with a detection limit of 0.5 ng mL-1 at a deposition potential of -1.2 V for 120 s. It also shows good reproducibility with a relative standard deviation of 8.6% of six times determination of 25 ng mL-1 of BSA-OP. This QD-based electrochemical immunoassay offers great promise for simple and cost-effective analysis of protein biomarkers.

  5. Epicutaneous immunization with DNP-BSA induces CD4+ CD25+ Treg cells that inhibit Tc1-mediated CS.

    PubMed

    Majewska-Szczepanik, Monika; Zemelka-Wiącek, Magdalena; Ptak, Włodzimierz; Wen, Li; Szczepanik, Marian

    2012-09-01

    As we have shown previously that protein antigen applied epicutaneously (EC) in mice inhibits TNP-specific Th1-mediated contact sensitivity (CS), we postulated that the maneuver of EC immunization might also suppress Tc1-dependent CS response. Here we showed that EC immunization of normal mice with 2,4-dinitrophenylated bovine serum albumin (DNP-BSA) applied on the skin in the form of a patch induces a state of subsequent unresponsiveness due to regulatory T cells (Treg) that inhibited sensitization and elicitation of effector T-cell responses. Suppression is transferable in vivo by TCRαβ(+) CD4(+) CD25(+) lymphocytes harvested from lymph nodes (LNs) of skin-patched animals. Flow cytometry revealed that EC immunization with DNP-BSA increased TCRαβ(+) CD4(+) CD25(+) FoxP3(+) lymphocytes in subcutaneous LNs, suggesting that observed suppression was mediated by Treg cells. Further, in vitro experiments showed that EC immunization with DNP-BSA prior to 1-fluoro-2,4-dinitrobenzen sensitization suppressed LN cell proliferation and inhibited production of TNF-α, IL-12 and IFN-γ. Using a transwell system or anti-CTLA-4 mAb, we found that EC induced suppression required direct Treg-effector cell contact and is CTLA-4-dependent.

  6. Preferential binding of fisetin to the native state of bovine serum albumin: spectroscopic and docking studies.

    PubMed

    Singha Roy, Atanu; Pandey, Nitin Kumar; Dasgupta, Swagata

    2013-04-01

    We have investigated the binding of the biologically important flavonoid fisetin with the carrier protein bovine serum albumin using multi-spectroscopic and molecular docking methods. The binding constants were found to be in the order of 10(4) M(-1) and the number of binding sites was determined as one. MALDI-TOF analyses showed that one fisetin molecule binds to a single bovine serum albumin (BSA) molecule which is also supported by fluorescence quenching studies. The negative Gibbs free energy change (∆G°) values point to a spontaneous binding process which occurs through the presence of electrostatic forces with hydrophobic association that results in a positive entropy change (+51.69 ± 1.18 J mol(-1) K(-1)). The unfolding and refolding of BSA in urea have been studied in absence and presence of fisetin using steady-state fluorescence and lifetime measurements. Urea denaturation studies indicate that fisetin is gradually released from its binding site on the protein. In the absence of urea, an increase in temperature that causes denaturation of the protein results in the release of fisetin from its bound state indicating that fisetin binds only to the native state of the protein. The circular dichroism (CD) and Fourier transform infrared (FTIR) spectroscopic studies showed an increase in % α-helix content of BSA after binding with fisetin. Site marker displacement studies in accordance with the molecular docking results suggested that fisetin binds in close proximity of the hydrophobic cavity in site 1 (subdomain IIA) of the protein. The PEARLS (Program of Energetic Analysis of Receptor Ligand System) has been used to estimate the interaction energy of fisetin with BSA and the results are in good correlation with the experimental findings.

  7. Ionic Liquid Surfactant Mediated Structural Transitions and Self-Assembly of Bovine Serum Albumin in Aqueous Media: Effect of Functionalization of Ionic Liquid Surfactants.

    PubMed

    Singh, Gurbir; Kang, Tejwant Singh

    2015-08-20

    The self-assembly of globular protein bovine serum albumin (BSA) has been investigated in aqueous solutions of ionic liquid surfactants (ILSs), 1-dodecyl-3-methyl imidazolium chloride, [C12mim][Cl], and its amide, [C12Amim][Cl], and ester, [C12Emim][Cl], functionalized counterparts. Dynamic light scattering (DLS) has provided insights into the alterations in hydrodynamic radii (D(h)) of BSA as a function of concentration of ILSs establishing the presence of different types of BSA-ILS complexes in different concentration regimes of ILSs. Isothermal titration calorimetry (ITC) has been exploited to quantify the ILSs interacting with BSA in dilute concentration regime of ILSs. The zeta-potential measurements shed light on changes in the charged state of BSA. The morphology of various self-assembled structures of BSA in different concentration regimes of ILSs have been explored using confocal laser scanning microscopy (CLSM) and scanning electron microscopy. The structural variations in ILSs have been found to produce remarkable effect on the nature and morphology of self-assembled structures of BSA. The presence of nonfunctionalized [C12mim][Cl] IL at all investigated concentrations has led to the formation of unordered large self-assembled structures of BSA. On the other hand, in specific concentration regimes, ordered self-assembled structures such as long rods and right-handedly twisted helical amyloid fibers have been observed in the presence of functionalized [C12Amim][Cl] and [C12Emim][Cl] ILSs, respectively. The nature of the formed helical fibers as amyloid ones has been confirmed using FTIR spectroscopy. Steady-state fluorescence and circular dichroism (CD) spectroscopy have provided insights into folding and unfolding of BSA as fashioned by interactions with ILSs in different concentration regimes supporting the observations made from other studies.

  8. Evaluation of Ischemia-Modified Albumin, Malondialdehyde, and Advanced Oxidative Protein Products as Markers of Vascular Injury in Diabetic Nephropathy

    PubMed Central

    Ahmad, Afzal; Manjrekar, Poornima; Yadav, Charu; Agarwal, Ashish; Srikantiah, Rukmini Mysore; Hegde, Anupama

    2016-01-01

    AIM This study aimed at evaluation of ischemia-modified albumin (IMA), malondialdehyde (MDA), and advanced oxidative protein products (AOPP) as markers of vascular injury in diabetic nephropathy (DN) with derivation of cutoff values for the same. MATERIALS AND METHODS Study population comprised 60 diabetes patients and 30 controls, with diabetes patients further categorized into three groups based on urine albumin/creatinine ratio (UACR) of <30 mg/g (diabetes without microalbuminuria), 30–300 mg/g (early DN), and >300 mg/g of creatinine (overt DN). Serum IMA, MDA, and AOPP were estimated by enzyme-linked immunosorbent assay; HbA1c, serum creatinine, urine albumin, and urine creatinine were estimated using automated analyzers. Statistical analysis was done using analysis of variance, Pearson’s correlation coefficient, and receiver-operating characteristic curve. RESULTS A statistically significant difference was found in the levels of IMA among patients with early DN (154 ng/mL), diabetes without nephropathy (109.4 ng/mL), and healthy controls (45.7 ng/mL), with highest levels in early DN cases. Similar increase was seen in AOPP as well. A significant correlation was observed between IMA and UACR in diabetes without nephropathy (r = 0.448). CONCLUSION The present study postulates serum IMA as a novel biomarker for the assessment of disease progression in diabetes even before microalbuminuria, and a cutoff point ≥99 ng/mL can be used for detection of early DN. PMID:27158221

  9. Structure of Serum Albumin

    NASA Technical Reports Server (NTRS)

    Carter, Daniel C.; Ho, Joseph X.

    1994-01-01

    Because of its availability, low cost, stability, and unusual ligand-binding properties, serum albumin has been one of the mst extensively studied and applied proteins in biochemistry. However, as a protein, albumin is far from typical, and the widespread interest in and application of albumin have not been balanced by an understanding of its molecular structure. Indeed, for more than 30 years structural information was surmised based solely on techniques such as hydrodynamics, low-angle X-ray scattering, and predictive methods.

  10. Protein nanoparticle interaction: A spectrophotometric approach for adsorption kinetics and binding studies

    NASA Astrophysics Data System (ADS)

    Vaishanav, Sandeep K.; Chandraker, Kumudini; Korram, Jyoti; Nagwanshi, Rekha; Ghosh, Kallol K.; Satnami, Manmohan L.

    2016-08-01

    Investigating the protein nanoparticle interaction is crucial to understand how to control the biological interactions of nanoparticles. In this work, Model protein Bovine serum albumin (BSA) was used to evaluate the process of protein adsorption to the gold nanoparticles (GNPs) surface. The binding of a model protein (BSA) to GNPs was investigated through fluorescence quenching measurements. The strong affinities of BSA for GNPs were confirmed by the high value of binding constant (Ks) which was calculated to be 2.2 × 1011 L/mol. In this consequence, we also investigated the adsorption behavior of BSA on GNPs surface via UV-Vis spectroscopy. The effect of various operational parameters such as pH, contact time, initial BSA concentration, and temperature on adsorption of BSA was investigated using batch adsorption experiments. Kinetics of adsorption was found to follow the pseudo-second order rate equation. The suitability of Freundlich and Langmuir adsorption models to the equilibrium data was investigated. The equilibrium adsorption was well described by the Freundlich isotherm model. The maximum adsorption capacity for BSA adsorbed on GNPs was 58.71 mg/g and equilibrium constant was 0.0058 calculated by the Langmuir model at 298 K and pH = 11.0. Thermodynamic parameters showed that the adsorption of BSA onto GNPs was feasible, spontaneous, and exothermic.

  11. Determination of Protein by Fluorescence Enhancement of Curcumin in Lanthanum-Curcumin-Sodium Dodecyl Benzene Sulfonate-Protein System

    SciTech Connect

    Wang, Feng; Huang, Wei; Zhang, Yunfeng; Wang, Mingyin; Sun, Lina; Tang, Bo; Wang, Wei

    2011-01-01

    We found that the fluorescence intensity of the lanthanum (La(3+))-curcumin (CU) complex can be highly enhanced by proteins in the presence of sodium dodecyl benzene sulphonate (SDBS). Based on this finding, a new fluorimetric method for the determination of protein was developed. Under optimized conditions, the enhanced intensities of fluorescence are quantitatively in proportion to the concentrations of proteins in the range 0.0080-20.0 g mL(-1) for bovine serum albumin (BSA) and 0.00080-20.0 g mL(-1) for human serum albumin (HSA) with excitation of 425 nm, and 0.00020-20.0 g mL(-1) for bovine serum albumin (BSA) and 0.00080-20.0 g mL(-1)for human serum albumin (HSA) with excitation of 280 nm, while corresponding qualitative detection limits (S/N 3) are as low as 5.368, 0.573, 0.049, 0.562 g mL(-1), respectively. Study on reaction mechanism reveals that proteins can bind with La(3+), CU and SDBS through self-assembling function with electrostatic attraction, hydrogen bonding, hydrophobic interaction and van der Waals forces, etc. The proteins form a supermolecular association with multilayer structure, in which La(3+)-CU is clamped between BSA and SDBS. The unique high fluorescence enhancement of CU is resulted through synergic effects of favorable hydrophobic microenvironment provided by BSA and SDBS, and efficient intermolecular energy transfer among BSA, SDBS and CU. In energy transfer process, La(3+) plays a crucial role because it not only shortens the distance between SDBS and CU, but also acts as a "bridge" for transferring the energy from BSA to CU.

  12. Caveolae-mediated albumin transcytosis is enhanced in dengue-infected human endothelial cells: A model of vascular leakage in dengue hemorrhagic fever.

    PubMed

    Chanthick, Chanettee; Kanlaya, Rattiyaporn; Kiatbumrung, Rattanaporn; Pattanakitsakul, Sa-Nga; Thongboonkerd, Visith

    2016-01-01

    Vascular leakage is a life-threatening complication of dengue virus (DENV) infection. Previously, association between "paracellular" endothelial hyperpermeability and plasma leakage had been extensively investigated. However, whether "transcellular" endothelial leakage is involved in dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS) remained unknown. We thus investigated effects of DENV (serotype 2) infection on transcellular transport of albumin, the main oncotic plasma protein, through human endothelial cell monolayer by Western blotting, immunofluorescence staining, fluorescence imaging, and fluorometry. The data showed that Alexa488-conjugated bovine serum albumin (Alexa488-BSA) was detectable inside DENV2-infected cells and its level was progressively increased during 48-h post-infection. While paracellular transport could be excluded using FITC-conjugated dextran, Alexa488-BSA was progressively increased and decreased in lower and upper chambers of Transwell, respectively. Pretreatment with nystatin, an inhibitor of caveolae-dependent endocytic pathway, significantly decreased albumin internalization into the DENV2-infected cells, whereas inhibitors of other endocytic pathways showed no significant effects. Co-localization of the internalized Alexa488-BSA and caveolin-1 was also observed. Our findings indicate that DENV infection enhances caveolae-mediated albumin transcytosis through human endothelial cells that may ultimately induce plasma leakage from intravascular compartment. Further elucidation of this model in vivo may lead to effective prevention and better therapeutic outcome of DHF/DSS. PMID:27546060

  13. Caveolae-mediated albumin transcytosis is enhanced in dengue-infected human endothelial cells: A model of vascular leakage in dengue hemorrhagic fever

    PubMed Central

    Chanthick, Chanettee; Kanlaya, Rattiyaporn; Kiatbumrung, Rattanaporn; Pattanakitsakul, Sa-nga; Thongboonkerd, Visith

    2016-01-01

    Vascular leakage is a life-threatening complication of dengue virus (DENV) infection. Previously, association between “paracellular” endothelial hyperpermeability and plasma leakage had been extensively investigated. However, whether “transcellular” endothelial leakage is involved in dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS) remained unknown. We thus investigated effects of DENV (serotype 2) infection on transcellular transport of albumin, the main oncotic plasma protein, through human endothelial cell monolayer by Western blotting, immunofluorescence staining, fluorescence imaging, and fluorometry. The data showed that Alexa488-conjugated bovine serum albumin (Alexa488-BSA) was detectable inside DENV2-infected cells and its level was progressively increased during 48-h post-infection. While paracellular transport could be excluded using FITC-conjugated dextran, Alexa488-BSA was progressively increased and decreased in lower and upper chambers of Transwell, respectively. Pretreatment with nystatin, an inhibitor of caveolae-dependent endocytic pathway, significantly decreased albumin internalization into the DENV2-infected cells, whereas inhibitors of other endocytic pathways showed no significant effects. Co-localization of the internalized Alexa488-BSA and caveolin-1 was also observed. Our findings indicate that DENV infection enhances caveolae-mediated albumin transcytosis through human endothelial cells that may ultimately induce plasma leakage from intravascular compartment. Further elucidation of this model in vivo may lead to effective prevention and better therapeutic outcome of DHF/DSS. PMID:27546060

  14. Biodegradable synthetic polymer scaffolds for reinforcement of albumin protein solders used for laser-assisted tissue repair.

    PubMed

    Hoffman, Grant T; Soller, Eric C; McNally-Heintzelman, Karen M

    2002-01-01

    Laser tissue soldering has been investigated for several years by researchers in our laboratory as an alternative to conventional tissue fasteners, including sutures, staples and clips. Laser tissue soldering is a bonding technique in which protein solder is applied to the tissue surfaces to be joined, and laser energy is used to bond the solder to the tissue surfaces. Over the past four years we have been investigating the use of synthetic polymer membranes as a means for reinforcing the strength of tissue repairs formed using traditional laser tissue soldering techniques. The purpose of this study was to assess the influence of various processing parameters on the strength of tissue repairs formed using the reinforced solder. Biodegradable polymer membranes of specific porosity were fabricated by means of a solvent-casting and particulate-leaching technique, using three different poly(alpha ester)s: polyglycolic acid (PGA), polylactic acid (PLA) and poly(L-lactic-co-glycolic acid) (PLGA). In addition, several membranes were also prepared with poly(ethylene glycol) (PEG). The membranes were then doped with the traditional protein solder mixture of serum albumin and indocyanine green dye. Varied processing parameters included the polymer type, the PLGA copolymer blend ratio, the polymer/PEG blend ratio, the porosity of the polymer membrane and the initial albumin weight fraction. Variation of the polymer type had negligible effect on the strength of the repairs. Although it is known that alteration of the copolymer blend ratio of PLGA influences the degradation rate of the polymer, this variation also had no significant effect on the strength of the repairs formed. Increased membrane flexibility was observed when PEG was added during the casting stage. An increase in the porosity of the polymer membranes led to a subsequent increase in the final concentration of protein contained within the membranes, hence aiding in strengthening the resultant repairs. Likewise

  15. The conformation of serum albumin in solution: a combined phosphorescence depolarization-hydrodynamic modeling study.

    PubMed Central

    Ferrer, M L; Duchowicz, R; Carrasco, B; de la Torre, J G; Acuña, A U

    2001-01-01

    There is a striking disparity between the heart-shaped structure of human serum albumin (HSA) observed in single crystals and the elongated ellipsoid model used for decades to interpret the protein solution hydrodynamics at neutral pH. These two contrasting views could be reconciled if the protein were flexible enough to change its conformation in solution from that found in the crystal. To investigate this possibility we recorded the rotational motions in real time of an erythrosin-bovine serum albumin complex (Er-BSA) over an extended time range, using phosphorescence depolarization techniques. These measurements are consistent with the absence of independent motions of large protein segments in solution, in the time range from nanoseconds to fractions of milliseconds, and give a single rotational correlation time phi(BSA, 1 cP, 20 degrees C) = 40 +/- 2 ns. In addition, we report a detailed analysis of the protein hydrodynamics based on two bead-modeling methods. In the first, BSA was modeled as a triangular prismatic shell with optimized dimensions of 84 x 84 x 84 x 31.5 A, whereas in the second, the atomic-level structure of HSA obtained from crystallographic data was used to build a much more refined rough-shell model. In both cases, the predicted and experimental rotational diffusion rate and other hydrodynamic parameters were in good agreement. Therefore, the overall conformation in neutral solution of BSA, as of HSA, should be rigid, in the sense indicated above, and very similar to the heart-shaped structure observed in HSA crystals. PMID:11325741

  16. The protein structure determines the sensitizing capacity of Brazil nut 2S albumin (Ber e1) in a rat food allergy model

    PubMed Central

    2013-01-01

    It is not exactly known why certain food proteins are more likely to sensitize. One of the characteristics of most food allergens is that they are stable to the acidic and proteolytic conditions in the digestive tract. This property is thought to be a risk factor in allergic sensitization. The purpose of the present study was to investigate the contribution of the protein structure of 2S albumin (Ber e1), a major allergen from Brazil nut, on the sensitizing capacity in vivo using an oral Brown Norway rat food allergy model. Disulphide bridges of 2S albumin were reduced and alkylated resulting in loss of protein structure and an increased pepsin digestibility in vitro. Both native 2S albumin and reduced/alkylated 2S albumin were administered by daily gavage dosing (0.1 and 1 mg) to Brown Norway rats for 42 days. Intraperitoneal administration was used as a positive control. Sera were analysed by ELISA and passive cutaneous anaphylaxis. Oral exposure to native or reduced/alkylated 2S albumin resulted in specific IgG1 and IgG2a responses whereas only native 2S albumin induced specific IgE in this model, which was confirmed by passive cutaneous anaphylaxis. This study has shown that the disruption of the protein structure of Brazil nut 2S albumin decreased the sensitizing potential in a Brown Norway rat food allergy model, whereas the immunogenicity of 2S albumin remained preserved. This observation may open possibilities for developing immunotherapy for Brazil nut allergy. PMID:24180644

  17. The protein structure determines the sensitizing capacity of Brazil nut 2S albumin (Ber e1) in a rat food allergy model.

    PubMed

    Van Bilsen, Jolanda Hm; Knippels, Léon Mj; Penninks, André H; Nieuwenhuizen, Willem F; De Jongh, Harmen Hj; Koppelman, Stef J

    2013-11-04

    : It is not exactly known why certain food proteins are more likely to sensitize. One of the characteristics of most food allergens is that they are stable to the acidic and proteolytic conditions in the digestive tract. This property is thought to be a risk factor in allergic sensitization. The purpose of the present study was to investigate the contribution of the protein structure of 2S albumin (Ber e1), a major allergen from Brazil nut, on the sensitizing capacity in vivo using an oral Brown Norway rat food allergy model. Disulphide bridges of 2S albumin were reduced and alkylated resulting in loss of protein structure and an increased pepsin digestibility in vitro. Both native 2S albumin and reduced/alkylated 2S albumin were administered by daily gavage dosing (0.1 and 1 mg) to Brown Norway rats for 42 days. Intraperitoneal administration was used as a positive control. Sera were analysed by ELISA and passive cutaneous anaphylaxis. Oral exposure to native or reduced/alkylated 2S albumin resulted in specific IgG1 and IgG2a responses whereas only native 2S albumin induced specific IgE in this model, which was confirmed by passive cutaneous anaphylaxis. This study has shown that the disruption of the protein structure of Brazil nut 2S albumin decreased the sensitizing potential in a Brown Norway rat food allergy model, whereas the immunogenicity of 2S albumin remained preserved. This observation may open possibilities for developing immunotherapy for Brazil nut allergy.

  18. Adsorption of serum albumin to thin films of poly(lactide-co-glycolide).

    PubMed

    Butler, S M; Tracy, M A; Tilton, R D

    1999-04-19

    Protein adsorption has been implicated in the variability of drug release from biodegradable microspheres. We used optical reflectometry to measure the extent and kinetics of bovine serum albumin (BSA) adsorption to smooth spin-cast films prepared from two poly(lactide-co-glycolide) (PLG) samples that have different end-groups, one being a hydrophilic carboxylic end group and the other a hydrophobic ester end group. One of us has previously shown that these end-groups influence microsphere degradation (Tracy et al. , 1998, Factors affecting the degradation rate of poly(lactide-co-glycolide) microspheres in vivo and in vitro. Biomaterials: submitted for publication.). Both films were moderately hydrophobic, and their wettability was independent of the type of end-group. BSA adsorbed readily to both native PLG films, attaining as much as 50% surface coverage by area and was insensitive to the type of end-group. Aging the films in water for 24 h prior to BSA exposure decreased the hydrophobicity of the films and this in turn correlated with a significant decrease in the initial BSA adsorption rate. This was consistent with the often-observed trend that surface hydrophobicity favors protein adsorption. In spite of the lower adsorption affinity revealed by this decreased initial adsorption rate, the final adsorbed amounts on the aged films exceeded those attained on native films, presumably due to the increase in total surface area produced by partial PLG erosion.

  19. Elucidating the interaction of limonene with bovine serum albumin: a multi-technique approach.

    PubMed

    Chaturvedi, Sumit Kumar; Ahmad, Ejaz; Khan, Javed Masood; Alam, Parvez; Ishtikhar, Mohd; Khan, Rizwan Hasan

    2015-01-01

    The interaction of Bovine Serum Albumin (BSA) with limonene has been studied by UV-visible spectroscopy, fluorescence spectroscopy and molecular docking, and its effects on protein conformation, topology and stability were determined by Circular Dichroism (CD), Dynamic Light Scattering (DLS) and Differential Scanning Calorimetry (DSC). A gradual decrease in Stern-Volmer quenching constants with the increase in temperature showed the static mode of fluorescence quenching. The obtained binding constant (Kb) was ∼10(4) M(-1). The temperature dependent Kb, Gibbs free energy (ΔG), enthalpy (ΔH) and entropy (ΔS) changes were calculated, which revealed that the reaction is spontaneous and exothermic. The UV-visible spectra showed a change in the peaks within the aromatic region indicating hydrophobic interactions with Trp, Tyr and Phe in the protein. Moreover, limonene induced an increase in α-helical contents probably on the cost of random coils or/and β-sheets of BSA, as observed from the far-UV CD spectra. The topology of BSA in the presence of limonene was slightly altered, as obtained from DLS results. The stability was also enhanced as revealed through thermal denaturation study by DSC and CD. Molecular docking study depicted that limonene fits into the hydrophobic pocket close to Sudlow site I in domain IIA of BSA. The present study will be helpful in understanding the binding mechanism of limonene and associated stability and conformational changes.

  20. Adsorption of bovine serum albumin on silver surfaces enhances the release of silver at pH neutral conditions.

    PubMed

    Wang, X; Herting, G; Wallinder, I Odnevall; Blomberg, E

    2015-07-28

    Metallic biomaterials are widely used to replace and/or restore the function of damaged bodily parts. The use of silver as antibacterial coatings onto implants has recently gained large interest in medical applications. The extent of silver that can be released into different biological fluids from such coatings is, except for the surface characteristics of the coating, governed by parameters such as protein characteristics, adsorbed layer properties, formation of silver-protein complexes as well as concentrations of proteins in the solution. This study aims to relate the structure of adsorbed net negatively charged bovine serum albumin (BSA), which is the most abundant protein in serum, to the release of silver from metallic silver surfaces in order to elucidate if the net charge of the protein has any effect of the silver release. Simultaneous adsorption measurements were performed in real time on the very same surface using combined ellipsometry and quartz crystal microbalance with dissipation monitoring (QCM-D) measurements to provide a more comprehensive understanding on adsorption kinetics and layer structures. The amount of released silver into solution was measured by means of graphite furnace atomic absorption spectroscopy (GF-AAS). The structure of the adsorbed BSA layer largely influenced the amount of released silver, an enhancement that increased with BSA concentration. These observations are in complete contrast to the effect of net positively charged lysozyme (LSZ) adsorbed on silver, previously studied by the authors, for which a complete surface coverage suppressed the possibility for silver release. The underlying mechanisms behind the enhanced release of silver in the presence of BSA were mainly attributed to surface complexation between BSA and silver followed by an enhanced exchange rate of these surface complexes with BSA molecules in the solution, which in turn increase the amount of released silver in solution. PMID:26111372

  1. Multi-spectral characterization & effect of metal ions on the binding of bovine serum albumin upon interaction with a lincosamide antibiotic drug, clindamycin phosphate.

    PubMed

    Meti, Manjunath D; Byadagi, Kirthi S; Nandibewoor, Sharanappa T; Chimatadar, Shivamurti A

    2014-09-01

    The interaction of clindamycin phosphate (CP) with bovine serum albumin (BSA) is studied by using fluorescence spectra, UV-visible absorption, synchronous fluorescence spectra (SFS), CD, 3D fluorescence spectra and lifetime measurements under simulated physiological conditions. CP effectively quenched intrinsic fluorescence of BSA. The binding constants KA values are 2.540×10(5), 4.960×10(5), 7.207×10(5) L mol(-1), the number of binding sites n and corresponding thermodynamic parameters ΔG(o), ΔH(o) and ΔS(o) between CP and BSA were calculated at different temperatures. The interaction between CP and BSA occurs through dynamic quenching and the effect of CP on the conformation of BSA was also analyzed using SFS. The average binding distance r between the donor (BSA) and acceptor (CP) was determined based on Förster's theory. The results of fluorescence spectra, UV-vis absorption spectra and SFS show that the secondary structure of the protein has been changed in the presence of CP.

  2. A Spectroscopic Approach to Investigate the Molecular Interactions between the Newly Approved Irreversible ErbB blocker "Afatinib" and Bovine Serum Albumin

    PubMed Central

    2016-01-01

    The interaction of afatinib (AFB) with bovine serum albumin (BSA) was examined via fluorescence and UV-Vis spectroscopy. Spectrofluorimetric measurements revealed that AFB can strongly quench the BSA intrinsic fluorescence through producing a non-fluorescent complex. This quenching mechanism was thoroughly investigated with regard to the type of quenching, binding constant, number of binding locations and the fundamental thermodynamic parameters. Subsequently, the association constant of AFB with BSA was computed at three different temperatures and was found to range from 7.34 to 13.19 x105 L mol-1. Thermodynamic parameters calculations demonstrated a positive ΔSƟvalue with both negative ΔHϴand ΔGϴvalues for AFB–BSA complex, which in turn infers thata spontaneous binding is taking place with both electrostatic bonding and hydrophobic interactions participating in the binding of AFB and BSA. Similarly, the UV absorption spectra of AFB-BSA system were studied and confirmed the interaction. Conformational alteration of the protein upon binding to AFB was elaborated with the aid of three dimensional fluorescence measurements as well as synchronous fluorescence spectra. PMID:26751077

  3. A Spectroscopic Approach to Investigate the Molecular Interactions between the Newly Approved Irreversible ErbB blocker "Afatinib" and Bovine Serum Albumin.

    PubMed

    Alanazi, Amer M; Abdelhameed, Ali Saber

    2016-01-01

    The interaction of afatinib (AFB) with bovine serum albumin (BSA) was examined via fluorescence and UV-Vis spectroscopy. Spectrofluorimetric measurements revealed that AFB can strongly quench the BSA intrinsic fluorescence through producing a non-fluorescent complex. This quenching mechanism was thoroughly investigated with regard to the type of quenching, binding constant, number of binding locations and the fundamental thermodynamic parameters. Subsequently, the association constant of AFB with BSA was computed at three different temperatures and was found to range from 7.34 to 13.19 x10(5) L mol(-1). Thermodynamic parameters calculations demonstrated a positive ΔSƟ value with both negative ΔHϴ and ΔGϴ values for AFB-BSA complex, which in turn infers that a spontaneous binding is taking place with both electrostatic bonding and hydrophobic interactions participating in the binding of AFB and BSA. Similarly, the UV absorption spectra of AFB-BSA system were studied and confirmed the interaction. Conformational alteration of the protein upon binding to AFB was elaborated with the aid of three dimensional fluorescence measurements as well as synchronous fluorescence spectra. PMID:26751077

  4. Photodynamic performance of zinc phthalocyanine in HeLa cells: A comparison between DPCC liposomes and BSA as delivery systems.

    PubMed

    M Garcia, Angélica; de Alwis Weerasekera, Hasitha; Pitre, Spencer P; McNeill, Brian; Lissi, Eduardo; Edwards, Ana M; Alarcon, Emilio I

    2016-10-01

    Comparable intracellular concentrations (≈30pmol/10(6) cells) of bovine serum albumin-ZnPc (BSA) adduct outperformed dipalmitoyl-phosphatidyl-choline (DPPC) liposomes containing ZnPc at photodynamic-killing of human cervical cancer cells (HeLa) after only 15min of irradiation using red light (λ>620nm, 30W/cm(2)). This result could not be simply explained in terms of dye aggregation within the carrier, since in the liposomes the dye was considerably less aggregated than in bovine serum albumin, formulation that was capable to induce cell apoptosis upon red light exposure. Thus, using specific organelle staining, our cumulative data points towards intrinsic differences in intra-cellular localization depending on the cargo vehicle used, being ZnPc:BSA preferentially located in the near vicinity of the nucleus and in the Golgi structures, while the liposomal formulation ZnPc:DPPC was preferentially located in cellular membrane and cytoplasm. In addition to those differences, using real-time advanced fluorescence lifetime imaging of HeLa cells loaded with the photosensitizer contained in the different vehicles, we have found that only for the ZnPc:BSA formulation, there was no significant changes in the fluorescence lifetime of the photosensitizer inside the cells. This contrasts with the in situ≈two-fold reduction of the fluorescence lifetime measured for the liposomal ZnPc formulation. Those observations point towards the superiority of the protein to preserve dye aggregation, and its photochemical activity, post-cell uptake, demonstrating the pivotal role of the delivery vehicle at determining the ultimate fate of a photosensitizer. PMID:27614847

  5. Photodynamic performance of zinc phthalocyanine in HeLa cells: A comparison between DPCC liposomes and BSA as delivery systems.

    PubMed

    M Garcia, Angélica; de Alwis Weerasekera, Hasitha; Pitre, Spencer P; McNeill, Brian; Lissi, Eduardo; Edwards, Ana M; Alarcon, Emilio I

    2016-10-01

    Comparable intracellular concentrations (≈30pmol/10(6) cells) of bovine serum albumin-ZnPc (BSA) adduct outperformed dipalmitoyl-phosphatidyl-choline (DPPC) liposomes containing ZnPc at photodynamic-killing of human cervical cancer cells (HeLa) after only 15min of irradiation using red light (λ>620nm, 30W/cm(2)). This result could not be simply explained in terms of dye aggregation within the carrier, since in the liposomes the dye was considerably less aggregated than in bovine serum albumin, formulation that was capable to induce cell apoptosis upon red light exposure. Thus, using specific organelle staining, our cumulative data points towards intrinsic differences in intra-cellular localization depending on the cargo vehicle used, being ZnPc:BSA preferentially located in the near vicinity of the nucleus and in the Golgi structures, while the liposomal formulation ZnPc:DPPC was preferentially located in cellular membrane and cytoplasm. In addition to those differences, using real-time advanced fluorescence lifetime imaging of HeLa cells loaded with the photosensitizer contained in the different vehicles, we have found that only for the ZnPc:BSA formulation, there was no significant changes in the fluorescence lifetime of the photosensitizer inside the cells. This contrasts with the in situ≈two-fold reduction of the fluorescence lifetime measured for the liposomal ZnPc formulation. Those observations point towards the superiority of the protein to preserve dye aggregation, and its photochemical activity, post-cell uptake, demonstrating the pivotal role of the delivery vehicle at determining the ultimate fate of a photosensitizer.

  6. Structural studies on serum albumins under green light irradiation.

    PubMed

    Comorosan, Sorin; Polosan, Silviu; Popescu, Irinel; Ionescu, Elena; Mitrica, Radu; Cristache, Ligia; State, Alina Elena

    2010-10-01

    This paper presents two new experimental results: the protective effect of green light (GL) on ultraviolet (UV) denaturation of proteins, and the effect of GL on protein macromolecular structures. The protective effect of GL was revealed on two serum albumins, bovine (BSA) and human (HSA), and recorded by electrophoresis, absorption, and circular dichroism spectra. The effect of GL irradiation on protein structure was recorded by using fluorescence spectroscopy and electrophoresis. These new effects were modeled by quantum-chemistry computation using Gaussian 03 W, leading to good fit between theoretical and experimental absorption and circular dichroism spectra. A mechanism for these phenomena is suggested, based on a double-photon absorption process. This nonlinear effect may lead to generation of long-lived Rydberg macromolecular systems, capable of long-range interactions. These newly suggested systems, with macroscopic quantum coherence behaviors, may block the UV denaturation processes. PMID:20473754

  7. Probing the interaction of a new synthesized CdTe quantum dots with human serum albumin and bovine serum albumin by spectroscopic methods.

    PubMed

    Bardajee, Ghasem Rezanejade; Hooshyar, Zari

    2016-05-01

    A novel CdTe quantum dots (QDs) were prepared in aqueous phase via a facile method. At first, poly (acrylic amide) grafted onto sodium alginate (PAAm-g-SA) were successfully synthesized and then TGA capped CdTe QDs (CdTe-TGA QDs) were embed into it. The prepared CdTe-PAAm-g-SA QDs were optimized and characterized by transmission electron microscopy (TEM), thermo-gravimetric (TG) analysis, Fourier transform infrared (FT-IR), UV-vis and fluorescence spectroscopy. The characterization results indicated that CdTe-TGA QDs, with particles size of 2.90 nm, were uniformly dispersed on the chains of PAAm-g-SA biopolymer. CdTe-PAAm-g-SA QDs also exhibited excellent UV-vis absorption and high fluorescence intensity. To explore biological behavior of CdTe-PAAm-g-SA QDs, the interactions between CdTe-PAAm-g-SA QDs and human serum albumin (HSA) (or bovine serum albumin (BSA)) were investigated by cyclic voltammetry, FT-IR, UV-vis, and fluorescence spectroscopic. The results confirmed the formation of CdTe-PAAm-g-SA QDs-HSA (or BSA) complex with high binding affinities. The thermodynamic parameters (ΔG<0, ΔH<0 and ΔS<0) were indicated that binding reaction was spontaneous and van der Waals interactions and hydrogen-bond interactions played a major role in stabilizing the CdTe-PAAm-g-SA QDs-HSA (or BSA) complexes. The binding distance between CdTe-PAAm-g-SA QDs and HSA (or BSA)) was calculated about 1.37 nm and 1.27 nm, respectively, according to Forster non-radiative energy transfer theory (FRET). Analyzing FT-IR spectra showed that the formation of QDs-HSA and QDs-BSA complexes led to conformational changes of the HSA and BSA proteins. All these experimental results clarified the effective transportation and elimination of CdTe-PAAm-g-SA QDs in the body by binding to HSA and BSA, which could be a useful guideline for the estimation of QDs as a drug carrier.

  8. BSA-imprinted synthetic receptor for reversible template recognition.

    PubMed

    Wang, Huafang; He, Yunhua; He, Xiwen; Li, Wenyou; Chen, Langxing; Zhang, Yukui

    2009-06-01

    A novel approach to the manufacturing of protein-responsive imprints on a home-made chitosan substrate was established together with m-aminophenylboronic acid (APBA) as a functional monomer. The produced polymers were characterized using both (1) equilibrium adsorption assays and (2) high performance liquid chromatography analysis. Results confirmed that the synthesized BSA-MIP (molecularly imprinted polymer) has a high affinity towards its template compared to the determined control proteins. The produced BSA-MIP featured largely in its good adsorption reversibility, especially in competitive binding assays, which is of great biological significance in separations. Non-specific binding was reduced to almost zero in a BSA/BHb competitive binding event. An excellent HPLC profile of template recognition was found for BSA-MIP, even under harsh mobile phase conditions. In the present work, the adopted trapped-template-release method permits recovery of bound BSA [1]. The strategy of making an artificial protein-receptor with high adsorption affinity and reversibility is promising in on-line isolation of target protein from complicated biological environments.

  9. On the study of BSA-loaded calcium-deficient hydroxyapatite nano-carriers for controlled drug delivery.

    PubMed

    Liu, Tse-Ying; Chen, San-Yuan; Liu, Dean-Mo; Liou, Sz-Chian

    2005-09-20

    Calcium-deficient hydroxyapatite (CDHA) nano-crystals incorporated with bovine serum albumin (BSA) to form BSA-loaded nano-carriers were synthesized via both in-situ and ex-situ processes. Amount of BSA uptake by the CDHA nano-crystals and subsequent release behaviors of the BSA-loaded nano-carriers were investigated. The amount of BSA uptake by CDHA decreases with increasing pH but a larger amount was observed in the ex-situ compared to in-situ process above pH=8.0. The release profile showed a bursting behavior for the nano-carrier prepared via the ex-situ process, which is probably due to the desorption of BSA molecules. In contrast, for the sample synthesized via the in-situ process at a higher pH level, a slower release profile without bursting behavior due to the dissolution of the BSA-incorporated CDHA crystal is seen from high solution TEM that indicates different extent of interaction between BSA and CDHA. On the other hand, for the nano-carriers prepared via the same process at lower pH level, a two-stage release profile was detected. An initial bursting release is due to the desorption of BSA from the CDHA surface, followed by a slow release as a result of the dissolution of the BSA-incorporated nano-crystals along its c-axis direction.

  10. Evidence for Water-Tuned Structural Differences in Proteins: An Approach Emphasizing Variations in Local Hydrophilicity

    PubMed Central

    Hinderberger, Dariush

    2012-01-01

    We present experimental evidence for the significant effect that water can have on the functional structure of proteins in solution. Human (HSA) and Bovine Serum Albumin (BSA) have an amino acid sequence identity of 75.52% and are chosen as model proteins. We employ EPR-based nanoscale distance measurements using double electron-electron resonance (DEER) spectroscopy and both albumins loaded with long chain fatty acids (FAs) in solution to globally (yet indirectly) characterize the tertiary protein structures from the bound ligands’ points of view. The complete primary structures and crystal structures of HSA and as of recently also BSA are available. We complement the picture as we have recently determined the DEER-derived solution structure of HSA and here present the corresponding BSA solution structure. The characteristic asymmetric FA distribution in the crystal structure of HSA can surprisingly be observed by DEER in BSA in solution. This indicates that the BSA conformational ensemble in solution seems to be narrow and close to the crystal structure of HSA. In contrast, for HSA in solution a much more symmetric FA distribution was found. Thus, conformational adaptability and flexibility dominate in the HSA solution structure while BSA seems to lack these properties. We further show that differences in amino acid hydropathies of specific structural regions in both proteins can be used to correlate the observed difference in the global (tertiary) solution structures with the differences on the primary structure level. PMID:23049837

  11. Depolymerization of insulin amyloid fibrils by albumin-modified magnetic fluid

    NASA Astrophysics Data System (ADS)

    Siposova, Katarina; Kubovcikova, Martina; Bednarikova, Zuzana; Koneracka, Martina; Zavisova, Vlasta; Antosova, Andrea; Kopcansky, Peter; Daxnerova, Zuzana; Gazova, Zuzana

    2012-02-01

    Pathogenesis of amyloid-related diseases is associated with the presence of protein amyloid deposits. Insulin amyloids have been reported in a patient with diabetes undergoing treatment by injection of insulin and causes problems in the production and storage of this drug and in application of insulin pumps. We have studied the interference of insulin amyloid fibrils with a series of 18 albumin magnetic fluids (MFBSAs) consisting of magnetite nanoparticles modified by different amounts of bovine serum albumin (w/w BSA/Fe3O4 from 0.005 up to 15). We have found that MFBSAs are able to destroy amyloid fibrils in vitro. The extent of fibril depolymerization was affected by nanoparticle physical-chemical properties (hydrodynamic diameter, zeta potential and isoelectric point) determined by the BSA amount present in MFBSAs. The most effective were MFBSAs with lower BSA/Fe3O4 ratios (from 0.005 to 0.1) characteristic of about 90% depolymerizing activity. For the most active magnetic fluids (ratios 0.01 and 0.02) the DC50 values were determined in the range of low concentrations, indicating their ability to interfere with insulin fibrils at stoichiometric concentrations. We assume that the present findings represent a starting point for the application of the active MFBSAs as therapeutic agents targeting insulin amyloidosis.

  12. Comprehensive study on the structure of the BSA from extended-to aged form in wide (2-12) pH range.

    PubMed

    Varga, N; Hornok, V; Sebők, D; Dékány, I

    2016-07-01

    In this work we studied the structure of the bovine serum albumin (BSA) and the protein-ligand interactions since researchers prefer to use them as carriers in drug delivery systems. Systematic study (between pH 2-12, in double distilled water and physiological salt solution) was carried out to determine the changes in the secondary and the tertiary structures of the BSA, the apparent molecular weight (Mw), the size (dLS) and the electrokinetic potential (ζ). At pH 7, the BSA has higher stability in the absence (ζ=-69mV, dLS=2.2nm, A2=1.4×10(-3)mlmol/g(2)) than in the presence of salt solution (ζ=-2.4mV, dLS=5.3nm, A2=-3.2×10(-4)mlmol/g(2)). The Mw strongly depends on the pH and the ionic strength (at pH 3 in the absence of salt, the Mw is 54.6kDa while in the presence of salt is 114kDa) which determines the geometry of the protein. The protein-ligand interactions were characterized by fluorescence (FL) and isothermal microcalorimetry (ITC) methods; these independent techniques provided similar thermodynamic parameters such as the binding constant (K) and the Gibbs free energy (ΔG). PMID:26995614

  13. Comprehensive study on the structure of the BSA from extended-to aged form in wide (2-12) pH range.

    PubMed

    Varga, N; Hornok, V; Sebők, D; Dékány, I

    2016-07-01

    In this work we studied the structure of the bovine serum albumin (BSA) and the protein-ligand interactions since researchers prefer to use them as carriers in drug delivery systems. Systematic study (between pH 2-12, in double distilled water and physiological salt solution) was carried out to determine the changes in the secondary and the tertiary structures of the BSA, the apparent molecular weight (Mw), the size (dLS) and the electrokinetic potential (ζ). At pH 7, the BSA has higher stability in the absence (ζ=-69mV, dLS=2.2nm, A2=1.4×10(-3)mlmol/g(2)) than in the presence of salt solution (ζ=-2.4mV, dLS=5.3nm, A2=-3.2×10(-4)mlmol/g(2)). The Mw strongly depends on the pH and the ionic strength (at pH 3 in the absence of salt, the Mw is 54.6kDa while in the presence of salt is 114kDa) which determines the geometry of the protein. The protein-ligand interactions were characterized by fluorescence (FL) and isothermal microcalorimetry (ITC) methods; these independent techniques provided similar thermodynamic parameters such as the binding constant (K) and the Gibbs free energy (ΔG).

  14. Synthesis of bovine serum albumin-protected high fluorescence Pt16-nanoclusters and their application to detect sulfide ions in solutions

    NASA Astrophysics Data System (ADS)

    Xu, Na; Li, Hong-Wei; Yue, Yuan; Wu, Yuqing

    2016-10-01

    Highly fluorescent (quantum yield, QY = 17%) Pt16-nanoclusters (Pt16-NCs@BSA) have been prepared via a one-step ultrasonic-assistance method by using cheap and easily available ascorbic acid as reductant and bovine serum albumin (BSA) as a stabilizing agent in aqueous solution. The fluorescence properties of the Pt-NCs@BSA can be easily controlled by optimizing conditions, and the products are extremely stable and could be used for the detection of sulfide ions (S2-) in solutions as a specific luminescence sensor. The present synthesis method is performed in one step, being cost-effective with a particularly short reaction time, which could be extended to the synthesis of other kinds of protein-protected Pt-NCs.

  15. Synthesis of bovine serum albumin-protected high fluorescence Pt16-nanoclusters and their application to detect sulfide ions in solutions.

    PubMed

    Xu, Na; Li, Hong-Wei; Yue, Yuan; Wu, Yuqing

    2016-10-21

    Highly fluorescent (quantum yield, QY = 17%) Pt16-nanoclusters (Pt16-NCs@BSA) have been prepared via a one-step ultrasonic-assistance method by using cheap and easily available ascorbic acid as reductant and bovine serum albumin (BSA) as a stabilizing agent in aqueous solution. The fluorescence properties of the Pt-NCs@BSA can be easily controlled by optimizing conditions, and the products are extremely stable and could be used for the detection of sulfide ions (S(2-)) in solutions as a specific luminescence sensor. The present synthesis method is performed in one step, being cost-effective with a particularly short reaction time, which could be extended to the synthesis of other kinds of protein-protected Pt-NCs. PMID:27631174

  16. Synthesis of bovine serum albumin-protected high fluorescence Pt16-nanoclusters and their application to detect sulfide ions in solutions.

    PubMed

    Xu, Na; Li, Hong-Wei; Yue, Yuan; Wu, Yuqing

    2016-10-21

    Highly fluorescent (quantum yield, QY = 17%) Pt16-nanoclusters (Pt16-NCs@BSA) have been prepared via a one-step ultrasonic-assistance method by using cheap and easily available ascorbic acid as reductant and bovine serum albumin (BSA) as a stabilizing agent in aqueous solution. The fluorescence properties of the Pt-NCs@BSA can be easily controlled by optimizing conditions, and the products are extremely stable and could be used for the detection of sulfide ions (S(2-)) in solutions as a specific luminescence sensor. The present synthesis method is performed in one step, being cost-effective with a particularly short reaction time, which could be extended to the synthesis of other kinds of protein-protected Pt-NCs.

  17. NO forms an adduct with serum albumin that has endothelium-derived relaxing factor-like properties.

    PubMed Central

    Keaney, J F; Simon, D I; Stamler, J S; Jaraki, O; Scharfstein, J; Vita, J A; Loscalzo, J

    1993-01-01

    Recent evidence suggests that sulfhydryl species can react with oxides of nitrogen under physiologic conditions and thereby stabilize endothelium-derived relaxing factor (EDRF) activity, but the presence of a specific in vivo thiol carrier for nitric oxide (NO) remains controversial. The single free sulfhydryl of serum albumin is the most abundant thiol species in plasma (approximately 0.5 mM) and is particularly reactive towards NO. To examine the potential role of serum albumin in endogenous nitric oxide metabolism, we synthesized S-nitroso-BSA (S-NO-BSA), a model S-nitroso-protein, and examined its effects on platelet function and coronary and systemic vascular tone in 16 mongrel dogs. Intravenous bolus S-NO-BSA markedly reduced mean arterial pressure in a dose-dependent manner and proved seven and a half-fold less potent than intravenous nitroglycerin and 10-fold less potent than intravenous S-nitroso-cysteine (half-maximal response of 75 nmol/kg compared to 10 and 7.5 nmol/kg, respectively; P < 0.05); when given by intravenous infusion (half-maximal response = 10 nmol/kg per min), however, S-NO-BSA and nitroglycerin were equipotent. Intravenous bolus S-NO-BSA had a greater duration of action than either nitroglycerin or S-nitroso-cysteine and produced marked prolongation of the template bleeding time associated with dose-dependent inhibition of ex vivo platelet aggregation (half-maximal response approximately 70 nmol/kg). Intracoronary S-NO-BSA increased coronary blood flow (mean +/- SEM) less effectively than nitroprusside, acetylcholine, or S-nitroso-cysteine (165% +/- 24% vs. 315% +/- 82%, 483% +/- 55%, or 475% +/- 66%, respectively; P < 0.05) although with much longer duration of action. On a molar basis, S-nitroso-cysteine proved more effective than S-nitroso-BSA, nitroprusside, or acetylcholine as an epicardial coronary vasodilator. Thus, serum albumin reacts with oxides of nitrogen to form a stable S-nitroso-thiol with properties reminiscent of

  18. Impact by condensed tannins with different mean degrees of polymerization on protein precipitation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Condensed tannins (CTs) isolated from white clover (Trifolium repens) flowers and big trefoil (Lotus pedunculatus) leaves were evaluated in precipitation studies with bovine serum albumin (BSA), lysozyme (LYS), and alfalfa leaf protein (ALF). The CTs were of similar compositions, but differed in the...

  19. Effects of Gold Salt Speciation and Structure of Human and Bovine Serum Albumin on the Synthesis and Stability of Gold Nanostructures

    NASA Astrophysics Data System (ADS)

    Miranda, Érica; Tofanello, Aryane; Brito, Adrianne; Lopes, David; Giacomelli, Fernando; Albuquerque, Lindomar; Costa, Fanny; Ferreira, Fabio; Araujo-Chaves, Juliana; de Castro, Carlos; Nantes, Iseli

    2016-03-01

    The present study aimed to investigate the influence of albumin structure and gold speciation on the synthesis of gold nanoparticles (GNPs). The strategy of synthesis was the addition of HAuCl4 solutions at different pH values (3-12) to solutions of human and bovine serum albumins (HSA and BSA) at the same corresponding pH values. Different pH values influence the GNP synthesis due to gold speciation. Besides the inherent effect of pH on the native structure of albumins, the use N-ethylmaleimide (NEM)-treated and heat-denaturated forms of HSA and BSA provided additional insights about the influence of protein structure, net charge, and thiol group approachability on the GNP synthesis. NEM treatment, heating, and the extreme values of pH promoted loss of the native albumin structure. The formation of GNPs indicated by the appearance of surface plasmon resonance (SPR) bands became detectable from fifteen days of the synthesis processes that were carried out with native, NEM-treated and heat-denaturated forms of HSA and BSA, exclusively at pH 6 and 7. After two months of incubation, SPR band was also detected for all synthesis carried out at pH 8.0. The mean values of the hydrodynamic radius (RH) were 24 and 34 nm for GNPs synthesized with native HSA and BSA, respectively. X-ray diffraction (XRD) revealed crystallites of 13 nm. RH, XRD, and zeta potential values were consistent with GNP capping by the albumins. However, the GNPs produced with NEM-treated and heat-denaturated albumins exhibited loss of protein capping by lowering the ionic strength. This result suggests a significant contribution of non-electrostatic interactions of albumins with the GNP surface, in these conditions. The denaturation of proteins exposes hydrophobic groups to the solvent, and these groups could interact with the gold surface. In these conditions, the thiol blockage or oxidation, the latter probably favored upon heating, impaired the formation of a stable capping by thiol coordination

  20. Complexation of bovine serum albumin and sugar beet pectin: stabilising oil-in-water emulsions.

    PubMed

    Li, Xiangyang; Fang, Yapeng; Al-Assaf, Saphwan; Phillips, Glyn O; Jiang, Fatang

    2012-12-15

    In a previous study (Langmuir 28 (2012) 10164-10176.), we investigated the complexation of bovine serum albumin (BSA) with sugar beet pectin (SBP). A pH-composition phase diagram was established and structural transitions in relation to the phase diagram during complexation were identified. The present study examines the implications of these interactions on the emulsifying performance of BSA/SBP mixtures. Middle-chain triglycerides (MCTs) in water emulsions were prepared using conditions corresponding to different regions of the phase diagram. At high pHs and in the stable region of mixed individual soluble polymers where complexation is absent, there is no improved emulsifying performance, compared with the individual protein and polysaccharide. For these mixtures, the emulsion characteristics are controlled by the major component in the solutions, as determined by the competitive adsorption of the two components at the oil-water interface. At low pHs and low BSA/SBP ratios, and so mainly within the stable region of intramolecular soluble complexes, BSA/SBP mixtures greatly improve the stability of emulsions. Here, stabilisation is controlled by the cooperative adsorption of the two components at the oil-water interface. Through electrostatic complexation BSA promotes the adsorption of SBP on to interfaces to form a thick steric layer around emulsion droplets and thus providing better stability. At low pHs and high BSA/SBP ratios, that is, mainly within the unstable region of intermolecular insoluble complexes, emulsions prepared are extremely unstable due to bridging flocculation between emulsion droplets.

  1. Expanded and packed bed albumin adsorption on fluoride modified zirconia.

    PubMed

    Mullick, A; Griffith, C M; Flickinger, M C

    1998-11-01

    The expanded bed characteristics of 75-103microm fluoride-modified zirconia (FmZr) particles synthesized by a fed batch oil emulsion process were investigated. These particles are distinguished from commercially available expanded-bed adsorbents by virtue of their high density (2.8 g/cc) and the mixed mode protein retention mechanism which allows for the retention of both cationic and anionic proteins. The linear velocity versus bed porosity data agree with the Richardson-Zaki relationship with the terminal velocity in infinite medium of 2858.4 cm/h and a bed expansion index of 5.1. Residence time distribution (RTD) studies and bovine serum albumin (BSA) adsorption studies were performed as a function of the height of the settled bed to the column diameter (H:D) ratio and degree of bed expansion with superficial velocities of 440 to 870 cm/h. The settled bed, a 2x expanded bed, and a 3x expanded bed were studied for the H:D ratios of 1:1, 2:1, and 3:1. The dynamic binding capacity (DBC) at 5% breakthrough was low (2-8 mg BSA/mL settled bed) and was independent of the H:D ratio or the degree of bed expansion. The saturation DBC was 32.3 +/- 7.0 mg BSA/mL settled bed. The adsorption-desorption kinetics and intraparticle diffusion for protein adsorption on FmZr (38-75 micrometer) were investigated by studying the packed bed RTD and BSA adsorption as a function of temperature and flow rate. The data show that the adsorption-desorption kinetics along with intraparticle diffusion significantly influence protein adsorption on FmZr. Low residence times ( approximately 0.8 min) of BSA result in a DBC at 5% breakthrough which is 3.5-fold lower compared to that at 6-fold higher protein residence time. At low linear velocity (45 cm/h) the breakthrough curve is nearly symmetrical and becomes asymmetrical and more dispersed at higher linear velocity (270 cm/h) due to the influence of slow adsorption-desorption kinetics and intraparticle diffusion. Bioeng 60: 333-340, 1998. PMID

  2. Quantitative proteomic analysis of Burkholderia pseudomallei Bsa type III secretion system effectors using hypersecreting mutants.

    PubMed

    Vander Broek, Charles W; Chalmers, Kevin J; Stevens, Mark P; Stevens, Joanne M

    2015-04-01

    Burkholderia pseudomallei is an intracellular pathogen and the causative agent of melioidosis, a severe disease of humans and animals. One of the virulence factors critical for early stages of infection is the Burkholderia secretion apparatus (Bsa) Type 3 Secretion System (T3SS), a molecular syringe that injects bacterial proteins, called effectors, into eukaryotic cells where they subvert cellular functions to the benefit of the bacteria. Although the Bsa T3SS itself is known to be important for invasion, intracellular replication, and virulence, only a few genuine effector proteins have been identified and the complete repertoire of proteins secreted by the system has not yet been fully characterized. We constructed a mutant lacking bsaP, a homolog of the T3SS "gatekeeper" family of proteins that exert control over the timing and magnitude of effector protein secretion. Mutants lacking BsaP, or the T3SS translocon protein BipD, were observed to hypersecrete the known Bsa effector protein BopE, providing evidence of their role in post-translational control of the Bsa T3SS and representing key reagents for the identification of its secreted substrates. Isobaric Tags for Relative and Absolute Quantification (iTRAQ), a gel-free quantitative proteomics technique, was used to compare the secreted protein profiles of the Bsa T3SS hypersecreting mutants of B. pseudomallei with the isogenic parent strain and a bsaZ mutant incapable of effector protein secretion. Our study provides one of the most comprehensive core secretomes of B. pseudomallei described to date and identified 26 putative Bsa-dependent secreted proteins that may be considered candidate effectors. Two of these proteins, BprD and BapA, were validated as novel effector proteins secreted by the Bsa T3SS of B. pseudomallei.

  3. The antifungal properties of a 2S albumin-homologous protein from passion fruit seeds involve plasma membrane permeabilization and ultrastructural alterations in yeast cells.

    PubMed

    Agizzio, Ana Paula; Da Cunha, Maura; Carvalho, André O; Oliveira, Marco Antônio; Ribeiro, Suzanna F F; Gomes, Valdirene M

    2006-10-01

    Different types of antimicrobial proteins were purified from plant seeds, including chitinases, β-1,3-glucanases, defensins, thionins, lipid transfer proteins and 2S albumins. It has become clear that these groups of proteins play an important role in the protection of plants from microbial infection. Recent results from our laboratory have shown that the defense-related proteins from passion fruit seeds, named Pf1 and Pf2 (which show sequence homology with 2S albumins), inhibit fungal growth and glucose-stimulated acidification of the medium by Saccharomyces cerevisiae cells. The aim of this study was to determine whether 2S albumins from passion fruit seeds induce plasma membrane permeabilization and cause morphological alterations in yeast cells. Initially, we used an assay based on the uptake of SYTOX Green, an organic compound that fluoresces upon interaction with nucleic acids and penetrates cells with compromised plasma membranes, to investigate membrane permeabilization in S. cerevisiae cells. When viewed with a confocal laser microscope, S. cervisiae cells showed strong SYTOX Green fluorescence in the cytosol, especially in the nuclei. 2S albumins also inhibited glucose-stimulated acidification of the medium by S. cerevisiae cells, which indicates a probable impairment of fungal metabolism. The microscopical analysis of the yeast cells treated with 2S albumins demonstrated several morphological alterations in cell shape, cell surface, cell wall and bud formation, as well as in the organization of intracellular organelles. PMID:25193649

  4. Spectroscopic investigations of the interactions of tramadol hydrochloride and 5-azacytidine drugs with human serum albumin and human hemoglobin proteins.

    PubMed

    Tunç, Sibel; Cetinkaya, Ahmet; Duman, Osman

    2013-03-01

    The interactions of tramadol hydrochloride (THC) and 5-azacytidine (AZA) drugs with human serum albumin (HSA) and human hemoglobin (HMG) proteins were investigated by fluorescence, UV absorption and circular dichroism (CD) spectroscopy at pH 7.4 and different temperatures. The UV absorption spectra and the fluorescence quenching of HSA and HMG proteins indicated the formation of HSA-THC and HMG-THC complexes via static quenching mechanism. AZA did not interact with HSA and HMG proteins. It was found that the formation of HMG-THC complex was stronger than that of HSA-THC complex. The stability of HSA-THC and HMG-THC complexes decreased with increasing temperature. The number of binding site was found as one for HSA-THC and HMG-THC systems. Negative enthalpy change (ΔH) and Gibbs free energy change (ΔG) and positive entropy change (ΔS) values were obtained for these systems. The binding of THC-HSA and HMG proteins was spontaneous and exothermic. In addition, electrostatic interactions between protein and drug molecules played an important role in the binding processes. The results of CD analysis revealed that the addition of THC led to a significant conformational change in the secondary structure of HSA protein, on the contrary to HMG protein. PMID:23428887

  5. Albumin-Mediated Biomineralization of Paramagnetic NIR Ag2S QDs for Tiny Tumor Bimodal Targeted Imaging in Vivo.

    PubMed

    Zhang, Jing; Hao, Guangyu; Yao, Chenfei; Yu, Jiani; Wang, Jun; Yang, Weitao; Hu, Chunhong; Zhang, Bingbo

    2016-07-01

    Bimodal imaging has captured increasing interests due to its complementary characteristics of two kinds of imaging modalities. Among the various dual-modal imaging techniques, MR/fluorescence imaging has been widely studied owing to its high 3D resolution and sensitivity. There is, however, still a strong demand to construct biocompatible MR/fluorescence contrast agents with near-infrared (NIR) fluorescent emissions and high relaxivities. In this study, BSA-DTPA(Gd) derived from bovine serum albumin (BSA) as a novel kind of biotemplate is employed for biomineralization of paramagnetic NIR Ag2S quantum dots (denoted as Ag2S@BSA-DTPA(Gd) pQDs). This synthetic strategy is found to be bioinspired, environmentally benign, and straightforward. The obtained Ag2S@BSA-DTPA(Gd) pQDs have fine sizes (ca. 6 nm) and good colloidal stability. They exhibit unabated NIR fluorescent emission (ca. 790 nm) as well as high longitudinal relaxivity (r1 = 12.6 mM(-1) s(-1)) compared to that of commercial Magnevist (r1 = 3.13 mM(-1) s(-1)). In vivo tumor-bearing MR and fluorescence imaging both demonstrate that Ag2S@BSA-DTPA(Gd) pQDs have pronounced tiny tumor targeting capability. In vitro and in vivo toxicity study show Ag2S@BSA-DTPA(Gd) pQDs are biocompatible. Also, biodistribution analysis indicates they can be cleared from body mainly via liver metabolism. This protein-mediated biomineralized Ag2S@BSA-DTPA(Gd) pQDs presents great potential as a novel bimodal imaging contrast agent for tiny tumor diagnosis. PMID:27300300

  6. Kinetics of Thermal Denaturation and Aggregation of Bovine Serum Albumin

    PubMed Central

    Borzova, Vera A.; Markossian, Kira A.; Chebotareva, Natalia A.; Kleymenov, Sergey Yu.; Poliansky, Nikolay B.; Muranov, Konstantin O.; Stein-Margolina, Vita A.; Shubin, Vladimir V.; Markov, Denis I.; Kurganov, Boris I.

    2016-01-01

    Thermal aggregation of bovine serum albumin (BSA) has been studied using dynamic light scattering, asymmetric flow field-flow fractionation and analytical ultracentrifugation. The studies were carried out at fixed temperatures (60°C, 65°C, 70°C and 80°C) in 0.1 M phosphate buffer, pH 7.0, at BSA concentration of 1 mg/ml. Thermal denaturation of the protein was studied by differential scanning calorimetry. Analysis of the experimental data shows that at 65°C the stage of protein unfolding and individual stages of protein aggregation are markedly separated in time. This circumstance allowed us to propose the following mechanism of thermal aggregation of BSA. Protein unfolding results in the formation of two forms of the non-native protein with different propensity to aggregation. One of the forms (highly reactive unfolded form, Uhr) is characterized by a high rate of aggregation. Aggregation of Uhr leads to the formation of primary aggregates with the hydrodynamic radius (Rh,1) of 10.3 nm. The second form (low reactive unfolded form, Ulr) participates in the aggregation process by its attachment to the primary aggregates produced by the Uhr form and possesses ability for self-aggregation with formation of stable small-sized aggregates (Ast). At complete exhaustion of Ulr, secondary aggregates with the hydrodynamic radius (Rh,2) of 12.8 nm are formed. At 60°C the rates of unfolding and aggregation are commensurate, at 70°C the rates of formation of the primary and secondary aggregates are commensurate, at 80°C the registration of the initial stages of aggregation is complicated by formation of large-sized aggregates. PMID:27101281

  7. Insights into the interactions between tetracycline, its degradation products and bovine serum albumin.

    PubMed

    Tong, Xingyu; Mao, Manfei; Xie, Jingqian; Zhang, Kefeng; Xu, Dongmei

    2016-01-01

    Tetracyclines (TCs) are the most widely used antibiotics in the world. Because antibiotics have low bioavailability and are difficult to completely remove using current sewage treatment facilities, residual TCs and their degradation products in the environment, animal and plant foodstuffs and personal care products may enter the body through the food chain, thus causing unpredictable effects on human health. We studied bovine serum albumin (BSA) (a functional protein) as a target of tetracycline-induced toxicity by examining its interactions with TC, anhydrotetracycline (ATC) and epitetracycline (ETC), based on a fluorescence spectroscopy and molecular docking method under simulated physiological conditions. The interaction mechanism was elucidated at the molecular level. The results show that TC, ATC and ETC bind at site II of BSA and interact mainly through hydrogen bonding interactions and van der Waals interactions. The binding affinities can be ranked in the order ATC > TC > ETC. PMID:27462521

  8. Influence of metal ions on phosphatidylcholine bovine serum albumin model membrane, an FTIR study

    NASA Astrophysics Data System (ADS)

    Wang, Fan; Yang, Zhanlan; Zhou, Yong; Weng, Shifu; Zhang, Li; Wu, Jinguang

    2006-08-01

    FTIR spectroscopy was used to study the interaction of K +, Ca 2+ and Eu 3+ ions and the Phosphatidylcholine (PC)-bovine serum albumin (BSA) complex. First, a PC-BSA interaction system was constructed. The analytical results of transmission electron microscope (TEM), quasi-elastic light scattering (QELS) techniques and FTIR-ATR spectroscopy indicated that PC molecules interacted with BSA in aqueous solutions. However, IR inspection was limited for aqueous solutions. Solid experimental condition was then employed, and FTIR spectra showed that the PC and BSA molecules incorporated with each other, which could represent their interactions in solutions. Then, the influence of metal ions on PC-BSA system was studied in solid experimental conditions, and FTIR spectroscopy was used in this study. The spectral results showed that: (1) K +, Ca 2+ and Eu 3+ ions all decreased the rigidities of acyl chains of PC in PC-BSA systems. (2) The interactions between Ca 2+, Eu 3+ ions and the hydrophilic phosphate ester and carbonyl ester groups of PC were stronger than that of K + ions, while the influent modes of Ca 2+ and Eu 3+ ions on these regions were different. (3) When the relative molar content of Eu 3+ ions to PC ( Ri/p) reached 2, the coordination effect between Eu 3+ ions and PO2- groups of PC was saturated. (4) The addition of these ions increased the content of α-helix structures of BSA, and decreased the content of β-turn structures. By comparing these results with the interactions of K +, Ca 2+, Eu 3+ ions with phospholipid system in the absence of protein, some special characters were discovered in the acyl regions of PC, while their interactions results with the hydrophilic regions of PC were alike. It might be interpreted that these metal ions influenced the acyl chains of PC mediated from BSA molecules, and coordinated directly with the hydrophilic regions of PC. As for biological membrane was a system included both phospholipid and proteins, these characters

  9. The metallomics approach: use of Fe(II) and Cu(II) footprinting to examine metal binding sites on serum albumins.

    PubMed

    Duff, Michael R; Kumar, Challa V

    2009-11-01

    Metal binding to serum albumins is examined by oxidative protein-cleavage chemistry, and relative affinities of multiple metal ions to particular sites on these proteins were identified using a fast and reliable chemical footprinting approach. Fe(ii) and Cu(ii), for example, mediate protein cleavage at their respective binding sites on serum albumins, in the presence of hydrogen peroxide and ascorbate. This metal-mediated protein-cleavge reaction is used to evaluate the binding of metal ions, Na(+), Mg(2+), Ca(2+), Al(3+), Cr(3+), Mn(2+), Co(2+), Ni(2+), Zn(2+), Cd(2+), Hg(2+), Pb(2+), and Ce(3+) to albumins, and the relative affinities (selectivities) of the metal ions are rapidly evaluated by examining the extent of inhibition of protein cleavage. Four distinct systems Fe(II)/BSA, Cu(II)/BSA, Fe(II)/HSA and Cu(II)/HSA are examined using the above strategy. This metallomics approach is novel, even though the cleavage of serum albumins by Fe(II)/Cu(II) has been reported previously by this laboratory and many others. The protein cleavage products were analyzed by SDS PAGE, and the intensities of the product bands quantified to evaluate the extent of inhibition of the cleavage and thereby evaluate the relative binding affinities of specific metal ions to particular sites on albumins. The data show that Co(II) and Cr(III) showed the highest degree of inhibition, across the table, followed by Mn(II) and Ce(III). Alakali metal ions and alkaline earth metal ions showed very poor affinity for these metal sites on albumins. Thus, metal binding profiles for particular sites on proteins can be obtained quickly and accurately, using the metallomics approach.

  10. Protein fouling in microfiltration: deposition mechanism as a function of pressure for different pH.

    PubMed

    Velasco, C; Ouammou, M; Calvo, J I; Hernández, A

    2003-10-01

    The influence of applied pressure on the fouling mechanism during bovine serum albumin (BSA) dead-end microfiltration (MF) has been investigated for a polyethersulfone acidic negatively charged membrane (ICE-450) from Pall Co. BSA solutions at pH values of 4, 5 (almost equal to the protein isoelectric point, IEP), and 6 were microfiltered through the membrane at different applied transmembrane pressures. Results have been analyzed in terms of the usual blocking filtration laws and a substantial change in the fouling mechanism was observed as the pressure was increased, this change can be related to the specific membrane-protein and protein-protein interactions. PMID:12957593

  11. Protein fouling in microfiltration: deposition mechanism as a function of pressure for different pH.

    PubMed

    Velasco, C; Ouammou, M; Calvo, J I; Hernández, A

    2003-10-01

    The influence of applied pressure on the fouling mechanism during bovine serum albumin (BSA) dead-end microfiltration (MF) has been investigated for a polyethersulfone acidic negatively charged membrane (ICE-450) from Pall Co. BSA solutions at pH values of 4, 5 (almost equal to the protein isoelectric point, IEP), and 6 were microfiltered through the membrane at different applied transmembrane pressures. Results have been analyzed in terms of the usual blocking filtration laws and a substantial change in the fouling mechanism was observed as the pressure was increased, this change can be related to the specific membrane-protein and protein-protein interactions.

  12. Controlling the taste receptor accessible structure of rebaudioside A via binding to bovine serum albumin.

    PubMed

    Mudgal, Samriddh; Keresztes, Ivan; Feigenson, Gerald W; Rizvi, S S H

    2016-04-15

    We illustrate a method that uses bovine serum albumin (BSA) to control the receptor-accessible part of rebaudioside A (Reb A). The critical micelle concentration (CMC) of Reb A was found to be 4.5 mM and 5 mM at pH 3 and 6.7 respectively. NMR studies show that below its CMC, Reb A binds weakly to BSA to generate a Reb A-protein complex ("RPC"), which is only modestly stable under varying conditions of pH (3.0-6.7) and temperature (4-40°C) with its binding affinities determined to be in the range of 5-280 mM. Furthermore, saturation transfer difference (STD) NMR experiments confirm that the RPC has fast exchange of the bitterness-instigating diterpene of Reb A into the binding sites of BSA. Our method can be used to alter the strength of Reb A-receptor interaction, as a result of binding of Reb A to BSA, which may ultimately lead to moderation of its taste.

  13. Controlling the taste receptor accessible structure of rebaudioside A via binding to bovine serum albumin.

    PubMed

    Mudgal, Samriddh; Keresztes, Ivan; Feigenson, Gerald W; Rizvi, S S H

    2016-04-15

    We illustrate a method that uses bovine serum albumin (BSA) to control the receptor-accessible part of rebaudioside A (Reb A). The critical micelle concentration (CMC) of Reb A was found to be 4.5 mM and 5 mM at pH 3 and 6.7 respectively. NMR studies show that below its CMC, Reb A binds weakly to BSA to generate a Reb A-protein complex ("RPC"), which is only modestly stable under varying conditions of pH (3.0-6.7) and temperature (4-40°C) with its binding affinities determined to be in the range of 5-280 mM. Furthermore, saturation transfer difference (STD) NMR experiments confirm that the RPC has fast exchange of the bitterness-instigating diterpene of Reb A into the binding sites of BSA. Our method can be used to alter the strength of Reb A-receptor interaction, as a result of binding of Reb A to BSA, which may ultimately lead to moderation of its taste. PMID:26616927

  14. Conjugation of bovine serum albumin and glucose under combined high pressure and heat.

    PubMed

    Buckow, Roman; Wendorff, Johannes; Hemar, Yacine

    2011-04-27

    The effect of combined heat and pressure on the Maillard reaction between bovine serum albumin (BSA) and glucose was investigated. The effects in the range of 60-132 °C and at 0.1-600 MPa on the lysine availability of BSA were investigated at isothermal/isobaric conditions. The kinetic results showed that the protein-sugar conjugation rate increased with increasing temperature, whereas it decreased with increasing pressure. The reaction followed 1.4th order kinetics at most conditions investigated. A mathematical model describing BSA-glucose conjugation kinetics as a function of pressure and temperature is proposed. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry were used to verify BSA-glucose conjugation and to identify the glucosylated sites. These indicated that the application of combined high pressure and high temperature resulted in significant differences in the progression of the Maillard reaction as compared to heat treatments at atmospheric pressure. PMID:21395313

  15. Moringa oleifera aqueous leaf extract inhibits reducing monosaccharide-induced protein glycation and oxidation of bovine serum albumin.

    PubMed

    Nunthanawanich, Pornpimon; Sompong, Weerachat; Sirikwanpong, Sukrit; Mäkynen, Kittana; Adisakwattana, Sirichai; Dahlan, Winai; Ngamukote, Sathaporn

    2016-01-01

    Advanced glycation end products (AGEs) play an important factor for pathophysiology of diabetes and its complications. Moringa oleifera is one of the medicinal plants that have anti-hyperglycemic activity. However, anti-glycation property of Moringa oleifera leaf extract on the different types of reducing monosaccharides-induced protein glycation has not been investigated. Therefore, the aim of this study was to examine the protective effect of Moringa oleifera aqueous leaf extract (MOE) on reducing sugars-induced protein glycation and protein oxidation. Total phenolic content of MOE was measured using the Folin-Ciocalteu method. Bovine serum albumin was incubated with 0.5 M of reducing sugars (glucose or fructose) with or without MOE (0.5-2.0 mg/mL) for 1, 2, 3 and 4 weeks. The results found that total phenolic content was 38.56 ± 1.50 mg gallic acid equivalents/g dry extract. The formation of fluorescent and non-fluorescent AGEs [N (ε)-(carboxymethyl) lysine (CML)] and the level of fructosamine were determined to indicate protein glycation, whereas the level of protein carbonyl content and thiol group were examined for protein oxidation. MOE (0.5-2.0 mg/mL) significantly inhibited the formation of fluorescent, N (ε)-CML and markedly decreased fructosamine level (P < 0.05). Moreover, MOE significantly prevented protein oxidation manifested by reducing protein carbonyl and the depletion of protein thiol in a dose-dependent manner (P < 0.05). Thus, the findings indicated that polyphenols containing in MOE have high potential for decreasing protein glycation and protein oxidation that may delay or prevent AGE-related diabetic complications. PMID:27468399

  16. Moringa oleifera aqueous leaf extract inhibits reducing monosaccharide-induced protein glycation and oxidation of bovine serum albumin.

    PubMed

    Nunthanawanich, Pornpimon; Sompong, Weerachat; Sirikwanpong, Sukrit; Mäkynen, Kittana; Adisakwattana, Sirichai; Dahlan, Winai; Ngamukote, Sathaporn

    2016-01-01

    Advanced glycation end products (AGEs) play an important factor for pathophysiology of diabetes and its complications. Moringa oleifera is one of the medicinal plants that have anti-hyperglycemic activity. However, anti-glycation property of Moringa oleifera leaf extract on the different types of reducing monosaccharides-induced protein glycation has not been investigated. Therefore, the aim of this study was to examine the protective effect of Moringa oleifera aqueous leaf extract (MOE) on reducing sugars-induced protein glycation and protein oxidation. Total phenolic content of MOE was measured using the Folin-Ciocalteu method. Bovine serum albumin was incubated with 0.5 M of reducing sugars (glucose or fructose) with or without MOE (0.5-2.0 mg/mL) for 1, 2, 3 and 4 weeks. The results found that total phenolic content was 38.56 ± 1.50 mg gallic acid equivalents/g dry extract. The formation of fluorescent and non-fluorescent AGEs [N (ε)-(carboxymethyl) lysine (CML)] and the level of fructosamine were determined to indicate protein glycation, whereas the level of protein carbonyl content and thiol group were examined for protein oxidation. MOE (0.5-2.0 mg/mL) significantly inhibited the formation of fluorescent, N (ε)-CML and markedly decreased fructosamine level (P < 0.05). Moreover, MOE significantly prevented protein oxidation manifested by reducing protein carbonyl and the depletion of protein thiol in a dose-dependent manner (P < 0.05). Thus, the findings indicated that polyphenols containing in MOE have high potential for decreasing protein glycation and protein oxidation that may delay or prevent AGE-related diabetic complications.

  17. Conjugates of folic acids with BSA-coated quantum dots for cancer cell targeting and imaging by single-photon and two-photon excitation.

    PubMed

    Meng, He; Chen, Ji-Yao; Mi, Lan; Wang, Pei-Nan; Ge, Mei-Ying; Yue, Yang; Dai, Ning

    2011-01-01

    Bovine serum albumin (BSA)-coated CdTe/ZnS quantum dots (BSA-QDs) were selected to conjugate with folic acid (FA), forming FA-BSA-QDs. This study aims to develop these small FA-BSA-QDs (less than 10 nm) for the diagnosis of cancers in which the FA receptor (FR) is overexpressed. The enhancement of cellular uptake in FR-positive human nasopharyngeal carcinoma cells (KB cells) for FA-BSA-QDs was found by means of confocal fluorescence microscopy under single-photon and two-photon excitation. The uptake enhancement for FA-BSA-QDs was further evaluated by flow-cytometric analysis in 10(4) KB cells, and was about 3 times higher than for BSA-QDs on average. The uptake enhancement was suppressed when KB cells had been pretreated with excess FA, reflecting that the enhancement was mediated by the association of FR at cell membranes with FA-BSA-QDs. When human embryonic kidney cells (293T) (FR-negative cells) and KB cells, respectively, were incubated with FA-BSA-QDs (1 μM) for 40 min, the FA-BSA-QD uptake by 293T cells was much weaker than that by KB cells, demonstrating that FA-BSA-QDs could undergo preferential binding on FR-positive cancer cells. These characteristics suggest that FA-BSA-QDs are potential candidates for cancer diagnosis.

  18. Influence of surface charge on the rate, extent, and structure of adsorbed Bovine Serum Albumin to gold electrodes.

    PubMed

    Beykal, Burcu; Herzberg, Moshe; Oren, Yoram; Mauter, Meagan S

    2015-12-15

    The objective of this work is to investigate the rate, extent, and structure of amphoteric proteins with charged solid surfaces over a range of applied potentials and surface charges. We use Electrochemical Quartz Crystal Microbalance with Dissipation Monitoring (E-QCM-D) to investigate the adsorption of amphoteric Bovine Serum Albumin (BSA) to a gold electrode while systematically varying the surface charge on the adsorbate and adsorbent by manipulating pH and applied potential, respectively. We also perform cyclic voltammetry-E-QCM-D on an adsorbed layer of BSA to elucidate conformational changes in response to varied applied potentials. We confirm previous results demonstrating that increasing magnitude of applied potential on the gold electrode is positively correlated with increasing mass adsorption when the protein and the surface are oppositely charged. On the other hand, we find that the rate of BSA adsorption is not governed by simple electrostatics, but instead depends on solution pH, an observation not well documented in the literature. Cyclic voltammetry with simultaneous E-QCM-D measurements suggest that BSA protein undergoes a conformational change as the surface potential varies.

  19. Interactions of two food colourants with BSA: Analysis by Debye-Hückel theory.

    PubMed

    Li, Tian; Cheng, Zhengjun; Cao, Lijun; Jiang, Xiaohui; Fan, Lei

    2016-11-15

    We have focused on exploring pH- and ionic strength-modulated binding of acid red 1 (AR1) and acid green 50 (AG50) with bovine serum albumin (BSA) by fluorescence, UV-vis absorption and FTIR spectra. The results implied that the quenching mechanism of BSA-AR1/AG50 system was a static quenching, electrostatic force dominated the formation of BSA-AR1/AG50 complex, and the binding affinity of AR1 was greater than that of AG50 on the subdomain IIA of BSA. Moreover, their true thermodynamic binding constant (Keq), true free energy change (ΔG(0)I→0), and effective charge (ZP) in the anion receptor pocket of BSA were calculated using Debye-Hückel limiting law. The local charge bound by AR1/AG50 rather than the overall or surface charge of BSA played a key role in determining their interaction strength. Besides, the thermal and structural stabilization of BSA was discussed by analyzing the changes of Tm and Hurea without/with the addition of AR1/AG50, respectively.

  20. BSA-templated MnO2 nanoparticles as both peroxidase and oxidase mimics.

    PubMed

    Liu, Xing; Wang, Qi; Zhao, Huihui; Zhang, Lichun; Su, Yingying; Lv, Yi

    2012-10-01

    Inorganic nanomaterials that mimic enzymes are fascinating as they potentially have improved properties relative to native enzymes, such as greater resistance to extremes of pH and temperature and lower sensitivity to proteases. Although many artificial enzymes have been investigated, searching for highly-efficient and stable catalysts is still of great interest. In this paper, we first demonstrated that bovine serum albumin (BSA)-stabilized MnO(2) nanoparticles (NPs) exhibited highly peroxidase-, oxidase-, and catalase-like activities. The activities of the BSA-MnO(2) NPs were evaluated using the typical horseradish peroxidase (HRP) substrates o-phenylenediamine (OPD) and 3,3',5,5'-tetramethylbenzidine (TMB) in the presence of either hydrogen peroxide or dissolved oxygen. These small-sized BSA-MnO(2) NPs with good dispersion, solubility and biocompatibility exhibited typical Michaelis-Menten kinetics and high affinity for H(2)O(2), OPD and TMB, indicating that BSA-MnO(2) NPs can be used as satisfactory enzyme mimics. Based on these findings, BSA-MnO(2) NPs were used as colorimetric immunoassay tags for the detection of goat anti-human IgG in place of HRP. The colorimetric immunoassay using BSA-MnO(2) NPs has the advantages of being fast, robust, inexpensive, easily prepared and with no HRP and H(2)O(2) being needed. These water-soluble BSA-MnO(2) NPs may have promising potential applications in biotechnology, bioassays, and biomedicine.

  1. Interactions of two food colourants with BSA: Analysis by Debye-Hückel theory.

    PubMed

    Li, Tian; Cheng, Zhengjun; Cao, Lijun; Jiang, Xiaohui; Fan, Lei

    2016-11-15

    We have focused on exploring pH- and ionic strength-modulated binding of acid red 1 (AR1) and acid green 50 (AG50) with bovine serum albumin (BSA) by fluorescence, UV-vis absorption and FTIR spectra. The results implied that the quenching mechanism of BSA-AR1/AG50 system was a static quenching, electrostatic force dominated the formation of BSA-AR1/AG50 complex, and the binding affinity of AR1 was greater than that of AG50 on the subdomain IIA of BSA. Moreover, their true thermodynamic binding constant (Keq), true free energy change (ΔG(0)I→0), and effective charge (ZP) in the anion receptor pocket of BSA were calculated using Debye-Hückel limiting law. The local charge bound by AR1/AG50 rather than the overall or surface charge of BSA played a key role in determining their interaction strength. Besides, the thermal and structural stabilization of BSA was discussed by analyzing the changes of Tm and Hurea without/with the addition of AR1/AG50, respectively. PMID:27283623

  2. The role of ribosylated-BSA in regulating PC12 cell viability.

    PubMed

    Kuo, Tsun-Yung; Huang, Chuen-Lin; Yang, Jung-Mou; Huang, Wei-Jung; Huang, Nai-Kuei; Chen, Yue-Wen; Lin, Ren-Jye; Yang, Ying-Chen

    2012-08-01

    Glycation, one of the post-translational modifications, is known to influence protein structure and biological function. Advanced glycation end products (AGEs) have been shown to cause pathologies of diabetes. Glycation levels in patients with Alzheimer's disease (AD) are higher than in normal people. However, whether the glycation of susceptible proteins is a triggering event for cell damage or simply a result remains to be elucidated. In this study, we demonstrated that ribose-conjugated BSA (Rib-BSA) directly induces PC12 cell death in a dose- and time-dependent manner. The IC(50) is 4.6 μM. Unlike glucose-incubated BSA, Rib-BSA rapidly forms cytotoxic AGEs. PC12 is vulnerable to Rib-BSA. However, fructose can induce AGE formation, although no effect on cell survival was observed. This effect of Rib-BSA is reversed by pretreatment of pioglitazone and rosiglitazone, which belongs to thiazolidinediones (TZDs) and are peroxisome proliferator-activated receptor (PPAR-γ) ligands. Moreover, Rib-BSA upregulates inducible nitric oxide synthase (iNOS), cycloxygenase 2 (COX-2) expression, and p-38 phosphorylation and leaves extracellular regulated protein1/2 (ERK1/2) phosphorylation unchanged. The Rib-BSA-induced signaling changes are blocked by rosiglitazone and confirmed by PPAR-γ small-interfering RNA transfection. The reduction of cell survival by Rib-BSA is blocked by the iNOS inhibitor and p38 inhibitor. No effect on cell survival was observed using the COX-2 inhibitor. Consequently, these results show that Rib-BSA directly inducing PC12 cell death is a triggering event and TZDs protect PC12 cell from Rib-BSA damage. Signaling molecules, such as PPAR-γ, P38, and iNOS, are involved in Rib-BSA-mediated cytotoxicity.

  3. Preparation, characterization and in vitro release study of BSA-loaded double-walled glucose-poly(lactide-co-glycolide) microspheres.

    PubMed

    Ansary, Rezaul H; Rahman, Mokhlesur M; Awang, Mohamed B; Katas, Haliza; Hadi, Hazrina; Mohamed, Farahidah; Doolaanea, Abd Almonem; Kamaruzzaman, Yunus B

    2016-09-01

    The aim of this study was to prepare a model protein, bovine serum albumin (BSA) loaded double-walled microspheres using a fast degrading glucose core, hydroxyl-terminated poly(lactide-co-glycolide) (Glu-PLGA) and a moderate-degrading carboxyl-terminated PLGA polymers to reduce the initial burst release and to eliminate the lag phase from the release profile of PLGA microspheres. The double-walled microspheres were prepared using a modified water-in-oil-in-oil-in-water (w/o/o/w) method and single-polymer microspheres were prepared using a conventional water-in-oil-in-water (w/o/w) emulsion solvent evaporation method. The particle size, morphology, encapsulation efficiency, thermal properties, in vitro drug release and structural integrity of BSA were evaluated in this study. Double-walled microspheres prepared with Glu-PLGA and PLGA polymers with a mass ratio of 1:1 were non-porous, smooth-surfaced, and spherical in shape. A significant reduction of initial burst release was achieved for the double-walled microspheres compared to single-polymer microspheres. In addition, microspheres prepared using Glu-PLGA and PLGA polymers in a mass ratio of 1:1 exhibited continuous BSA release after the small initial burst without any lag phase. It can be concluded that the double-walled microspheres made of Glu-PLGA and PLGA polymers in a mass ratio of 1:1 can be a potential delivery system for pharmaceutical proteins.

  4. Protein precipitation behavior of condensed tannins from Lotus pedunculatus and Trifolium repens with different mean degrees of polymerization

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The precipitation of bovine serum albumin (BSA), lysozyme (LYS), and alfalfa leaf protein (ALF) by two large- and two medium-sized condensed tannin (CT) fractions of similar flavan-3-ol subunit composition is described. CT fractions isolated from white clover flowers and big trefoil leaves exhibited...

  5. Kinetic regime of dithiothreitol-induced aggregation of bovine serum albumin.

    PubMed

    Borzova, Vera A; Markossian, Kira A; Kara, Dmitriy A; Kurganov, Boris

    2015-09-01

    A search for agents, which are capable of effectively suppressing protein aggregation, and elaboration of the appropriate test systems, are among important problems of modern biochemistry and biotechnology. One such test system is based on dithiothreitol (DTT)-induced aggregation of bovine serum albumin (BSA). Study of the kinetics of DTT-induced aggregation of BSA by asymmetric flow field flow fractionation showed that a decrease in the portion of the non-aggregated protein in time followed the exponential law, the rate constant of the first order remaining unchanged at varying protein concentration (0.1M Na-phosphate buffer, pH 7.0; 45 °C). The obtained results indicate that the rate-limiting stage of the general aggregation process is that of unfolding of the protein molecule. When studying the kinetics of DTT-induced aggregation of BSA by dynamic light scattering, we proposed to use parameter K(LS) as a measure of the initial rate of aggregation. Parameter K(LS) corresponds to the initial slope of the dependence of (I-I0)(0.5) on time (I0 and I are the initial and current values of the light scattering intensity, respectively). The K(LS) value has been applied to estimate anti-aggregation activity of chemical chaperones (arginine, its derivatives and proline). PMID:26116389

  6. Fluorescence study of bovine serum albumin and Ti and Sn oxide nanoparticles interactions

    NASA Astrophysics Data System (ADS)

    Togashi, Denisio M.; Ryder, Alan G.; Mc Mahon, Deirdre; Dunne, Peter; McManus, James

    2007-07-01

    Nanochemistry offers stimulating opportunities for a wide variety of applications in the biosciences. Understanding of the interaction of nanoparticles with biomolecules such as proteins is very important as it can help better design and fabricate nanocomposites for applications in diagnostics, drug delivery, and cell monitoring. In this work, the interaction of Bovine Serum Albumin (BSA) and two types of metal oxide nanoparticles (titanium and tin) have been studied using the intrinsic fluorescence of tryptophan residue from the proteins measured by steady state and time resolved fluorescence techniques. The nanoparticles which were fabricated using a novel synthetic process have average sizes of ~2 nm (SnO II) and ~6 nm (estimated for TiO II) and have very high solubilities in a variety of solvents. The Stern-Volmer plots indicate an effective quenching process by TiO II nanoparticles whereas SnO II nanoparticles have a lower quenching efficiency for BSA fluorescence. Static quenching is the major contribution in the overall process which may indicate a high degree of association between protein and nanoparticles. The difference in BSA fluorescence quenching efficiency between the two types of nanoparticles can be explained by the non-covalent interaction differences and the thermal stability of protein-nanoparticle associated species for both materials.

  7. The effect of albumin fusion structure on the production and bioactivity of the somatostatin-28 fusion protein in Pichia pastoris.

    PubMed

    Ding, Yuedi; Fan, Jun; Li, Wenxin; Peng, Ying; Yang, Runlin; Deng, Lili; Fu, Qiang

    2014-06-01

    Somatostatin, a natural inhibitor of growth hormone (GH), and its analogs have been used in clinical settings for the treatment of acromegaly, gigantism, thyrotropinoma, and other carcinoid syndromes. However, natural somatostatin is limited for clinical usage because of its short half-life in vivo. Albumin fusion technology was used to construct long-acting fusion proteins and Pichia pastoris was used as an expression system. Three fusion proteins (SS28)(2)-HSA, (SS28)(3)-HSA, and HSA-(SS28)(2), were constructed with different fusion copies of somatostatin-28 and fusion orientations. The expression level of (SS28)(3)-HSA was much lower than (SS28)(2)-HSA and HSA-(SS28)(2) due to the additional fusion of the somatostatin-28 molecule. MALDI-TOF mass spectrometry revealed that severe degradation occurred in the fermentation process. Similar to the standard, somatostatin-14, all three fusion proteins were able to inhibit GH secretion in blood, with (SS28)(2)-HSA being the most effective one. A pharmacokinetics study showed that (SS28)(2)-HSA had a prolonged half-life of 2 h. These results showed that increasing the number of small protein copies fused to HSA may not be a suitable method for improving protein bioactivity.

  8. The effect of albumin fusion structure on the production and bioactivity of the somatostatin-28 fusion protein in Pichia pastoris.

    PubMed

    Ding, Yuedi; Fan, Jun; Li, Wenxin; Peng, Ying; Yang, Runlin; Deng, Lili; Fu, Qiang

    2014-06-01

    Somatostatin, a natural inhibitor of growth hormone (GH), and its analogs have been used in clinical settings for the treatment of acromegaly, gigantism, thyrotropinoma, and other carcinoid syndromes. However, natural somatostatin is limited for clinical usage because of its short half-life in vivo. Albumin fusion technology was used to construct long-acting fusion proteins and Pichia pastoris was used as an expression system. Three fusion proteins (SS28)(2)-HSA, (SS28)(3)-HSA, and HSA-(SS28)(2), were constructed with different fusion copies of somatostatin-28 and fusion orientations. The expression level of (SS28)(3)-HSA was much lower than (SS28)(2)-HSA and HSA-(SS28)(2) due to the additional fusion of the somatostatin-28 molecule. MALDI-TOF mass spectrometry revealed that severe degradation occurred in the fermentation process. Similar to the standard, somatostatin-14, all three fusion proteins were able to inhibit GH secretion in blood, with (SS28)(2)-HSA being the most effective one. A pharmacokinetics study showed that (SS28)(2)-HSA had a prolonged half-life of 2 h. These results showed that increasing the number of small protein copies fused to HSA may not be a suitable method for improving protein bioactivity. PMID:24752560

  9. Surface-protein interactions on different stainless steel grades: effects of protein adsorption, surface changes and metal release.

    PubMed

    Hedberg, Y; Wang, X; Hedberg, J; Lundin, M; Blomberg, E; Wallinder, I Odnevall

    2013-04-01

    Implantation using stainless steels (SS) is an example where an understanding of protein-induced metal release from SS is important when assessing potential toxicological risks. Here, the protein-induced metal release was investigated for austenitic (AISI 304, 310, and 316L), ferritic (AISI 430), and duplex (AISI 2205) grades in a phosphate buffered saline (PBS, pH 7.4) solution containing either bovine serum albumin (BSA) or lysozyme (LSZ). The results show that both BSA and LSZ induce a significant enrichment of chromium in the surface oxide of all stainless steel grades. Both proteins induced an enhanced extent of released iron, chromium, nickel and manganese, very significant in the case of BSA (up to 40-fold increase), whereas both proteins reduced the corrosion resistance of SS, with the reverse situation for iron metal (reduced corrosion rates and reduced metal release in the presence of proteins). A full monolayer coverage is necessary to induce the effects observed.

  10. Affinity of rosmarinic acid to human serum albumin and its effect on protein conformation stability.

    PubMed

    Peng, Xin; Wang, Xiangchao; Qi, Wei; Su, Rongxin; He, Zhimin

    2016-02-01

    Rosmarinic acid (RA) is a natural polyphenol contained in many aromatic plants with promising biological activities. The interaction between RA and human serum albumin (HSA) was investigated by multi-spectroscopic, electrochemistry, molecular docking and molecular dynamics simulation methods. The fluorescence emission of HSA was quenched by RA through a combined static and dynamic quenching mechanism, but the static quenching was the major constituent. Fluorescence experiments suggested that RA was bound to HSA with moderately strong binding affinity through hydrophobic interaction. The probable binding location of RA was located near site I of HSA. Additionally, as shown by the Fourier transform infrared (FT-IR) and circular dichroism (CD) spectra, RA can result in conformational and structural alterations of HSA. Furthermore, the molecular dynamics studies were used to investigate the stability of the HSA and HSA-RA system. Altogether, the results can provide an important insight for the applications of RA in the food industry.

  11. Affinity of rosmarinic acid to human serum albumin and its effect on protein conformation stability.

    PubMed

    Peng, Xin; Wang, Xiangchao; Qi, Wei; Su, Rongxin; He, Zhimin

    2016-02-01

    Rosmarinic acid (RA) is a natural polyphenol contained in many aromatic plants with promising biological activities. The interaction between RA and human serum albumin (HSA) was investigated by multi-spectroscopic, electrochemistry, molecular docking and molecular dynamics simulation methods. The fluorescence emission of HSA was quenched by RA through a combined static and dynamic quenching mechanism, but the static quenching was the major constituent. Fluorescence experiments suggested that RA was bound to HSA with moderately strong binding affinity through hydrophobic interaction. The probable binding location of RA was located near site I of HSA. Additionally, as shown by the Fourier transform infrared (FT-IR) and circular dichroism (CD) spectra, RA can result in conformational and structural alterations of HSA. Furthermore, the molecular dynamics studies were used to investigate the stability of the HSA and HSA-RA system. Altogether, the results can provide an important insight for the applications of RA in the food industry. PMID:26304336

  12. Rate constant for reaction of vitamin C with protein radicals in γ-irradiated aqueous albumin solution at 295 K

    NASA Astrophysics Data System (ADS)

    Miyazaki, Tetsuo; Yoshimura, Toru; Mita, Kazuya; Suzuki, Keiji; Watanabe, Masami

    1995-02-01

    When an aqueous solution of albumin (0.1 kg dm -3) is irradiated by γ-rays at 295 K, albumin radicals with a long lifetime are observed by ESR. The reaction of vitamin C with the albumin radicals has been studied at 295 K in the albumin solution, which is considered as a model of cells. The rate constant for the reaction of vitamin C with the albumin radicals was measured as 0.014 dm 3 mol -1 s -1, which is much smaller than the reported rate constants (10 6-10 10 dm 3 mol -1 s -1) for the reaction of vitamin C with radicals in a dilute aqueous solution. The small rate constant for the reaction of vitamin C is ascribed to the reaction in polymer coils in the albumin solution, since vitamin C and albumin radicals diffuse very slowly in the coils.

  13. Ultrastable BSA-capped gold nanoclusters with a polymer-like shielding layer against reactive oxygen species in living cells.

    PubMed

    Zhou, Wenjuan; Cao, Yuqing; Sui, Dandan; Guan, Weijiang; Lu, Chao; Xie, Jianping

    2016-05-01

    The prevalence of reactive oxygen species (ROS) production and the enzyme-containing intracellular environment could lead to the fluorescence quenching of bovine serum albumin (BSA)-capped gold nanoclusters (AuNCs). Here we report an efficient strategy to address this issue, where a polymer-like shielding layer is designed to wrap around the Au core to significantly improve the stability of AuNCs against ROS and protease degradation. The key of our design is to covalently incorporate a thiolated AuNC into the BSA-AuNC via carbodiimide-activated coupling, leading to the formation of a AuNC pair inside the cross-linked BSA molecule. The as-designed paired AuNCs in BSA (or BSA-p-AuNCs for short) show improved performances in living cells.

  14. The ligand activity of AGE-proteins to scavenger receptors is dependent on their rate of modification by AGEs.

    PubMed

    Nagai, Ryoji; Mera, Katsumi; Nakajou, Keisuke; Fujiwara, Yukio; Iwao, Yasunori; Imai, Hiroki; Murata, Toshinori; Otagiri, Masaki

    2007-12-01

    The cellular interaction of proteins modified with advanced glycation end-products (AGEs) is believed to induce several different biological responses, which are involved in the development of diabetic vascular complications. We report here that the ratio of protein glycation is implicated in its ligand activity to scavenger receptors. Although highly-modified AGE-bovine serum albumin (high-AGE-BSA) was significantly recognized by human monocyte-derived macrophages and Chinese hamster ovary cells which overexpress such scavenger receptors as CD36, SR-BI (scavenger receptor class B type-I), and LOX-1 (Lectin-like Ox-LDL receptor-1), the mildly-modified-AGE-BSA (mild-AGE-BSA) did not show any ligand activity to these cells. Furthermore, when (111)In-labeled high- or mild-AGE-BSA were injected into the tail vein of mice, the high-AGE-BSA was rapidly cleared from the circulation whereas the clearance rate of the mild-AGE-BSA was very slow, similar to the native BSA. These results demonstrate the first evidence that the ligand activity of the AGE-proteins to the scavenger receptors and its pharmacokinetic properties depend on their rate of modification by AGEs, and we should carefully prepare the AGE-proteins in vitro to clarify the physiological significance of the interaction between the AGE-receptors and AGE-proteins.

  15. Kinetics of advanced glycation end products formation on bovine serum albumin with various reducing sugars and dicarbonyl compounds in equimolar ratios.

    PubMed

    Luers, Lars; Rysiewski, Karolina; Dumpitak, Christian; Birkmann, Eva

    2012-04-01

    Reducing sugars and reactive dicarbonyl compounds play a major role in glycation of proteins in vivo. Glycation of proteins is the first step in of a nonenzymatic reaction, resulting in advanced glycation end products (AGEs). AGEs can inactivate proteins or modify their biological activities. Therefore, it is important to understand the mechanism of AGE formation. Here, we systematically analyzed the kinetics of AGE formation in vitro by fluorescence and absorption measurements utilizing a microplate reader system and bovine serum albumin (BSA) as a model protein. Comparing different concentrations of BSA, we applied various reducing sugars and reactive dicarbonyl compounds as AGE-inducing agents at different concentrations. In summary, this experimental setup enabled us to measure the kinetics of AGE formation in an efficient and defined way.

  16. Kinetics of advanced glycation end products formation on bovine serum albumin with various reducing sugars and dicarbonyl compounds in equimolar ratios.

    PubMed

    Luers, Lars; Rysiewski, Karolina; Dumpitak, Christian; Birkmann, Eva

    2012-04-01

    Reducing sugars and reactive dicarbonyl compounds play a major role in glycation of proteins in vivo. Glycation of proteins is the first step in of a nonenzymatic reaction, resulting in advanced glycation end products (AGEs). AGEs can inactivate proteins or modify their biological activities. Therefore, it is important to understand the mechanism of AGE formation. Here, we systematically analyzed the kinetics of AGE formation in vitro by fluorescence and absorption measurements utilizing a microplate reader system and bovine serum albumin (BSA) as a model protein. Comparing different concentrations of BSA, we applied various reducing sugars and reactive dicarbonyl compounds as AGE-inducing agents at different concentrations. In summary, this experimental setup enabled us to measure the kinetics of AGE formation in an efficient and defined way. PMID:22533432

  17. Metformin-loaded BSA nanoparticles in cancer therapy: a new perspective for an old antidiabetic drug.

    PubMed

    Jose, Pinkybel; Sundar, K; Anjali, C H; Ravindran, Aswathy

    2015-03-01

    Clinical and experimental data suggest that there is a strong association between type II diabetic mellitus and pancreatic cancer. The present study focuses on exploring the anticancer and antidiabetic properties of metformin-loaded bovine serum albumin nanoparticles (BSA NPs) on (MiaPaCa-2) pancreatic carcinoma cell lines. Albumin nanoparticles were synthesized using coacervation method and the average size of the particles was found to be 97 nm. The particles were stable and showed a spherical morphology with narrow size distribution. We investigated the impact of two stages characterized in type II diabetes mellitus (hyperglycemia and hyperinsulinemia) on the proliferation of MiaPaCa-2 cells and compared the inhibitory effects of bare metformin to that of MET-BSA NPs. Further, different concentrations of insulin and glucose were added along with bare metformin, bare BSA, and metformin encapsulated BSA carrier on MiaPaCa-2 cells to check the strong association between type II diabetes and pancreatic cancer. The results revealed that MET-BSA NPs showed more toxicity when compared with drug and carrier individually.

  18. Effects of surface compositional and structural heterogeneity on nanoparticle-protein interactions: different protein configurations.

    PubMed

    Huang, Rixiang; Carney, Randy P; Ikuma, Kaoru; Stellacci, Francesco; Lau, Boris L T

    2014-06-24

    As nanoparticles (NPs) enter into biological systems, they are immediately exposed to a variety and concentration of proteins. The physicochemical interactions between proteins and NPs are influenced by the surface properties of the NPs. To identify the effects of NP surface heterogeneity, the interactions between bovine serum albumin (BSA) and gold NPs (AuNPs) with similar chemical composition but different surface structures were investigated. Different interaction modes and BSA conformations were studied by dynamic light scattering, circular dichroism spectroscopy, fluorescence quenching and isothermal titration calorimetry (ITC). Depending on the surface structure of AuNPs, BSA seems to adopt either a "side-on" or an "end-on" conformation on AuNPs. ITC demonstrated that the adsorption of BSA onto AuNPs with randomly distributed polar and nonpolar groups was primarily driven by electrostatic interaction, and all BSA were adsorbed in the same process. The adsorption of BSA onto AuNPs covered with alternating domains of polar and nonpolar groups was a combination of different interactions. Overall, the results of this study point to the potential for utilizing nanoscale manipulation of NP surfaces to control the resulting NP-protein interactions. PMID:24882660

  19. Molecular dynamics simulation of free and forced BSA adsorption on a hydrophobic graphite surface.

    PubMed

    Mücksch, Christian; Urbassek, Herbert M

    2011-11-01

    The adsorption of bovine serum albumin (BSA) onto a hydrophobic graphite surface is studied using molecular-dynamics simulation. In addition to the free, that is, unsteered, adsorption, we also investigate forced adsorption, in which the action of an AFM tip pushing the protein with constant force to the surface is modeled. Using an implicit inviscid water model, the adsorption dynamics and energetics are monitored for two different initial protein orientations toward the surface. In all cases, we find that the protein partially unfolds and spreads on the surface. The spreading is in agreement with the well-known high biocompatibility of graphite-based implants. The denaturation is, however, greatly enhanced in the case of forced adsorption. We follow the position of the so-called lipid-binding pocket found in subdomain IIIA (Sudlow site II) during adsorption and find that it is tilted and moved toward the graphite surface in all cases, in agreement with its hydrophobic character. The relevance of our findings for the common measurement procedure of studying protein adhesion using AFM experiments is discussed.

  20. Ultrastable BSA-capped gold nanoclusters with a polymer-like shielding layer against reactive oxygen species in living cells

    NASA Astrophysics Data System (ADS)

    Zhou, Wenjuan; Cao, Yuqing; Sui, Dandan; Guan, Weijiang; Lu, Chao; Xie, Jianping

    2016-05-01

    The prevalence of reactive oxygen species (ROS) production and the enzyme-containing intracellular environment could lead to the fluorescence quenching of bovine serum albumin (BSA)-capped gold nanoclusters (AuNCs). Here we report an efficient strategy to address this issue, where a polymer-like shielding layer is designed to wrap around the Au core to significantly improve the stability of AuNCs against ROS and protease degradation. The key of our design is to covalently incorporate a thiolated AuNC into the BSA-AuNC via carbodiimide-activated coupling, leading to the formation of a AuNC pair inside the cross-linked BSA molecule. The as-designed paired AuNCs in BSA (or BSA-p-AuNCs for short) show improved performances in living cells.The prevalence of reactive oxygen species (ROS) production and the enzyme-containing intracellular environment could lead to the fluorescence quenching of bovine serum albumin (BSA)-capped gold nanoclusters (AuNCs). Here we report an efficient strategy to address this issue, where a polymer-like shielding layer is designed to wrap around the Au core to significantly improve the stability of AuNCs against ROS and protease degradation. The key of our design is to covalently incorporate a thiolated AuNC into the BSA-AuNC via carbodiimide-activated coupling, leading to the formation of a AuNC pair inside the cross-linked BSA molecule. The as-designed paired AuNCs in BSA (or BSA-p-AuNCs for short) show improved performances in living cells. Electronic supplementary information (ESI) available: Detailed experimental materials, apparatus, experimental procedures and characterization data. See DOI: 10.1039/c6nr02178f

  1. Polarization properties of fluorescent BSA protected Au25 nanoclusters.

    PubMed

    Raut, Sangram; Chib, Rahul; Rich, Ryan; Shumilov, Dmytro; Gryczynski, Zygmunt; Gryczynski, Ignacy

    2013-04-21

    BSA protected gold nanoclusters (Au25) are attracting a great deal of attention due to their unique spectroscopic properties and possible use in biophysical applications. Although there are reports on synthetic strategies, spectroscopy and applications, little is known about their polarization behavior. In this study, we synthesized the BSA protected Au25 nanoclusters and studied their steady state and time resolved fluorescence properties including polarization behavior in different solvents: glycerol, propylene glycol and water. We demonstrated that the nanocluster absorption spectrum can be separated from the extinction spectrum by subtraction of Rayleigh scattering. The nanocluster absorption spectrum is well approximated by three Gaussian components. By a comparison of the emissions from BSA Au25 clusters and rhodamine B in water, we estimated the quantum yield of nanoclusters to be higher than 0.06. The fluorescence lifetime of BSA Au25 clusters is long and heterogeneous with an average value of 1.84 μs. In glycerol at -20 °C the anisotropy is high, reaching a value of 0.35. However, the excitation anisotropy strongly depends on the excitation wavelengths indicating a significant overlap of the different transition moments. The anisotropy decay in water reveals a correlation time below 0.2 μs. In propylene glycol the measured correlation time is longer and the initial anisotropy depends on the excitation wavelength. BSA Au25 clusters, due to long lifetime and high polarization, can potentially be used in studying large macromolecules such as protein complexes with large molecular weight.

  2. Properties of mixed monolayers of clinical lung surfactant, serum albumin and hydrophilic polymers.

    PubMed

    Minkov, I; Mircheva, K; Grozev, N; Tz, Ivanova; Panaiotov, I

    2013-01-01

    It is now established that the surface activity of the clinically used lung surfactant is reduced by serum proteins and can be restored by adding the hydrophilic polymers. The mechanisms of lung surfactant inactivation by serum proteins and restoring effect by the hydrophilic polymers remain not completely understood. In this paper the state and rheological dilatational properties of surface films formed from clinical lung surfactant Exosurf, Survanta, Curosurf and Alveofact in the presence of serum albumin (BSA) and hydrophilic polymers polyvinylpyrrolidone (PVP), polyethylene glycol (PEG) and Dextran were studied. The obtained results suggest that the lung surfactant and BSA mixtures spread at air-water interface form a DPPC/BSA mixed monolayers with lower content of DPPC. The presence of hydrophilic polymers PVP, PEG and Dextran restore the DPPC content in the surface film. The effectiveness of the DPPC spreading and formation of better compacted film increases in order Exosurf, Survanta, Curosurf, Alveofact. The obtained results are in accordance with the generally admitted ideas about the mechanisms of serum protein inactivation and restoring effect of hydrophilic polymers based on the previously studies of the lung surfactant adsorption rate.

  3. 19F nuclear magnetic resonance investigation of stereoselective binding of isoflurane to bovine serum albumin.

    PubMed Central

    Xu, Y; Tang, P; Firestone, L; Zhang, T T

    1996-01-01

    Whether proteins or lipids are the primary target sites for general anesthetic action has engendered considerable debate. Recent in vivo studies have shown that the S(+) and R(-) enantiomers of isoflurane are not equipotent, implying involvement of proteins. Bovine serum albumin (BSA), a soluble protein devoid of lipid, contains specific binding sites for isoflurane and other anesthetics. We therefore conducted 19F nuclear magnetic resonance measurements to determine whether binding of isoflurane to BSA was stereoselective. Isoflurane chemical shifts were measured as a function of BSA concentration to determine the chemical shift differences between the free and bound isoflurane. KD was determined by measuring the 19F transverse relaxation times (T2) as a function of isoflurane concentration. The binding duration was determined by assessing increases in 1/T2 as a result of isoflurane exchanging between the free and bound states. The S(+) and R(-) enantiomers exhibited no stereoselectivity in chemical shifts and KD values (KD = 1.3 +/- 0.2 mM, mean +/- SE, for S(+), R(-), and the racemic mixture). Nonetheless, stereoselectivity was observed in dynamic binding parameters; the S(+) enantiomer bound with slower association and dissociation rates than the R(-). Images FIGURE 1 PMID:8770230

  4. Long-circulating self-assembled cholesteryl albumin nanoparticles enhance tumor accumulation of hydrophobic anticancer drug.

    PubMed

    Battogtokh, Gantumur; Kang, Ji Hee; Ko, Young Tag

    2015-10-01

    The objective of this study was to develop an albumin nanoparticle with improved stability and drug loading capacity. Generation of nanomaterials having physiologically stable and high potential for drug delivery is still challenging. Herein we synthesized cholesteryl albumin conjugate using N,N-disuccinimidyl carbonate coupling reagent and prepared paclitaxel-loaded cholesteryl albumin nanoparticle (PTX-Chol-BSA) by self-assembly with the mean hydrodynamic diameter of 147.6±1.6nm and with high loading capacity. PTX-Chol-BSA nanoparticle showed much higher colloidal stability than a simple complex of PTX and BSA (PTX-BSA) and sustained release profile. PTX-Chol-BSA nanoparticles exhibited greater cellular uptake and cytotoxicity in B16F10 and MCF-7 cancer cell lines, as compared with PTX in Cremophor EL/ethanol (PTX-Cre/EtOH) and PTX-BSA formulations. A pharmacokinetic study in tumor-bearing mice showed that the area under the concentration-time curve (AUC0-8 h) following the administration of PTX-Chol-BSA was 1.6-2-fold higher than those following the administration of PTX-Cre/EtOH and PTX-BSA. In addition, the tumor AUC0-8 h of PTX-Chol-BSA was around 2-fold higher than that of PTX-BSA. Furthermore, in vivo antitumor efficacy results revealed that PTX-Chol-BSA nanoparticles have greater antitumor efficacy. In conclusion, we demonstrated the potential of PTX-Chol-BSA nanoparticles for anti-tumor chemotherapy, with enhanced in vitro and in vivo behaviors, as compared to PTX-BSA and PTX-Cre/EtOH.

  5. Albumin Test

    MedlinePlus

    ... to a variety of conditions in addition to malnutrition , a decrease in albumin needs to be evaluated ... can also be seen in inflammation , shock, and malnutrition . They may be seen with conditions in which ...

  6. Development and characterization of a novel nanoemulsion drug-delivery system for potential application in oral delivery of protein drugs

    PubMed Central

    Sun, Hongwu; Liu, Kaiyun; Liu, Wei; Wang, Wenxiu; Guo, Chunliang; Tang, Bin; Gu, Jiang; Zhang, Jinyong; Li, Haibo; Mao, Xuhu; Zou, Quanming; Zeng, Hao

    2012-01-01

    Background: The stability of protein drugs remains one of the key hurdles to their success in the market. The aim of the present study was to design a novel nanoemulsion drug-delivery system (NEDDS) that would encapsulate a standard-model protein drug – bovine serum albumin (BSA) – to improve drug stability. Methods: The BSA NEDDS was prepared using a phase-inversion method and pseudoternary phase diagrams. The following characteristics were studied: morphology, size, zeta potential, drug loading, and encapsulation efficiency. We also investigated the stability of the BSA NEDDS, bioactivity of BSA encapsulated within the NEDDS, the integrity of the primary, secondary, and tertiary structures, and specificity. Results: The BSA NEDDS consisted of Cremophor EL-35, propylene glycol, isopropyl myristate, and normal saline. The average particle diameter of the BSA NEDDS was about 21.8 nm, and the system showed a high encapsulation efficiency (>90%) and an adequate drug-loading capacity (45 mg/mL). The thermodynamic stability of the system was investigated at different temperatures and pH levels and in room-temperature conditions for 180 days. BSA NEDDS showed good structural integrity and specificity for the primary, secondary, and tertiary structures, and good bioactivity of the loaded BSA. Conclusions: BSA NEDDS showed the properties of a good nanoemulsion-delivery system. NEDDS can greatly enhance the stability of the protein drug BSA while maintaining high levels of drug bioactivity, good specificity, and integrity of the primary, secondary, and tertiary protein structures. These findings indicate that the nanoemulsion is a potential formulation for oral administration of protein drugs. PMID:23118537

  7. Albuminated Glycoenzymes: Enzyme Stabilization through Orthogonal Attachment of a Single-Layered Protein Shell around a Central Glycoenzyme Core.

    PubMed

    Ritter, Dustin W; Newton, Jared M; Roberts, Jason R; McShane, Michael J

    2016-05-18

    Here we demonstrate an approach to stabilize enzymes through the orthogonal covalent attachment of albumin on the single-enzyme level. Albuminated glycoenzymes (AGs) based upon glucose oxidase and catalase from Aspergillus niger were prepared in this manner. Gel filtration chromatography and dynamic light scattering support modification, with an increase in hydrodynamic radius of ca. 60% upon albumination. Both AGs demonstrate a marked resistance to aggregation during heating to 90 °C, but this effect is more profound in albuminated catalase. The functional characteristics of albuminated glucose oxidase vary considerably with exposure type. The AG's thermal inactivation is reduced more than 25 times compared to native glucose oxidase, and moderate stabilization is observed with one month storage at 37 °C. However, albumination has no effect on operational stability of glucose oxidase.

  8. Rapid Screening of Drug-Protein Binding Using High-Performance Affinity Chromatography with Columns Containing Immobilized Human Serum Albumin

    PubMed Central

    Li, Ying-Fei; Zhang, Xiao-Qiong; Hu, Wei-Yu; Li, Zheng; Liu, Ping-Xia; Zhang, Zhen-Qing

    2013-01-01

    For drug candidates, a plasma protein binding (PPB) more than 90% is more meaningful and deserves further investigation in development. In the study, a high-performance liquid chromatography method employing column containing immobilized human serum albumin (HSA) to screen in vitro PPB of leading compounds was established and successfully applied to tested compounds. Good correlation (a coefficient correlation of 0.96) was attained between the reciprocal values (X) of experimentally obtained retention time of reference compounds eluted through HSA column and the reported PPB values (Y) with a correlation equation of Y = 92.03 − 97.01X. The method was successfully applied to six test compounds, and the result was confirmed by the conventional ultrafiltration technique, and both yielded equal results. However, due to the particular protein immobilized to column, the method cannot be applied for all compounds and should be exploited judiciously based on the value of the logarithmic measure of the acid dissociation constant (pKa) as per the requirement. If α1-acid glycoprotein and other plasma proteins could be immobilized like HSA with their actual ratio in plasma to column simultaneously, the result attained using immobilized column may be more accurate, and the method could be applied to more compounds without pKa limitation. PMID:23607050

  9. Spectroscopic investigation of the interaction between riboflavin and bovine serum albumin

    NASA Astrophysics Data System (ADS)

    Guo, Xing-Jia; Sun, Xiu-Dan; Xu, Shu-Kun

    2009-08-01

    The mutual interaction of riboflavin (RF) with bovine serum albumin (BSA) was investigated using fluorescence spectroscopy under simulative physiological conditions. The fluorescence quenching mechanism of BSA by RF should belong to dynamic quenching according to the Stern-Volmer equation, but also the effect of ground complex formation and energy transfer could not be completely precluded in BSA-RF system. The binding constants and the corresponding thermodynamic parameters at different temperatures were calculated, which indicated the presence of hydrophobic forces between RF and BSA. The averaged binding distance between riboflavin and BSA was also obtained based on the theory of FÖrster's non-radiation energy transfer. Moreover, the effect of riboflavin on the conformation of BSA was analyzed using synchronous fluorescence. The effects of some common ions Cu 2+, Zn 2+, Ca 2+, and Mg 2+ on the binding constant between riboflavin and BSA were also examined.

  10. Molecular spectroscopic studies on the interaction of morin with bovine serum albumin.

    PubMed

    Hu, Yan-Jun; Yue, Hua-Li; Li, Xiao-Ling; Zhang, Si-Si; Tang, E; Zhang, Li-Ping

    2012-07-01

    The interaction between morin and bovine serum albumin (BSA) was studied using molecular spectroscopic approach at different temperatures under imitated physiological conditions. Quenching of intrinsic tryptophanyl fluorescence of BSA with increasing morin concentration is the actuating tool in the analysis. The obtained quenching mechanisms, binding constants, binding sites and corresponding thermodynamic parameters at different temperatures indicate that the hydrophobic interaction play a major role in the morin-BSA association. Binding affinity between morin and BSA was determined using Scatchard equation and the modified Stern-Volmer equation, and the corresponding Structure-affinity relationships of flavonoids were discussed. Site marker competitive displacement experiments demonstrated that morin binds with high affinity to site II (subdomain IIIA) of BSA. Furthermore, the circular dichroism spectral results indicated that the conformation of BSA changed in the presence of morin. In addition, the effect of some common metal ions on the binding constant between morin and BSA was examined.

  11. Albumin stimulates p44/p42 extracellular-signal-regulated mitogen-activated protein kinase in opossum kidney proximal tubular cells.

    PubMed

    Dixon, R; Brunskill, N J

    2000-03-01

    The presence of protein in the urine of patients with renal disease is an adverse prognostic feature. It has therefore been suggested that proteinuria per se may be responsible for the development of renal tubulo-interstitial scarring and fibrosis, and disturbances in tubular cell growth and proliferation. We have used the opossum kidney proximal tubular cell line to investigate the effects of albumin on cell growth. The effect of albumin on cell proliferation was investigated by cell counting and measurement of [(3)H]thymidine incorporation. We studied the effect of recombinant human albumin on the activity of p44/p42 extracellular-signal-regulated mitogen-activated protein kinase (MAP kinase ) using an in vitro kinase assay, and immunoblotting with antibodies against active extracellular-signal-regulated kinase (ERK). The effects of the ERK inhibitor PD98059 were also examined. Recombinant human albumin was found to stimulate proliferation of opossum kidney cells in a dose-dependent manner, with maximal stimulation at a concentration of 1 mg/ml. In addition, recombinant human albumin activated ERK in a time-dependent (maximal after 5 min) and dose-dependent (maximal at 1 mg/ml) fashion. These effects on cell proliferation and ERK activity were inhibited by PD98059, and were not reproduced by ovalbumin or mannitol. The data therefore indicate that albumin is able to stimulate growth and proliferation of proximal tubular cells that is dependent on the ERK family of MAP kinases. The potential importance of this pathway in the development of renal disease is discussed. PMID:10677388

  12. Effect of temperature on the metronidazole BSA interaction: Multi-spectroscopic method

    NASA Astrophysics Data System (ADS)

    Chen, Jun; Jiang, Xin Yu; Chen, Xiao Qing; Chen, Yue

    2008-03-01

    The interaction between metronidazole and bovine serum albumin (BSA) was investigated using fluorescence spectroscopy (FS) and resonance light scattering spectroscopy (RLS). The apparent binding constants ( Ka) between metronidazole and BSA were 3.42 × 10 4 (20 °C), 5.78 × 10 4 (30 °C) and 8.23 × 10 4 L mol -1 (40 °C), and the binding sites values ( n) were 1.48 ± 0.03. The experimental results showed that the metronidazole could be inserted into the BSA, quenching the inner fluorescence by forming the metronidazole-BSA complex. The addition of increasing metronidazole to BSA solution leads to the gradual enhancement in RLS intensity, exhibiting the formation of the aggregate in solution. It was found that both static quenching and non-radiation energy transfer were the main reasons for the fluorescence quenching. The entropy change and enthalpy change were positive, which indicated that the interaction of metronidazole and BSA was driven mainly by hydrophobic forces. The process of binding was a spontaneous process in which Gibbs free energy change was negative.

  13. Theoretical model to investigate the alkyl chain and anion dependent interactions of gemini surfactant with bovine serum albumin.

    PubMed

    Vishvakarma, Vijay K; Kumari, Kamlesh; Patel, Rajan; Dixit, V S; Singh, Prashant; Mehrotra, Gopal K; Chandra, Ramesh; Chakrawarty, Anand Kumar

    2015-05-15

    Surfactants are used to prevent the irreversible aggregation of partially refolded proteins and they also assist in protein refolding. We have reported the design and screening of gemini surfactant to stabilize bovine serum albumin (BSA) with the help of computational tool (iGEMDOCK). A series of gemini surfactant has been designed based on bis-N-alkyl nicotinate dianion via varying the alkyl group and anion. On changing the alkyl group and anion of the surfactant, the value of Log P changes means polarity of surfactant can be tuned. Further, the virtual screening of the gemini surfactant has been carried out based on generic evolutionary method. Herein, thermodynamic data was studied to determine the potential of gemini surfactant as BSA stabilizer. Computational tools help to find out the efficient gemini surfactant to stabilize the BSA rather than to use the surfactant randomly and directionless for the stabilization. It can be confirmed through the experimental techniques. Previously, researcher synthesized one of the designed and used gemini surfactant to stabilize the BSA and their interactions were confirmed through various techniques and computational docking. But herein, the authors find the most competent gemini surfactant to stabilize BSA using computational tools on the basis of energy score. Different from the single chain surfactant, the gemini surfactants exhibit much stronger electrostatic and hydrophobic interactions with the protein and are thus effective at much lower concentrations. Based on the present study, it is expected that gemini surfactants may prove useful in the protein stabilization operations and may thus be effectively employed to circumvent the problem of misfolding and aggregation.

  14. DLS microrheology at the onset of weak elasticity during thermal denaturation of BSA

    NASA Astrophysics Data System (ADS)

    Nobbmann, Ulf; Rega, Carlos A.; Jankevics, Hanna; Amin, Samiul

    2011-03-01

    The ability to precisely detect the onset of protein aggregation to draw insights into microstructural characteristics plays a critical role in a variety of biotechnological applications such as therapeutic protein stability. Rheological techniques are very sensitive to evolution of an aggregating network but have been limited in biotechnology, due to large sample volume and moderately high viscosity requirements in traditional mechanical rheometry. Dynamic Light Scattering (DLS) overcomes these limitations as experiments can be carried out on very dilute samples and small volumes. We present a method based on optical microrheology to study the onset of bovine serum albumin (BSA) aggregation to develop an understanding of the evolving network structure. The exponent of the tracer mean squared displacement power law fit and the elastic modulus G' emerge as two key parameters. The impact of probe chemistry and probe size on the extracted microrheological response is discussed. A Saluja et al., ``Ultrasonic rheology of a monoclonal antibody (IgG 2) solution: implication for physical stability of proteins in high concentration formulations'' J. of Pharm. Sci. (2007) 96, 3181-3195.

  15. [Albumin in sepsis].

    PubMed

    Tamion, F

    2010-09-01

    Human serum albumin is a small (66kD) globular protein representing over 60 % of the total plasma protein content. It is made up of 585 amino 6 acids and contains 35 cysteine residues forming disulfide bridges that contribute to its overall tertiary structure. It has a free cysteine-derived thiol group at Cys-34, which accounts for 80 % of its redox activity. Physiologically, serum albumin exists in a reduced form with a free thiol contributing to its antioxidant properties. It is synthesized primarily in the liver and is an acute-phase protein. It is a multifunctional plasma protein ascribed ligand-binding and transport properties as well as antioxidants and enzymatic functions. It maintains colloid osmotic pressure, modulates inflammatory response and may influence oxidative damage. Hypoalbuminemia is common in the intensive care unit and may be due to decreased synthesis by the liver and/or to increased losses or increased proteolysis and clearance. Although albumin was long used to control vascular collapse in critically ill patients, the evidence suggests that it does not offer a benefit over crystalloid solutions in vascular collapse. However, human serum albumin is an important circulating antioxidant and it may be beneficial in critically ill patients to limit oxidative damage. A number of studies suggest that in specific groups of hypoalbuminemic critically ill patients, albumin administration may have beneficial effects on organ function, although the exact mechanisms remain undefined. Further trials are needed to confirm theses observations and to clearly demonstrate whether albumin should be administered in critically ill patients with hypoalbuminemia. PMID:20675098

  16. [Study of Reaction Dynamics between Bovine Serum Albumin and Folic Acid by Stopped-Flow/Fluorescence].

    PubMed

    Ye, San-xian; Luo, Yun-jing; Qiao, Shu-liang; Li, Li; Liu, Cai-hong; Shi, Jian-long; An, Xue-jing

    2016-01-01

    As a kind of coenzyme of one-carbon enzymes in vivo, folic acid belongs to B vitamins, which can interact with other vitamins and has great significance for converting among amino acids, dividing growth of cells and protein synthesis reactions. Half-life, concentration and reaction rate constant of drugs are important parameters in pharmacokinetic study. In this paper, by utilizing fluorescence spectrophotometer and stopped-flow spectrum analyzer, reaction kinetic parameters between bovine serum albumin(BSA) and folic acid in a bionic system have been investigated, which provide references for parameters of drug metabolism related to folic acid. By using Stern-Volmer equation dealing with fluorescence quenching experiments data, we concluded that under 25, 30, and 37 degrees C, the static quenching constants of folic acid to intrinsic fluorescence from bovine serum albumin were 2.455 x 10(10), 4.900 x 10(10) and 6.427 x 10(10) L x mol(-1) x s(-1) respectively; The results of kinetic reaction rate have shown that the reaction rate of BSA and folic acid are greater than 100 mol x L(-1) x s(-1) at different temperatures, pH and buffering media, illustrating that the quenching mechanism between BSA and folic acid is to form composite static quenching process. Reaction concentration of bovine serum albumin and its initial concentration were equal to the secondary reaction formula, and the correlation coefficient was 0.998 7, while the half-life (t1/2) was 0.059 s at physiological temperature. With the increase of folic acid concentration, the apparent rate constant of this reaction had a linear increasing trend, the BSA fluorescence quenching rate constant catalyzed by folic acid was 3.174 x 10(5) mol x L(-1) x s(-1). Furthermore, with different buffer, the apparent rate constant and reaction rate constant of BSA interacting with folic acid were detected to explore the influence on the reaction under physiological medium, which is of great significance to determine the

  17. [Study of Reaction Dynamics between Bovine Serum Albumin and Folic Acid by Stopped-Flow/Fluorescence].

    PubMed

    Ye, San-xian; Luo, Yun-jing; Qiao, Shu-liang; Li, Li; Liu, Cai-hong; Shi, Jian-long; An, Xue-jing

    2016-01-01

    As a kind of coenzyme of one-carbon enzymes in vivo, folic acid belongs to B vitamins, which can interact with other vitamins and has great significance for converting among amino acids, dividing growth of cells and protein synthesis reactions. Half-life, concentration and reaction rate constant of drugs are important parameters in pharmacokinetic study. In this paper, by utilizing fluorescence spectrophotometer and stopped-flow spectrum analyzer, reaction kinetic parameters between bovine serum albumin(BSA) and folic acid in a bionic system have been investigated, which provide references for parameters of drug metabolism related to folic acid. By using Stern-Volmer equation dealing with fluorescence quenching experiments data, we concluded that under 25, 30, and 37 degrees C, the static quenching constants of folic acid to intrinsic fluorescence from bovine serum albumin were 2.455 x 10(10), 4.900 x 10(10) and 6.427 x 10(10) L x mol(-1) x s(-1) respectively; The results of kinetic reaction rate have shown that the reaction rate of BSA and folic acid are greater than 100 mol x L(-1) x s(-1) at different temperatures, pH and buffering media, illustrating that the quenching mechanism between BSA and folic acid is to form composite static quenching process. Reaction concentration of bovine serum albumin and its initial concentration were equal to the secondary reaction formula, and the correlation coefficient was 0.998 7, while the half-life (t1/2) was 0.059 s at physiological temperature. With the increase of folic acid concentration, the apparent rate constant of this reaction had a linear increasing trend, the BSA fluorescence quenching rate constant catalyzed by folic acid was 3.174 x 10(5) mol x L(-1) x s(-1). Furthermore, with different buffer, the apparent rate constant and reaction rate constant of BSA interacting with folic acid were detected to explore the influence on the reaction under physiological medium, which is of great significance to determine the

  18. Bioactivity of albumins bound to silver nanoparticles.

    PubMed

    Mariam, Jessy; Sivakami, S; Kothari, D C; Dongre, P M

    2014-06-01

    The last decade has witnessed a tremendous rise in the proposed applications of nanomaterials in the field of medicine due to their very attractive physiochemical properties and novel actions such as the ability to reach previously inaccessible targets such as brain. However biological activity of functional molecules bound to nanoparticles and its physiological consequences is still unclear and hence this area requires immediate attention. The functional properties of Human Serum Albumin (HSA) and Bovine Serum Albumin (BSA) bound to silver nanoparticles (~60 nm) have been studied under physiological environment. Esterase activity, binding of drugs (warfarin and ibuprofen), antioxidant activity and copper binding by albumins was evaluated. The catalytic efficiencies of HSA and BSA diminished upon binding to silver nanoparticles. Perturbation in binding of warfarin and ibuprofen, loss of free sulphydryls, antioxidant activity and enhancement of copper binding were observed in albumins bound to nanoparticles. These alterations in functional activity of nanoparticle bound albumins which will have important consequences should be taken into consideration while using nanoparticles for diagnostic and therapeutic purposes.

  19. Protein binding of fentanyl and its metabolite nor-fentanyl in human plasma, albumin and α-1 acid glycoprotein.

    PubMed

    Bista, Sudeep Raj; Haywood, Alison; Hardy, Janet; Lobb, Michael; Tapuni, Angela; Norris, Ross

    2015-03-01

    1.Fentanyl is a highly lipophilic opioid commonly used to treat cancer pain. Plasma protein binding (PPB) of fentanyl in human plasma is reported as 80-85%, however it is unclear whether fentanyl binds primarily to albumin (ALB) or α-1 acid glycoprotein (AAG) and no studies have been conducted on the metabolite, nor-fentanyl. Fentanyl is also known to bind to plasticware and ultrafiltration (UF) devices which impacts adversely on binding experiments. 2.PPB of fentanyl and nor-fentanyl to ALB and AAG in isotonic phosphate buffer solution and seeded human plasma was quantified. PPB was also performed in plasma samples obtained from cancer patients receiving transdermal fentanyl. The adsorption of fentanyl and nor-fentanyl to UF devices and plasticware commonly used in PPB studies was also assessed. 3.Fentanyl was shown to bind primarily to ALB as opposed to AAG, with nor-fentanyl exhibiting negligible binding to plasma proteins. Total PPB of fentanyl was 86-89% in seeded human plasma. PPB in 56 cancer patient samples was 95.1 ± 3.52% for fentanyl and 32.4 ± 21.9% for nor-fentanyl. 4.UF was shown to be a reliable and convenient method for PPB studies, thereby removing the need for complex testing for adsorption of the drug to plasticware during UF.

  20. Biocompatible Size-Defined Dendrimer-Albumin Binding Protein Hybrid Materials as a Versatile Platform for Biomedical Applications.

    PubMed

    Maly, Jan; Stanek, Ondrej; Frolik, Jan; Maly, Marek; Ennen, Franka; Appelhans, Dietmar; Semeradtova, Alena; Wrobel, Dominika; Stofik, Marcel; Knapova, Tereza; Kuchar, Milan; Stastna, Lucie Cervenkova; Cermak, Jan; Sebo, Peter; Maly, Petr

    2016-04-01

    For the design of a biohybrid structure as a ligand-tailored drug delivery system (DDS), it is highly sophisticated to fabricate a DDS based on smoothly controllable conjugation steps. This article reports on the synthesis and the characterization of biohybrid conjugates based on noncovalent conjugation between a multivalent biotinylated and PEGylated poly(amido amine) (PAMAM) dendrimer and a tetrameric streptavidin-small protein binding scaffold. This protein binding scaffold (SA-ABDwt) possesses nM affinity toward human serum albumin (HSA). Thus, well-defined biohybrid structures, finalized by binding of one or two HSA molecules, are available at each conjugation step in a controlled molar ratio. Overall, these biohybrid assemblies can be used for (i) a controlled modification of dendrimers with the HSA molecules to increase their blood-circulation half-life and passive accumulation in tumor; (ii) rendering dendrimers a specific affinity to various ligands based on mutated ABD domain, thus replacing tedious dendrimer-antibody covalent coupling and purification procedures.

  1. Biocompatible Size-Defined Dendrimer-Albumin Binding Protein Hybrid Materials as a Versatile Platform for Biomedical Applications.

    PubMed

    Maly, Jan; Stanek, Ondrej; Frolik, Jan; Maly, Marek; Ennen, Franka; Appelhans, Dietmar; Semeradtova, Alena; Wrobel, Dominika; Stofik, Marcel; Knapova, Tereza; Kuchar, Milan; Stastna, Lucie Cervenkova; Cermak, Jan; Sebo, Peter; Maly, Petr

    2016-04-01

    For the design of a biohybrid structure as a ligand-tailored drug delivery system (DDS), it is highly sophisticated to fabricate a DDS based on smoothly controllable conjugation steps. This article reports on the synthesis and the characterization of biohybrid conjugates based on noncovalent conjugation between a multivalent biotinylated and PEGylated poly(amido amine) (PAMAM) dendrimer and a tetrameric streptavidin-small protein binding scaffold. This protein binding scaffold (SA-ABDwt) possesses nM affinity toward human serum albumin (HSA). Thus, well-defined biohybrid structures, finalized by binding of one or two HSA molecules, are available at each conjugation step in a controlled molar ratio. Overall, these biohybrid assemblies can be used for (i) a controlled modification of dendrimers with the HSA molecules to increase their blood-circulation half-life and passive accumulation in tumor; (ii) rendering dendrimers a specific affinity to various ligands based on mutated ABD domain, thus replacing tedious dendrimer-antibody covalent coupling and purification procedures. PMID:26748571

  2. Malondialdehyde, carbonyl proteins and albumin-disulphide as useful oxidative markers in mild cognitive impairment and Alzheimer's disease.

    PubMed

    Greilberger, J; Koidl, C; Greilberger, M; Lamprecht, M; Schroecksnadel, K; Leblhuber, F; Fuchs, D; Oettl, K

    2008-07-01

    The question arises as to whether oxidative stress has a primary role in neurodegeneration or is a secondary end-stage epiphenomenon. The aim of the present study was to determine oxidative stress parameters like malondialdehyde (MDA), carbonyl proteins (CP) and Albumin-disulphide (Alb-SSR) and relate these parameters to the immune parameter neopterin, folic acid and vitamin B12 as vitamins and homocysteine in patients with neuro-degenerative diseases (NDD), namely mild cognitive impairment (MCI) and Alzheimer's disease (AD) compared to an aged matched control group. MDA, CP and Alb-SSR were significantly increased in the NDD group compared to controls, but not vitamin B12, folic acid and neopterin. Significant correlations were found between CP and Alb-SSR, CP and MDA and between MDA and Alb-SSR including patients with NDD and the control group. These results support the hypothesis that oxidative damage to lipids and proteins is an important early event in the pathogenesis of neurodegenerative diseases.

  3. Denaturation of bovine serum albumin under the action of cetyltrimethylammonium bromide, according to data from fluorescence analysis

    NASA Astrophysics Data System (ADS)

    Vlasova, I. M.; Zhuravleva, V. V.; Saletskii, A. M.

    2013-06-01

    The tryptophan fluorescence of bovine serum albumin (BSA) in solutions with different concentrations of cationic detergent cetyltrimethylammonium bromide (CTAB) at different pH is investigated, providing information on BSA denaturation under the action of CTAB. It is found that BSA denaturation under the action of CTAB at all of the investigated pH values (3.5-8.0) is a single-stage process, as determined by BSA tryptophan fluorescence quenching, by an increased degree of the BSA tryptophan fluorescence polarization, and by the values of the parameters for the rotational diffusion of BSA molecules in CTAB solutions. It is shown that the cationic detergent CTAB is more efficient for BSA denaturation at pH values higher than the BSA isoelectric point (4.9).

  4. Spectroscopic investigation of interaction between bovine serum albumin and amine-functionalized silicon quantum dots.

    PubMed

    Chatterjee, Surajit; Mukherjee, Tushar Kanti

    2014-05-14

    We have investigated the dynamics and mechanistic details of the interaction between bovine serum albumin (BSA) and allylamine-capped silicon quantum dots (Si QDs) by means of fluorescence spectroscopy, circular dichroism (CD), and FTIR spectroscopy. The intrinsic fluorescence of BSA gets quenched in the presence of Si QDs due to ground-state complex formation. The binding stoichiometry and various thermodynamic parameters have been evaluated by using the van't Hoff equation. It has been observed that the association process is driven by a favourable negative enthalpy change with an unfavorable negative entropy change. These results have been explained by considering specific hydrogen bonding interactions between amine moieties (-NH2) of Si QDs and carboxylate groups (-COO(-)) of aspartate (Asp) and glutamate (Glu) residues of BSA. Circular dichroism (CD) and FTIR spectroscopy revealed nominal changes in the secondary structure of the adsorbed proteins due to partial unfolding of the native protein upon surface adsorption while the overall tertiary structure remains close to that of the native state.

  5. Spectroscopic investigation of interaction between bovine serum albumin and amine-functionalized silicon quantum dots.

    PubMed

    Chatterjee, Surajit; Mukherjee, Tushar Kanti

    2014-05-14

    We have investigated the dynamics and mechanistic details of the interaction between bovine serum albumin (BSA) and allylamine-capped silicon quantum dots (Si QDs) by means of fluorescence spectroscopy, circular dichroism (CD), and FTIR spectroscopy. The intrinsic fluorescence of BSA gets quenched in the presence of Si QDs due to ground-state complex formation. The binding stoichiometry and various thermodynamic parameters have been evaluated by using the van't Hoff equation. It has been observed that the association process is driven by a favourable negative enthalpy change with an unfavorable negative entropy change. These results have been explained by considering specific hydrogen bonding interactions between amine moieties (-NH2) of Si QDs and carboxylate groups (-COO(-)) of aspartate (Asp) and glutamate (Glu) residues of BSA. Circular dichroism (CD) and FTIR spectroscopy revealed nominal changes in the secondary structure of the adsorbed proteins due to partial unfolding of the native protein upon surface adsorption while the overall tertiary structure remains close to that of the native state. PMID:24663102

  6. Interaction of bovine serum albumin with N-acyl amino acid based anionic surfactants: Effect of head-group hydrophobicity.

    PubMed

    Ghosh, Subhajit; Dey, Joykrishna

    2015-11-15

    The function of a protein depends upon its structure and surfactant molecules are known to alter protein structure. For this reason protein-surfactant interaction is important in biological, pharmaceutical, and cosmetic industries. In the present work, interactions of a series of anionic surfactants having the same hydrocarbon chain length, but different amino acid head group, such as l-alanine, l-valine, l-leucine, and l-phenylalanine with the transport protein, bovine serum albumin (BSA), were studied at low surfactant concentrations using fluorescence and circular dichroism (CD) spectroscopy, and isothermal titration calorimetry (ITC). The results of fluorescence measurements suggest that the surfactant molecules bind simultaneously to the drug binding site I and II of the protein subdomain IIA and IIIA, respectively. The fluorescence as well as CD spectra suggest that the conformation of BSA goes to a more structured state upon surfactant binding at low concentrations. The binding constants of the surfactants were determined by the use of fluorescence as well as ITC measurements and were compared with that of the corresponding glycine-derived surfactant. The binding constant values clearly indicate a significant head-group effect on the BSA-surfactant interaction and the interaction is mainly hydrophobic in nature.

  7. Evidence of Energy Transfer from Tryptophan to BSA/HSA Protected Gold Nanoclusters

    PubMed Central

    Raut, Sangram; Chib, Rahul; Butler, Susan; Borejdo, Julian; Gryczynski, Zygmunt; Gryczynski, Ignacy

    2015-01-01

    This work reports on the chromophores interactions within protein-protected gold nanoclusters. We conducted spectroscopic studies of fluorescence emissions originated from gold nanoclusters and intrinsic tryptophan (Trp) in BSA or HSA proteins. Both, steady state fluorescence and lifetime measurements show a significant Forster resonance energy transfer (FRET) from Trp to the gold nanocluster. Tryptophan lifetimes in the case of protein-protected gold nanoclusters are 2.6ns and 2.3ns for BSA and HSA Au clusters while 5.8ns for native BSA and 5.6 for native HSA. The apparent distances from Trp to gold nanocluster emission center, we estimated as 24.75A0 for BSA and 23.80A0 for HSA. We also studied a potassium iodide (KI) quenching of protein-protected gold nanoclusters and compared with the quenching of BSA and HAS alone. The rates of Trp quenching were smaller in BSA-Au and HSA-Au nanoclusters than in the case of free proteins, which is consistent with shorter lifetime of quenched Trp(s) and lower accessibility for KI. While Trp residues were quenched by KI, the emissions originated from nanoclusters were practically unquenched. In summary, for BSA and HSA Au clusters, we found 55% and 59% energy transfer efficiency respectively from tryoptophan to gold clusters. We believe this interaction can be used to our advantage in terms of developing resonance energy transfer based sensing applications. PMID:26767113

  8. Evidence of energy transfer from tryptophan to BSA/HSA protected gold nanoclusters

    NASA Astrophysics Data System (ADS)

    Raut, Sangram; Chib, Rahul; Butler, Susan; Borejdo, Julian; Gryczynski, Zygmunt; Gryczynski, Ignacy

    2014-09-01

    This work reports on the chromophores interactions within protein-protected gold nanoclusters. We conducted spectroscopic studies of fluorescence emissions originated from gold nanoclusters and intrinsic tryptophan (Trp) in BSA or HSA proteins. Both steady state fluorescence and lifetime measurements showed a significant Forster Resonance Energy Transfer (FRET) from Trp to the gold nanocluster. Tryptophan lifetimes in the case of protein-protected gold nanoclusters are 2.6 ns and 2.3 ns for BSA and HSA Au clusters while 5.8 ns for native BSA and 5.6 for native HSA. The apparent distances from Trp to gold nanocluster emission center, we estimated as 24.75 Å for BSA and 23.80 Å for HSA. We also studied a potassium iodide (KI) quenching of protein-protected gold nanoclusters and compared with the quenching of BSA and HSA alone. The rates of Trp quenching were smaller in BSA-Au and HSA-Au nanoclusters than in the case of free proteins, which is consistent with shorter lifetime of quenched Trp(s) and lower accessibility for KI. While Trp residues were quenched by KI, the emissions originated from nanoclusters were practically unquenched. In summary, for BSA and HSA Au clusters, we found 55% and 59% energy transfer efficiency respectively from tryoptophan to gold clusters. We believe this interaction can be used to our advantage in terms of developing resonance energy transfer based sensing applications.

  9. The interaction of human serum albumin with selected lanthanide and actinide ions: Binding affinities, protein unfolding and conformational changes.

    PubMed

    Ali, Manjoor; Kumar, Amit; Kumar, Mukesh; Pandey, Badri N

    2016-04-01

    Human serum albumin (HSA), the most abundant soluble protein in blood plays critical roles in transportation of biomolecules and maintenance of osmotic pressure. In view of increasing applications of lanthanides- and actinides-based materials in nuclear energy, space, industries and medical applications, the risk of exposure with these metal ions is a growing concern for human health. In present study, binding interaction of actinides/lanthanides [thorium: Th(IV), uranium: U(VI), lanthanum: La(III), cerium: Ce(III) and (IV)] with HSA and its structural consequences have been investigated. Ultraviolet-visible, Fourier transform-infrared, Raman, Fluorescence and Circular dichroism spectroscopic techniques were applied to study the site of metal ions interaction, binding affinity determination and the effect of metal ions on protein unfolding and HSA conformation. Results showed that these metal ions interacted with carbonyl (CO..:)/amide(N..-H) groups and induced exposure of aromatic residues of HSA. The fluorescence analysis indicated that the actinide binding altered the microenvironment around Trp214 in the subdomain IIA. Binding affinity of U(VI) to HSA was slightly higher than that of Th(IV). Actinides and Ce(IV) altered the secondary conformation of HSA with a significant decrease of α-helix and an increase of β-sheet, turn and random coil structures, indicating a partial unfolding of HSA. A correlation was observed between metal ion's ability to alter HSA conformation and protein unfolding. Both cationic effects and coordination ability of metal ions seemed to determine the consequences of their interaction with HSA. Present study improves our understanding about the protein interaction of these heavy ions and their impact on its secondary structure. In addition, binding characteristics may have important implications for the development of rational antidote for the medical management of health effects of actinides and lanthanides.

  10. The interaction of human serum albumin with selected lanthanide and actinide ions: Binding affinities, protein unfolding and conformational changes.

    PubMed

    Ali, Manjoor; Kumar, Amit; Kumar, Mukesh; Pandey, Badri N

    2016-04-01

    Human serum albumin (HSA), the most abundant soluble protein in blood plays critical roles in transportation of biomolecules and maintenance of osmotic pressure. In view of increasing applications of lanthanides- and actinides-based materials in nuclear energy, space, industries and medical applications, the risk of exposure with these metal ions is a growing concern for human health. In present study, binding interaction of actinides/lanthanides [thorium: Th(IV), uranium: U(VI), lanthanum: La(III), cerium: Ce(III) and (IV)] with HSA and its structural consequences have been investigated. Ultraviolet-visible, Fourier transform-infrared, Raman, Fluorescence and Circular dichroism spectroscopic techniques were applied to study the site of metal ions interaction, binding affinity determination and the effect of metal ions on protein unfolding and HSA conformation. Results showed that these metal ions interacted with carbonyl (CO..:)/amide(N..-H) groups and induced exposure of aromatic residues of HSA. The fluorescence analysis indicated that the actinide binding altered the microenvironment around Trp214 in the subdomain IIA. Binding affinity of U(VI) to HSA was slightly higher than that of Th(IV). Actinides and Ce(IV) altered the secondary conformation of HSA with a significant decrease of α-helix and an increase of β-sheet, turn and random coil structures, indicating a partial unfolding of HSA. A correlation was observed between metal ion's ability to alter HSA conformation and protein unfolding. Both cationic effects and coordination ability of metal ions seemed to determine the consequences of their interaction with HSA. Present study improves our understanding about the protein interaction of these heavy ions and their impact on its secondary structure. In addition, binding characteristics may have important implications for the development of rational antidote for the medical management of health effects of actinides and lanthanides. PMID:26821345

  11. Lipid-rich bovine serum albumin improves the viability and hatching ability of porcine blastocysts produced in vitro

    PubMed Central

    SUZUKI, Chie; SAKAGUCHI, Yosuke; HOSHI, Hiroyoshi; YOSHIOKA, Koji

    2015-01-01

    The effects of lipid-rich bovine serum albumin (LR-BSA) on the development of porcine blastocysts produced in vitro were examined. Addition of 0.5 to 5 mg/ml LR-BSA to porcine blastocyst medium (PBM) from Day 5 (Day 0 = in vitro fertilization) significantly increased the hatching rates of blastocysts on Day 7 and the total cell numbers in Day-7 blastocysts. When Day-5 blastocysts were cultured with PBM alone, PBM containing LR-BSA, recombinant human serum albumin or fatty acid-free BSA, addition of LR-BSA significantly enhanced hatching rates and the cell number in blastocysts that survived compared with other treatments. The diameter, ATP content and numbers of both inner cell mass and total cells in Day-6 and Day-7 blastocysts cultured with PBM containing LR-BSA were significantly higher than in blastocysts cultured with PBM alone, whereas LR-BSA had no effect on mitochondrial membrane potential. The mRNA levels of enzymes involved in fatty acid metabolism and β-oxidation (ACSL1, ACSL3, CPT1, CPT2 and KAT) in Day-7 blastocysts were significantly upregulated by the addition of LR-BSA. The results indicated that LR-BSA enhanced hatching ability and quality of porcine blastocysts produced in vitro, as determined by ATP content, blastocyst diameter and expression levels of the specific genes, suggesting that the stimulatory effects of LR-BSA arise from lipids bound to albumin. PMID:26582048

  12. Förster resonance energy transfer between pyrene and bovine serum albumin: Effect of the hydrophobic pockets of cyclodextrins

    NASA Astrophysics Data System (ADS)

    Maity, Arnab; Mukherjee, Puspal; Das, Tarasankar; Ghosh, Prasun; Purkayastha, Pradipta

    The phenomenon of Förster resonance energy transfer (FRET) between pyrene and bovine serum albumin (BSA) protein in presence of cyclodextrins (CDs) is explored in the present work. CDs provide hydrophobic environment and thus the aromatic molecules get encapsulated in them depending on the relative size and space. In this work we revealed that along with pyrene monomer, the side chains of amino acids in BSA can get trapped partly in the hydrophobic cavities of CDs if space permits. While being encapsulated by β-CD as pyrene monomer, it can interact with the BSA tryptophan moiety exposed toward the aqueous environment to form a dimer through π-π interaction. This, in turn, affects the energy transfer process by reducing the efficiency. On the other hand, pyrene excimer gets encapsulated in a γ-CD molecule due to availability of enough space. The excimer shows a new band at a higher wavelength. This further reduces FRET efficiency due to scarcity of acceptor for the tryptophan moieties in BSA.

  13. Mechanistic Studies on the Photoallergy Mediated by Fenofibric Acid: Photoreactivity with Serum Albumins.

    PubMed

    Vayá, Ignacio; Andreu, Inmaculada; Monje, Vicente T; Jiménez, M Consuelo; Miranda, Miguel A

    2016-01-19

    The photoreactivity of fenofibric acid (FA) in the presence of human and bovine serum albumins (HSA and BSA, respectively) has been investigated by steady-state irradiation, fluorescence, and laser flash photolysis (LFP). Spectroscopic measurements allowed for the determination of a 1:1 stoichiometry for the FA/SA complexes and pointed to a moderate binding of FA to the proteins; by contrast, the FA photoproducts were complexed more efficiently with SAs. Covalent photobinding to the protein, which is directly related to the photoallergic properties of the drug, was detected after long irradiation times and was found to be significantly higher in the case of BSA. Intermolecular FA-amino acid and FA-albumin irradiations resulted in the formation of photoproducts arising from coupling between both moieties, as indicated by mass spectrometric analysis. Mechanistic studies using model drug-amino acid linked systems indicated that the key photochemical step involved in photoallergy is formal hydrogen atom transfer from an amino acid residue to the excited benzophenone chromophore of FA or (more likely) its photoproducts. This results in the formation of caged radical pairs followed by C-C coupling to give covalent photoaducts.

  14. Fabrication Of Nano-Silver Thin Films Using Self Assembly And Its Interaction With Proteins

    SciTech Connect

    Verma, Gunjan; Choudhury, Sipra; Hassan, P. A.

    2010-12-01

    The silver nanoparticle thin films were prepared with an aim to use them for sensing of biomolecules. The monolayers of arachidic acid were deposited on glass plates by Langmuir Blodgett (LB) technique and silver nanoparticles thin films were deposited within the arachidic acid films. Small angle XRD studies confirm the formation of ordered array of nanoparticles. These thin films were treated with a model protein, bovine serum albumin (BSA a natural protein). From the optical absorption spectra a shift in the intensity as well as lambda max ({lambda}max) could be observed when silver thin films were treated with BSA.

  15. FTIR Examination Of Thermal Denaturation And Gel-Formation In Whey Proteins

    NASA Astrophysics Data System (ADS)

    Byler, D. M.; Purcell, James M.

    1989-12-01

    Second derivative Fourier-transform infrared [DR2-FTIR] spectra of β-lactoglobulin [RIG], serum albumin [BSA], and a-lactalbumin [aLA], three proteins found in bovine whey, are markedly different before and after thermal denaturation. In no case, however, do the heat-treated proteins unfold as completely as does alkaline-denatured RLG [1]. The spectra also suggest that, for RLG and BSA, formation of intermolecularly hydrogen-bonded (β-strands precedes the onset of heat-induced gelation.

  16. The preparation and use of fluorescent-protein conjugates for microvascular research.

    PubMed

    McDonagh, P F; Williams, S K

    1984-01-01

    A procedure is described for making large quantities (100 ml) of fluorochrome-labeled albumin. Chromatographic techniques are described for the purification of commercial albumin (BSA) and the purification of albumin from serum. We report experimentally determined optimal conditions for the covalent attachment of fluorescent dyes (rhodamine isothiocyanate (RITC) and fluorescein isothiocyanate (FITC] to albumin. Subsequent removal of all unreacted fluorescent material (UFM) was achieved using charcoal adsorption. We observed no loss of protein following charcoal treatment. The final protein conjugate was analyzed by polyacrylamide gel electrophoresis, gel chromatography, and isoelectric focusing. The conjugates were determined to be free of UFM and homogeneous with respect to molecular weight. However, FITC conjugation lowered the average isoelectric point of albumin by 0.1 to 0.3 pH units. Illustrations of combining fluorescence microscopy with FITC-BSA and RITC-BSA to view microvascular phenomena in skeletal muscle and the heart are given. Knowledge of the biochemical characteristics of the fluorochrome employed is important for proper interpretation of experimental results using this technique.

  17. Preparation and characterization of folate-poly(ethylene glycol)-grafted-trimethylchitosan for intracellular transport of protein through folate receptor-mediated endocytosis.

    PubMed

    Zheng, Yu; Song, Xiangrong; Darby, Michael; Liang, Yufeng; He, Ling; Cai, Zheng; Chen, Qiuhong; Bi, Yueqi; Yang, Xiaojuan; Xu, Jiapeng; Li, Yuanbo; Sun, Yiyi; Lee, Robert J; Hou, Shixiang

    2010-01-01

    To develop a receptor-mediated intracellular delivery system that can transport therapeutic proteins to specific tumor cells, folate-poly(ethylene glycol)-grafted-trimethylchitosan (folate-PEG-g-TMC) was synthesized. Nano-scaled spherical polyelectrolyte complexes between the folate-PEG-g-TMC and fluorescein isothiocyanate conjugated bovine serum albumin (FITC-BSA) were prepared under suitable weight ratio of copolymer to FITC-BSA by ionic interaction between the positively charged copolymers and the negatively charged FITC-BSA. Intracellular uptake of FITC-BSA was specifically enhanced in SKOV3 cells (folate receptor over-expressing cell line) through folate receptor-mediated endocytosis compared with A549 cells (folate receptor deficient cell line). Folate-PEG-g-TMC shows promise for intracellular transport of negatively charged therapeutic proteins into folate receptor over-expressing tumor cells.

  18. Interaction of Di-2-pyridylketone 2-pyridine Carboxylic Acid Hydrazone and Its Copper Complex with BSA: Effect on Antitumor Activity as Revealed by Spectroscopic Studies.

    PubMed

    Li, Cuiping; Huang, Tengfei; Fu, Yun; Liu, Youxun; Zhou, Sufeng; Qi, Zhangyang; Li, Changzheng

    2016-04-28

    The drug, di-2-pyridylketone-2-pyridine carboxylic acid hydrazone (DPPCAH) and its copper complex (DPPCAH-Cu) exhibit significant antitumor activity. However, the mechanism of their pharmacological interaction with the biological molecule bovine serum albumin (BSA) remains poorly understood. The present study elucidates the interactions between the drug and BSA through MTT assays, spectroscopic methods and molecular docking analysis. Our results indicate that BSA could attenuate effect on the cytotoxicity of DPPCAH, but not DPPCAH-Cu. Data from fluorescence quenching measurements demonstrated that both DPPCAH and DPPCAH-Cu could bind to BSA, with a reversed effect on the environment of tryptophan residues in polarity. CD spectra revealed that the DPPCAH-Cu exerted a slightly stronger effect on the secondary structure of BSA than DPPCAH. The association constant of DPPCAH with BSA was greater than that of DPPCAH-Cu. Docking studies indicated that the binding of DPPCAH to BSA involved a greater number of hydrogen bonds compared to DPPCAH-Cu. The calculated distances between bound ligands and tryptophans in BSA were in agreement with fluorescence resonance energy transfer results. Thus, the binding affinity of the drug (DPPCAH or DPPCAH-Cu) with BSA partially contributes to its antitumor activity; the greater the drug affinity is to BSA, the less is its antitumor activity.

  19. Interaction of Di-2-pyridylketone 2-pyridine Carboxylic Acid Hydrazone and Its Copper Complex with BSA: Effect on Antitumor Activity as Revealed by Spectroscopic Studies.

    PubMed

    Li, Cuiping; Huang, Tengfei; Fu, Yun; Liu, Youxun; Zhou, Sufeng; Qi, Zhangyang; Li, Changzheng

    2016-01-01

    The drug, di-2-pyridylketone-2-pyridine carboxylic acid hydrazone (DPPCAH) and its copper complex (DPPCAH-Cu) exhibit significant antitumor activity. However, the mechanism of their pharmacological interaction with the biological molecule bovine serum albumin (BSA) remains poorly understood. The present study elucidates the interactions between the drug and BSA through MTT assays, spectroscopic methods and molecular docking analysis. Our results indicate that BSA could attenuate effect on the cytotoxicity of DPPCAH, but not DPPCAH-Cu. Data from fluorescence quenching measurements demonstrated that both DPPCAH and DPPCAH-Cu could bind to BSA, with a reversed effect on the environment of tryptophan residues in polarity. CD spectra revealed that the DPPCAH-Cu exerted a slightly stronger effect on the secondary structure of BSA than DPPCAH. The association constant of DPPCAH with BSA was greater than that of DPPCAH-Cu. Docking studies indicated that the binding of DPPCAH to BSA involved a greater number of hydrogen bonds compared to DPPCAH-Cu. The calculated distances between bound ligands and tryptophans in BSA were in agreement with fluorescence resonance energy transfer results. Thus, the binding affinity of the drug (DPPCAH or DPPCAH-Cu) with BSA partially contributes to its antitumor activity; the greater the drug affinity is to BSA, the less is its antitumor activity. PMID:27136517

  20. Studies of interaction between colchicine and bovine serum albumin by fluorescence quenching method

    NASA Astrophysics Data System (ADS)

    Hu, Yan-Jun; Liu, Yi; Zhang, Li-Xia; Zhao, Ru-Ming; Qu, Song-Sheng

    2005-08-01

    We investigated the interaction between colchicine and bovine serum albumin (BSA) by fluorescence and UV-Vis absorption spectroscopy. In the mechanism discussion, it was proved that the fluorescence quenching of BSA by colchicine is a result of the formation of colchicine-BSA complex; van der Waals interactions and hydrogen bonds play a major role in stabilizing the complex. The modified Stern-Volmer quenching constant Ka and corresponding thermodynamic parameters ΔH, ΔG, ΔS at different temperatures were calculated. The distance r between donor (BSA) and acceptor (colchicine) was obtained according to fluorescence resonance energy transfer (FRET).

  1. Scavenger receptor for aldehyde-modified proteins.

    PubMed

    Horiuchi, S; Murakami, M; Takata, K; Morino, Y

    1986-04-15

    This paper describes an unexpectedly broad ligand specificity of a scavenger receptor of sinusoidal liver cells that is responsible for endocytic uptake of formaldehyde-treated bovine serum albumin (f-Alb). Binding of 125I-f-Alb to the isolated cells was effectively inhibited by bovine serum albumin (BSA) modified with aliphatic aldehydes such as glycolaldehye, DL-glyceraldehyde, and propionaldehyde whereas albumin preparations modified by aromatic aldehydes such as pyridoxal, pyridoxal phosphate, salicylaldehyde, and benzaldehyde did not affect this binding process. Binding of 125I-glycolaldehyde-treated BSA to the cells exhibited a saturation kinetics with an apparent Kd = 3.3 micrograms of the ligand/ml. This binding process was inhibited by unlabeled f-Alb as well as by the antibody raised against the f-Alb receptor. Indeed, 125I-glycolaldehyde-treated BSA underwent a rapid plasma clearance (t1/2 approximately 2 min) which was markedly retarded by unlabeled f-Alb. Upon treatment by these aldehydes, other proteins such as ovalbumin, soybean trypsin inhibitor, and hemoglobin were also converted to active ligands for the f-Alb receptor, while no ligand activity was generated with gamma-globulin and RNase A. These results clearly show that the f-Alb receptor, originally described as being specific for f-Alb, exhibits a broad ligand specificity in terms of both aldehydes and proteins and, hence, should be described as a scavenger receptor for aldehyde-modified proteins.

  2. Effect of proteins with different isoelectric points on the gene transfection efficiency mediated by stearic acid grafted chitosan oligosaccharide micelles.

    PubMed

    Yan, Jingjing; Du, Yong-Zhong; Chen, Feng-Ying; You, Jian; Yuan, Hong; Hu, Fu-Qiang

    2013-07-01

    A stearic acid-grafted chitosan oligosaccharide (CS-SA) micelle has been demonstrated as an effective gene carrier in vitro and in vivo. Although being advantageous for DNA package, protection, and excellent cellular internalization, a CS-SA based delivery system may lead to difficulties in the dissociation of polymer/DNA complexes in intracells. In this research, bovine serum albumin (BSA) with a different isoelectric point value (4.7, 6.0 and 9.3) was synthesized and incorporated into a CS-SA based gene delivery system. CS-SA/DNA binary complexes and CS-SA/BSA/DNA ternary complexes were then prepared and characterized. The binding ability of the CS-SA vector with DNA was not affected by the incorporation of BSA. However, referring to the transfection activity, the BSA of different isoelectric point value (pI) had a distinct influence on the CS-SA/BSA/DNA complexes. CS-SA/BSA(4.7)/DNA and CS-SA/BSA(6.0)/DNA complexes had better transfection efficiency than binary complexes, especially CS-SA/BSA(4.7)/DNA complexes which showed the highest transfection efficiency. On the contrary, CS-SA/BSA(9.3)/DNA complexes had undesirable performances. Interestingly, the incorporation of BSA(4.7) in CS-SA/DNA complexes significantly enhanced the dissociation of polymer/DNA complexes and improved the release of DNA intracellular without influencing their cellular uptake. The aforementioned results indicated that the acid group in protein played an important role in enhancing the transfection efficiency of CS/BSA/DNA complexes, and the study provided guidelines in the design of an efficient vector for DNA transfection.

  3. Effect of proteins with different isoelectric points on the gene transfection efficiency mediated by stearic acid grafted chitosan oligosaccharide micelles.

    PubMed

    Yan, Jingjing; Du, Yong-Zhong; Chen, Feng-Ying; You, Jian; Yuan, Hong; Hu, Fu-Qiang

    2013-07-01

    A stearic acid-grafted chitosan oligosaccharide (CS-SA) micelle has been demonstrated as an effective gene carrier in vitro and in vivo. Although being advantageous for DNA package, protection, and excellent cellular internalization, a CS-SA based delivery system may lead to difficulties in the dissociation of polymer/DNA complexes in intracells. In this research, bovine serum albumin (BSA) with a different isoelectric point value (4.7, 6.0 and 9.3) was synthesized and incorporated into a CS-SA based gene delivery system. CS-SA/DNA binary complexes and CS-SA/BSA/DNA ternary complexes were then prepared and characterized. The binding ability of the CS-SA vector with DNA was not affected by the incorporation of BSA. However, referring to the transfection activity, the BSA of different isoelectric point value (pI) had a distinct influence on the CS-SA/BSA/DNA complexes. CS-SA/BSA(4.7)/DNA and CS-SA/BSA(6.0)/DNA complexes had better transfection efficiency than binary complexes, especially CS-SA/BSA(4.7)/DNA complexes which showed the highest transfection efficiency. On the contrary, CS-SA/BSA(9.3)/DNA complexes had undesirable performances. Interestingly, the incorporation of BSA(4.7) in CS-SA/DNA complexes significantly enhanced the dissociation of polymer/DNA complexes and improved the release of DNA intracellular without influencing their cellular uptake. The aforementioned results indicated that the acid group in protein played an important role in enhancing the transfection efficiency of CS/BSA/DNA complexes, and the study provided guidelines in the design of an efficient vector for DNA transfection. PMID:23679858

  4. Protein-templated biomimetic silica nanoparticles.

    PubMed

    Jackson, Erienne; Ferrari, Mariana; Cuestas-Ayllon, Carlos; Fernández-Pacheco, Rodrigo; Perez-Carvajal, Javier; de la Fuente, Jesús M; Grazú, Valeria; Betancor, Lorena

    2015-03-31

    Biomimetic silica particles can be synthesized as a nanosized material within minutes in a process mimicked from living organisms such as diatoms and sponges. In this work, we have studied the effect of bovine serum albumin (BSA) as a template to direct the synthesis of silica nanoparticles (NPs) with the potential to associate proteins on its surface. Our approach enables the formation of spheres with different physicochemical properties. Particles using BSA as a protein template were smaller (∼250-380 nm) and were more monodisperse than those lacking the proteic core (∼700-1000 nm) as seen by dynamic light scattering (DLS), scanning electron microscopy (SEM), and environmental scanning electron microscopy (ESEM) analysis. The absence of BSA during synthesis produced silica nanoparticles without any porosity that was detectable by nitrogen adsorption, whereas particles containing BSA developed porosity in the range of 4 to 5 nm which collapsed on the removal of BSA, thus producing smaller pores. These results were in accordance with the pore size calculated by high-resolution transmission electron microscopy (HTEM). The reproducibility of the BSA-templated nanoparticle properties was determined by analyzing four batches of independent synthesizing experiments that maintained their properties. The high positive superficial charge of the nanoparticles facilitated adsorption under mild conditions of a range of proteins from an E. coli extract and a commercial preparation of laccase from Trametes versicolor. All of the proteins were quantitatively desorbed. Experiments conducted showed the reusability of the particles as supports for the ionic adsorption of the biomolecules. The protein loading capacity of the BSA-based biomimetic particles was determined using laccase as 98.7 ± 6.6 mg·g(-1) of particles.

  5. Characterization of the binding of chrysoidine, an illegal food additive to bovine serum albumin.

    PubMed

    Yang, Bingjun; Hao, Fang; Li, Jiarong; Wei, Kai; Wang, Wenyu; Liu, Rutao

    2014-03-01

    Chrysoidine is an industrial azo dye and the presence of chrysoidine in water and food has become an environmental concern due to its negative effects on human beings. Binding of dyes to serum albumins significantly influence their absorption, distribution, metabolism, and excretion properties. In this work, the interactions of chrysoidine with bovine serum albumin (BSA) were explored. Isothermal titration calorimetry results reveal the binding stoichiometry of chrysoidine to BSA is 1:15.5, and van der Waals and hydrogen bonding interactions are the major driving force in the binding of chrysoidine to BSA. Molecular docking simulations show that chrysoidine binds to BSA at a cavity close to Sudlow site I in domain IIA. However, no detectable conformational change of BSA occurs in the presence of chrysoidine as revealed by UV-vis absorption, circular dichroism and fluorescence spectroscopy studies.

  6. An improved method for removal of azo dye orange II from textile effluent using albumin as sorbent.

    PubMed

    Ohashi, Tadashi; Jara, Alícia M T; Batista, Anabelle C L; Franco, Luciana O; Barbosa Lima, Marcos A; Benachour, Mohand; Alves da Silva, Carlos A; Campos-Takaki, Galba M

    2012-11-30

    Azo dyes are generally resistant to biodegradation due to their complex structures. Acid orange II is one of the most widely used dyes in the textile industry. The influence of bovine serum albumin (BSA) in different concentrations, pH, and time of contact on Orange II was investigated using kinetics and adsorption-isotherm experiments. The results showed that the maximum colour removed from dye/albumin was 99.50% and that a stable dye-protein complex had been formed at pH 3.5 and in a proportion of 1:3 (v/v), respectively. The synthetic effluent did not show toxicity to the microcrustacean Artemia salina, and showed a CL₅₀ equal to 97 µg/mL to azo dye orange II. Additionally, the methodology was effective in removing the maximum of orange II using BSA by adsorption at pH 3.5 which mainly attracted ions to the azo dye during the adsorption process. This suggests that this form of treatment is economical and easy to use which potentially could lead to bovine serum albumin being used as a sorbent for azo dyes.

  7. Binding affinities of Schiff base Fe(II) complex with BSA and calf-thymus DNA: Spectroscopic investigations and molecular docking analysis.

    PubMed

    Rudra, Suparna; Dasmandal, Somnath; Patra, Chiranjit; Kundu, Arjama; Mahapatra, Ambikesh

    2016-09-01

    The binding interaction of a synthesized Schiff base Fe(II) complex with biological macromolecules viz., bovine serum albumin (BSA) and calf thymus(ct)-DNA have been investigated using different spectroscopic techniques coupled with viscosity measurements at physiological pH and 298K. Regular amendments in emission intensities of BSA upon the action of the complex indicate significant interaction between them, and the binding interaction have been characterized by Stern Volmer plots and thermodynamic binding parameters. On the basis of this quenching technique one binding site with binding constant (Kb=(7.6±0.21)×10(5)) between complex and protein have been obtained at 298K. Time-resolved fluorescence studies have also been encountered to understand the mechanism of quenching induced by the complex. Binding affinities of the complex to the fluorophores of BSA namely tryptophan (Trp) and tyrosine (Tyr) have been judged by synchronous fluorescence studies. Secondary structural changes of BSA rooted by the complex has been revealed by CD spectra. On the other hand, hypochromicity of absorption spectra of the complex with the addition of ct-DNA and the gradual reduction in emission intensities of ethidium bromide bound ct-DNA in presence of the complex indicate noticeable interaction between ct-DNA and the complex with the binding constant (4.2±0.11)×10(6)M(-1). Life-time measurements have been studied to determine the relative amplitude of binding of the complex to ct-DNA base pairs. Mode of binding interaction of the complex with ct-DNA has been deciphered by viscosity measurements. CD spectra have also been used to understand the changes in ct-DNA structure upon binding with the metal complex. Density functional theory (DFT) and molecular docking analysis have been employed in highlighting the interactive phenomenon and binding location of the complex with the macromolecules.

  8. Binding affinities of Schiff base Fe(II) complex with BSA and calf-thymus DNA: Spectroscopic investigations and molecular docking analysis

    NASA Astrophysics Data System (ADS)

    Rudra, Suparna; Dasmandal, Somnath; Patra, Chiranjit; Kundu, Arjama; Mahapatra, Ambikesh

    2016-09-01

    The binding interaction of a synthesized Schiff base Fe(II) complex with biological macromolecules viz., bovine serum albumin (BSA) and calf thymus(ct)-DNA have been investigated using different spectroscopic techniques coupled with viscosity measurements at physiological pH and 298 K. Regular amendments in emission intensities of BSA upon the action of the complex indicate significant interaction between them, and the binding interaction have been characterized by Stern Volmer plots and thermodynamic binding parameters. On the basis of this quenching technique one binding site with binding constant (Kb = (7.6 ± 0.21) × 105) between complex and protein have been obtained at 298 K. Time-resolved fluorescence studies have also been encountered to understand the mechanism of quenching induced by the complex. Binding affinities of the complex to the fluorophores of BSA namely tryptophan (Trp) and tyrosine (Tyr) have been judged by synchronous fluorescence studies. Secondary structural changes of BSA rooted by the complex has been revealed by CD spectra. On the other hand, hypochromicity of absorption spectra of the complex with the addition of ct-DNA and the gradual reduction in emission intensities of ethidium bromide bound ct-DNA in presence of the complex indicate noticeable interaction between ct-DNA and the complex with the binding constant (4.2 ± 0.11) × 106 M- 1. Life-time measurements have been studied to determine the relative amplitude of binding of the complex to ct-DNA base pairs. Mode of binding interaction of the complex with ct-DNA has been deciphered by viscosity measurements. CD spectra have also been used to understand the changes in ct-DNA structure upon binding with the metal complex. Density functional theory (DFT) and molecular docking analysis have been employed in highlighting the interactive phenomenon and binding location of the complex with the macromolecules.

  9. Reversible immobilization of BSA on Cu-chelated PAMAM dendrimer modified iron oxide nanoparticles

    NASA Astrophysics Data System (ADS)

    Demir, M.; Şenel, M.; Baykal, A.

    2014-09-01

    In this study, polyamidoamine (PAMAM) dendrimer coated superparamagnetite nanoparticles were synthesized by growing of PAMAM on amino-silane coated iron oxide nanoparticles. The PAMAM modified superparamagnetite nanoparticles were used as reversible protein immobilization host materials. During the reversible immobilization studies the effect of different metal ions such as; Cu+2, Zn+2, Co+2, Ni+2 on immobilization efficiency of BSA were evaluated. The maximum BSA adsorption capacity of the PAMAM-MNP- Cu+2 beads was observed to be 52.84 mg/g (BSA/PAMAM-MNP) at pH 7.0. Various characteristics of immobilized BSA such as; effect of generation, effect of pH, BSA concentration, temperature, salt concentration and reusability of PAMAM-MNP were evaluated.

  10. Facile synthesis of hairy core-shell structured magnetic polymer submicrospheres and their adsorption of bovine serum albumin.

    PubMed

    Yan, Xianming; Kong, Juan; Yang, Chongchong; Fu, Guoqi

    2015-05-01

    Highly magnetic polymer submicrospheres with a hairy core-shell structure were facilely synthesized by combining distillation-precipitation polymerization (DPP) with subsequent surface-initiated atom transfer radical polymerization (SI-ATRP), and then investigated for protein adsorption. A robust polymer shell consisting of poly(divinylbenzene-co-chloromethylstyrene) (P(DVB-co-CMS)) was coated on superparamagnetic submicrometer-sized magnetite colloid nanocrystal clusters (MCNCs) via DPP. With the benzyl chloride groups on the shell as initiator, poly(2-(dimethylamino) ethyl methacrylate) (PDMAEMA) hairs were grafted by SI-ATRP approach. The resulting hairy core-shell structured Fe3O4@ P(DVB-co-CMS)-PDMAEMA microspheres showed pH- and temperature-sensitivity, and high-magnetization. The composite microspheres were further investigated for adsorption of a typical acidic protein, i.e. bovine serum albumin (BSA). They exhibited a high binding capacity up to over 660 mg/g (corresponding to 158 DMAEMA monomer units cooperating for binding one BSA molecule) and could rapidly reach binding equilibrium within 5 min. Moreover, the adsorption of BSA was found to be remarkably dependent on the pH and salt concentration of the protein solutions, and the bound protein could be quantitatively desorbed by washing with a medium with lowered pH or raised salt concentration.

  11. Facile synthesis of hairy core-shell structured magnetic polymer submicrospheres and their adsorption of bovine serum albumin.

    PubMed

    Yan, Xianming; Kong, Juan; Yang, Chongchong; Fu, Guoqi

    2015-05-01

    Highly magnetic polymer submicrospheres with a hairy core-shell structure were facilely synthesized by combining distillation-precipitation polymerization (DPP) with subsequent surface-initiated atom transfer radical polymerization (SI-ATRP), and then investigated for protein adsorption. A robust polymer shell consisting of poly(divinylbenzene-co-chloromethylstyrene) (P(DVB-co-CMS)) was coated on superparamagnetic submicrometer-sized magnetite colloid nanocrystal clusters (MCNCs) via DPP. With the benzyl chloride groups on the shell as initiator, poly(2-(dimethylamino) ethyl methacrylate) (PDMAEMA) hairs were grafted by SI-ATRP approach. The resulting hairy core-shell structured Fe3O4@ P(DVB-co-CMS)-PDMAEMA microspheres showed pH- and temperature-sensitivity, and high-magnetization. The composite microspheres were further investigated for adsorption of a typical acidic protein, i.e. bovine serum albumin (BSA). They exhibited a high binding capacity up to over 660 mg/g (corresponding to 158 DMAEMA monomer units cooperating for binding one BSA molecule) and could rapidly reach binding equilibrium within 5 min. Moreover, the adsorption of BSA was found to be remarkably dependent on the pH and salt concentration of the protein solutions, and the bound protein could be quantitatively desorbed by washing with a medium with lowered pH or raised salt concentration. PMID:25594881

  12. Solute effects on the irreversible aggregation of serum albumin.

    PubMed

    Bagger, Heidi L; Øgendal, Lars H; Westh, Peter

    2007-10-01

    Thermal stress on bovine serum albumin (BSA) promotes protein aggregation through the formation of intermolecular beta-sheets. We have used light scattering and chromatography to study effects of (<1 M) Na(2)SO(4), NaSCN, sucrose, sorbitol and urea on the rate of the thermal aggregation. Both salts were strong inhibitors of BSA aggregation and they reduced both the size and number (concentration) of aggregate particles compared to non-ionic solutes (or pure buffer). Hence, the salts appear to suppress both nucleation- and growth rate. The non-electrolyte additives reduced the initial aggregation rate (compared to pure buffer), but did not significantly limit the extent of aggregation in samples quenched after 27 min. heat exposure (40-50% aggregation in all samples). The non-electrolytes did, however, modify the aggregation process as they consistently brought about smaller but more concentrated aggregates than pure buffer. The results are discussed along the lines of linkage- and transition state theories. In this framework, the rate of the aggregation process is governed by the equilibrium between a thermally denatured state (D) and the transition state D( not equal). Thus, the effect of a solute relies on its preferential interactions with respectively D and D( not equal). The current results do not show any correlation between the solutes' preferential interactions with native BSA and their effect on the rate of aggregation. This suggests that non-specific, "Hofmeister-type" interactions, which scale with the solvent accessible surface area, are of minor importance. Rather, salt induced suppression of aggregation is suggested to depend on the modulation of specific electrostatic forces in the D( not equal) state.

  13. Low protein catabolic rate and serum albumin correlate with increased mortality and abdominal complications in peritoneal dialysis patients.

    PubMed

    Germain, M; Harlow, P; Mulhern, J; Lipkowitz, G; Braden, G

    1992-01-01

    We retrospectively reviewed 167 consecutive peritoneal dialysis patients with regard to serum albumin (Alb), mortality and abdominal complications. In addition, 25 patients were studied with serial measurements of urea kinetics. The patients were divided into four groups based on their dialysis index (DI) and normalized protein catabolic rate (NPCR) (Table I). 12/167 patients were identified with abdominal catastrophes. Before these complications occurred, the M Alb in this group was 2.67 + 0.24 (compared to age, sex and disease matched controls of 3.55 + .11 P < .05). Six of these patients died from abdominal complications. In the 26 patients with serial urea kinetic studies, 4/11 patients in group IV died (low NPCR and low DI) (P < .05 compared to Group I, II or III). We conclude that urea kinetic modeling is predictive of outcome in those patients with presumed poor nutrition and inadequate dialysis and that abdominal catastrophes are more common in those patients with poor nutrition. Prospective interventional studies should be designed in an attempt to improve the poor outcome in this group of patients.

  14. Preparation and characterization of bovine serum albumin surface-imprinted thermosensitive magnetic polymer microsphere and its application for protein recognition.

    PubMed

    Li, Xiangjie; Zhang, Baoliang; Li, Wei; Lei, Xingfeng; Fan, Xinlong; Tian, Lei; Zhang, Hepeng; Zhang, Qiuyu

    2014-01-15

    A novel bovine serum albumin surface-imprinted thermosensitive magnetic composite microsphere was successfully prepared by the surface grafting copolymerization method in the presence of temperature-sensitive monomer N-isopropylacrylamide (NIPAM), functional monomer methacrylic acid (MAA) and cross-linking agent N,N'-methylenebisacrylamide (MBA). The structure and component of the thermosensitive magnetic molecularly imprinted microsphere were investigated by transmission electron microscopy (TEM), Fourier transform infrared (FT-IR), vibrating sample magnetometer (VSM) and thermogravimetric analysis (TGA). The results of thermosensitivity, adsorption capacity, selectivity and reusability showed the formation of a thermosensitivity grafting polymer layer P(NIPAM-MAA-MBA) on the surface of Fe3O4@SiO2 and the good adsorption capacity and specific recognition for template protein. When the adsorption temperature was higher than the lower critical solution temperature (LCST) of poly(N-isopropylacrylamide) (PNIPAM), shape memory effect of imprinted cavities would be more effective. In other words, it was more conducive to capture template molecules under this condition and the imprinting factor would be higher. On the other hand, when the desorption temperature was lower than LCST of PNIPAM, the decrease of shape memory effect between imprinted cavities and template molecules would facilitate the release of template molecules from the imprinted cavities. Based on this property, the adsorption and desorption of template molecules could be regulated by system temperature indirectly which benefited from the existence of thermosensitivity imprinting layer.

  15. Biological applications of zinc imidazole framework through protein encapsulation

    NASA Astrophysics Data System (ADS)

    Kumar, Pawan; Bansal, Vasudha; Paul, A. K.; Bharadwaj, Lalit M.; Deep, Akash; Kim, Ki-Hyun

    2016-10-01

    The robustness of biomolecules is always a significant challenge in the application of biostorage in biotechnology or pharmaceutical research. To learn more about biostorage in porous materials, we investigated the feasibility of using zeolite imidazolate framework (ZIF-8) with respect to protein encapsulation. Here, bovine serum albumin (BSA) was selected as a model protein for encapsulation with the synthesis of ZIF-8 using water as a media. ZIF-8 exhibited excellent protein adsorption capacity through successive adsorption of free BSA with the formation of hollow crystals. The loading of protein in ZIF-8 crystals is affected by the molecular weight due to diffusion-limited permeation inside the crystals and also by the affinity of the protein to the pendent group on the ZIF-8 surface. The polar nature of BSA not only supported adsorption on the solid surface, but also enhanced the affinity of crystal spheres through weak coordination interactions with the ZIF-8 framework. The novel approach tested in this study was therefore successful in achieving protein encapsulation with porous, biocompatible, and decomposable microcrystalline ZIF-8. The presence of both BSA and FITC-BSA in ZIF-8 was confirmed consistently by spectroscopy as well as optical and electron microscopy.

  16. Biological applications of zinc imidazole framework through protein encapsulation

    NASA Astrophysics Data System (ADS)

    Kumar, Pawan; Bansal, Vasudha; Paul, A. K.; Bharadwaj, Lalit M.; Deep, Akash; Kim, Ki-Hyun

    2015-12-01

    The robustness of biomolecules is always a significant challenge in the application of biostorage in biotechnology or pharmaceutical research. To learn more about biostorage in porous materials, we investigated the feasibility of using zeolite imidazolate framework (ZIF-8) with respect to protein encapsulation. Here, bovine serum albumin (BSA) was selected as a model protein for encapsulation with the synthesis of ZIF-8 using water as a media. ZIF-8 exhibited excellent protein adsorption capacity through successive adsorption of free BSA with the formation of hollow crystals. The loading of protein in ZIF-8 crystals is affected by the molecular weight due to diffusion-limited permeation inside the crystals and also by the affinity of the protein to the pendent group on the ZIF-8 surface. The polar nature of BSA not only supported adsorption on the solid surface, but also enhanced the affinity of crystal spheres through weak coordination interactions with the ZIF-8 framework. The novel approach tested in this study was therefore successful in achieving protein encapsulation with porous, biocompatible, and decomposable microcrystalline ZIF-8. The presence of both BSA and FITC-BSA in ZIF-8 was confirmed consistently by spectroscopy as well as optical and electron microscopy.

  17. Chemical conversion of benzo(e)pyrene by aqueous serum albumin to pyrene-like product: fluorescence method

    SciTech Connect

    Srinivasan, B.N.; Fujimori, E.

    1980-01-01

    The interaction of benzo(e)pyrene, an atmospheric pollutant formed during the burning of organic materials, and bovine serum albumin (BSA) was studied to determine a pyrene-type metabolite is formed. (ACR)

  18. Interaction of cationic surfactant cethyltrimethylammonium bromide with bovine serum albumin in dependence on pH: A study of tryptophan fluorescence

    NASA Astrophysics Data System (ADS)

    Vlasova, Irina M.; Zhuravleva, Valeria V.; Vlasov, Alexander A.; Saletsky, Alexander M.

    2013-02-01

    The interaction of cationic surfactant cethyltrimethylammonium bromide (CTAB) with bovine serum albumin (BSA) at various values of pH has been studied using steady-state non-polarized tryptophan fluorescence of BSA and polarized tryptophan fluorescence of BSA. By analysis of intensity of tryptophan fluorescence of BSA, by analysis of position of maximum of spectrum of BSA tryptophan fluorescence, by analysis of polarization of BSA tryptophan fluorescence the qualitative rearrangements of BSA globules at denaturation under action of CTAB are registered. The estimation of parameters of rotational diffusion of BSA molecules helps one to determine the quantitative changes of size of BSA at CTAB-induced denaturation. It is shown that denaturation of BSA, taking place at interaction of cationic surfactant CTAB with BSA, has one-stage mono-phase character. At interaction of CTAB with BSA the deepest denaturation of BSA is reached at 4 mM CTAB (at pH 3.5-8.0). More intensive denaturation of BSA under action of CTAB takes place at values of pH, higher than the isoelectric point of BSA.

  19. In vitro evaluation of doxorubicin-incorporated magnetic albumin nanospheres.

    PubMed

    Zeybek, Ayça; Şanlı-Mohamed, Gülşah; Ak, Güliz; Yılmaz, Habibe; Şanlıer, Şenay H

    2014-07-01

    Magnetic albumin nanospheres that incorporate doxorubicin (M-DOX-BSA-NPs) were prepared previously by our research group to develop magnetically responsive drug carrier system. This nanocarrier was synthesized as a drug delivery system for targeted chemotherapy. In this work, cytotoxic effects of doxorubicin (DOX)-loaded/unloaded or magnetic/non-magnetic nanoparticles and free DOX against PC-3 cells and A549 cells were determined with the MTT test and the results were compared with each other. DOX-loaded magnetic albumin nanospheres (M-DOX-BSA-NPs) were found more cytotoxic than other formulations. The quantitative data obtained from flow cytometry analysis further verified the higher targeting and killing ability of M-DOX-BSA-NPs than free DOX on both of the cancer cell lines. Additionally, the results of cell cycle analysis have showed that M-DOX-BSA-NPs affected G1 and G2 phases. Finally, cell images were obtained using spin-disk confocal microscopy, and cellular uptake of M-DOX-BSA-NPs was visualized. The findings of this study suggest that M-DOX-BSA-NPs represent a potential doxorubicin delivery system for targeted drug transport into prostate and lung cancer cells.

  20. Real-Time Trapping of Intact Singly-Charged Bovine Serum Albumin Proteins with a Big Frequency-Adjusted Quadrupole

    SciTech Connect

    Koizumi, Hideya; Whitten, William B; Reilly, Pete

    2008-01-01

    High-resolution real-time particle mass measurements have not been achievable because the enormous amount of kinetic energy imparted to the particles upon expansion into vacuum competes with and overwhelms the forces applied to the charged particles within the mass spectrometer. It is possible to reduce the kinetic energy of a collimated particulate ion beam through collisions with a buffer gas while radially constraining their motion using a quadrupole guide or trap over a limited mass range. Controlling the pressure drop of the final expansion into a quadrupole trap permits a much broader mass range at the cost of sacrificing collimation. To achieve high-resolution mass analysis of massive particulate ions, an efficient trap with a large tolerance for radial divergence of the injected ions was developed that permits trapping a large range of ions for on-demand injection into an awaiting mass analyzer. The design specifications required that frequency of the trapping potential be adjustable to cover a large mass range and the trap radius be increased to increase the tolerance to divergent ion injection. The large-radius linear quadrupole ion trap was demonstrated by trapping singly-charged bovine serum albumin ions for on-demand injection into a mass analyzer. Additionally, this work demonstrates the ability to measure an electrophoretic mobility cross section (or ion mobility) of singly-charged intact proteins in the low-pressure regime. This work represents a large step toward the goal of high-resolution analysis of intact proteins, RNA, DNA, and viruses.

  1. Study on the structural changes of bovine serum albumin with effects on polydatin binding by a multitechnique approach

    NASA Astrophysics Data System (ADS)

    Peng, Xialian; Yao, Di; Pan, Yingming; Yu, Qing; Ni, Shouhai; Bian, Hedong; Huang, Fuping; Liang, Hong

    2011-10-01

    Polydatin is a traditional Chinese medicine which shows effective biological activity as antimicrobial and antiviral agent. The secondary structure changes of bovine serum albumin (BSA) were investigated by the methods of Fourier transform infrared spectroscopy (FT-IR), circular dichroism (CD) and Raman spectroscopy. The experimental results indicated that polydatin changed the secondary structure of BSA. The presence of polydatin decreased α-helix content of BSA. The conformations of disulfide bridges and the microenvironment of Tyr, Trp residues were also changed.

  2. Adsorption of bovine serum albumin onto synthetic Fe-doped geomimetic chrysotile

    PubMed Central

    Adamiano, Alessio; Lesci, Isidoro Giorgio; Fabbri, Daniele; Roveri, Norberto

    2015-01-01

    Synthetic stoichiometric and Fe-doped geomimetic chrysotile nanocrystals represent a reference standard to investigate the health hazard associated with mineral asbestos fibres. Experimental evidence suggests that the generation of reactive oxygen species and other radicals, catalysed by iron ions at the fibre surface, plays an important role in asbestos-induced cytotoxicity and genotoxicity. In this study, structural modification of bovine serum albumin (BSA) adsorbed onto synthetic chrysotile doped with different amounts of Fe has been investigated by Fourier transform infrared spectroscopy (FT-IR), thermal gravimetric analysis (TGA) and analytical pyrolysis coupled with gas chromatography–mass spectrometry. FT-IR data evidenced a marked increase in disordered structures like random coil and β-turn of BSA–nanocrystal adduct with 0.52 wt% of Fe doped. The TGA profile of the BSA revealed that its interaction with the synthetic chrysotile surface was strongly affected by the substitution of Fe into the chrysotile structure. The 2,5-diketopiperazine yields, formed upon thermal degradation of the polypeptide chain (pyrolysis–gas chromatography), changed when the BSA was adsorbed on the nanofibres. In general, results suggested that minute amount (less than 1 wt%) of Fe doping in chrysotile affected the protein–nanofibre interactions, supporting the role that this element may play in asbestos toxicity. The catalytic role of iron and the consequent unfolding of protein due to the structural surface modification of nanofibres were also evaluated. PMID:26018963

  3. nanoparticles via a facile one-step solvothermal process for adsorption of bovine serum albumin

    NASA Astrophysics Data System (ADS)

    Shen, Mao; Yu, Yujing; Fan, Guodong; Chen, Guang; Jin, Ying min; Tang, Wenyuan; Jia, Wenping

    2014-06-01

    Preparation of magnetic nanoparticles coated with chitosan (CS-coated Fe3O4 NPs) in one step by the solvothermal method in the presence of different amounts of added chitosan is reported here. The magnetic property of the obtained magnetic composite nanoparticles was confirmed by X-ray diffraction (XRD) and magnetic measurements (VSM). Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) allowed the identification of spherical nanoparticles with about 150 nm in average diameter. Characterization of the products by Fourier transform infrared spectroscopy (FTIR) demonstrated that CS-coated Fe3O4 NPs were obtained. Chitosan content in the obtained nanocomposites was estimated by thermogravimetric analysis (TGA). The adsorption properties of the CS-coated Fe3O4 NPs for bovine serum albumin (BSA) were investigated under different concentrations of BSA. Compared with naked Fe3O4 nanoparticles, the CS-coated Fe3O4 NPs showed a higher BSA adsorption capacity (96.5 mg/g) and a fast adsorption rate (45 min) in aqueous solutions. This work demonstrates that the prepared magnetic nanoparticles have promising applications in enzyme and protein immobilization.

  4. Detection of Hg2+ based on the selective inhibition of peroxidase mimetic activity of BSA-Au clusters.

    PubMed

    Zhu, Rui; Zhou, Yan; Wang, Xi-Liang; Liang, Li-Ping; Long, Yi-Juan; Wang, Qin-Long; Zhang, Hai-Jie; Huang, Xiao-Xiao; Zheng, Hu-Zhi

    2013-12-15

    It was found that Hg(2+) can inhibit the peroxidase mimetic activity of bovine serum albumin (BSA) protected Au clusters (BSA-Au) due to the specific interaction between Hg(2+) and Au(+) existed onto the surface of BSA-Au clusters. By coupling with 3, 3', 5, 5'-tetramethylbenzidine (TMB)-H2O2 chromogenic reaction, a novel method for Hg(2+) detection was developed based on the inhibiting effect of Hg(2+) on BSA-Au clusters peroxidase-like activity. This method exhibited high selectivity and sensitivity. As low as 3 nM (0.6 ppb, 3σ) Hg(2+) could be detected with a linear range from 10 nM (2 ppb) to 10 µM (2 ppm) and this method was successfully applied for the determination of total mercury content in skin lightening products.

  5. In-vitro study on the competitive binding of diflunisal and uraemic toxins to serum albumin and human plasma using a potentiometric ion-probe technique.

    PubMed

    Davilas, A; Koupparis, M; Macheras, P; Valsami, G

    2006-11-01

    The competitive binding of diflunisal and three well-known uraemic toxins (3-indoxyl sulfate, indole-3-acetic acid and hippuric acid) to bovine serum albumin (BSA), human serum albumin (HSA) and human plasma was studied by direct potentiometry. The method used the potentiometric drug ion-probe technique with a home-made ion sensor (electrode) selective to the drug anion. The site-oriented Scatchard model was used to describe the binding of diflunisal to BSA, HSA and human plasma, while the general competitive binding model was used to calculate the binding parameters of the three uraemic toxins to BSA. Diflunisal binding parameters, number of binding sites, n(i) and association constants for each class of binding site, K(i), were calculated in the absence and presence of uraemic toxins. Although diflunisal exhibits high binding affinity for site I of HSA and the three uraemic toxins bind primarily to site II, strong interaction was observed between the drug and the three toxins, which were found to affect the binding of diflunisal on its primary class of binding sites on both BSA and HSA molecules and on human plasma. These results are strong evidence that the decreased binding of diflunisal that occurs in uraemic plasma may not be solely attributed to the lower albumin concentration observed in many patients with renal failure. The uraemic toxins that accumulate in uraemic plasma may displace the drug from its specific binding sites on plasma proteins, resulting in increased free drug plasma concentration in uraemic patients. PMID:17132209

  6. Binding interaction of a gamma-aminobutyric acid derivative with serum albumin: an insight by fluorescence and molecular modeling analysis.

    PubMed

    Pal, Uttam; Pramanik, Sumit Kumar; Bhattacharya, Baisali; Banerji, Biswadip; C Maiti, Nakul

    2016-01-01

    gamma-Aminobutyric acid (GABA) is a naturally occurring inhibitory neurotransmitter and some of its derivatives showed potential to act as neuroprotective agents. With the aim of developing potential leads for anti-Alzheimer's drugs, in this study we synthesized a novel GABA derivative, methyl 4-(4-((2-(tert-butoxy)-2-oxoethyl)(4-methoxyphenyl)amino)benzamido)butanoate by a unique method of Buchwald-Hartwig cross coupling synthesis; with some modification the yield was significant (97 %) and spectroscopic analysis confirmed that the compound was highly pure (98.8 % by HPLC). The druglikeness properties such as logP, logS, and polar surface area were 3.87, -4.86 and 94.17 Å(2) respectively and it satisfied the Lipinski's rule of five. We examined the binding behavior of the molecule to human serum albumin (HSA) and bovine serum albumin (BSA) which are known as universal drug carrier proteins. The molecule binds to the proteins with low micromolar efficiency and the calculated binding constants were 3.85 and 2.75 micromolar for BSA and HSA, respectively. Temperature dependent study using van't Hoff equation established that the binding was thermodynamically favorable and the changes in the Gibb's free energy, ΔG for the binding process was negative. However, the binding of the molecule to HSA was enthalpy driven and the change of enthalpy (ΔH) was -10.63 kJ/mol, whereas, the binding to BSA was entropy driven and the change in entropy ΔS was 222 J/mol. The molecular docking analysis showed that the binding sites of the molecule lie in the groove between domain I and domain III of BSA, whereas it is within the domain I in case of HSA, which also supported the different thermodynamic nature of binding with HSA and BSA. Molecular dynamics analysis suggested that the binding was stable with time and provided further details of the binding interaction. Molecular dynamics study also highlighted the effect of this ligand binding on the serum albumin structure. PMID

  7. Receptor-mediated hepatic uptake of M6P-BSA-conjugated triplex-forming oligonucleotides in rats.

    PubMed

    Ye, Zhaoyang; Cheng, Kun; Guntaka, Ramareddy V; Mahato, Ram I

    2006-01-01

    Excessive production of extracellular matrix, predominantly type I collagen, results in liver fibrosis. Earlier we synthesized mannose 6-phosphate-bovine serum albumin (M6P-BSA) and conjugated to the type I collagen specific triplex-forming oligonucleotide (TFO) for its enhanced delivery to hepatic stellate cells (HSCs), which is the principal liver fibrogenic cell. In this report, we demonstrate a time-dependent cellular uptake of M6P-BSA-33P-TFO by HSC-T6 cells. Both cellular uptake and nuclear deposition of M6P-BSA-33P-TFO were significantly higher than those of 33P-TFO, leading to enhanced inhibition of type I collagen transcription. Following systemic administration into rats, hepatic accumulation of M6P-BSA-33P-TFO increased from 55% to 68% with the number of M6P per BSA from 14 to 27. Unlike 33P-TFO, there was no significant decrease in the hepatic uptake of (M6P)20-BSA-33P-TFO in fibrotic rats. Prior administration of excess M6P-BSA decreased the hepatic uptake of (M6P)20-BSA-33P-TFO from 66% to 40% in normal rats, and from 60% to 15% in fibrotic rats, suggesting M6P/insulin-like growth factor II (M6P/IGF II) receptor-mediated endocytosis of M6P-BSA-33P-TFO by HSCs. Almost 82% of the total liver uptake in fibrotic rats was contributed by HSCs. In conclusion, by conjugation with M6P-BSA, the TFO could be potentially used for the treatment of liver fibrosis.

  8. Investigation of Cu(II) Binding to Bovine Serum Albumin by Potentiometry with an Ion Selective Electrode

    ERIC Educational Resources Information Center

    Jie Liu

    2004-01-01

    A laboratory project that investigates Cu(II) bind to bovine serum albumin (BSA) in an aqueous solution is developed to assist undergraduate students in gaining better understanding of the interaction of ligands with biological macromolecule. Thus, students are introduced to investigation of Cu(II) binding to BSA by potentiometry with the Cu(II)…

  9. Hollow hydroxyapatite microspheres as a device for controlled delivery of proteins

    PubMed Central

    Fu, Hailuo; Day, Delbert E.; Brown, Roger F.

    2011-01-01

    Hollow hydroxyapatite (HA) microspheres were prepared by reacting solid microspheres of Li2O–CaO–B2O3 glass (106–150 μm) in K2HPO4 solution, and evaluated as a controlled delivery device for a model protein, bovine serum albumin (BSA). Reaction of the glass microspheres for 2 days in 0.02 M K2HPO4 solution (pH = 9) at 37°C resulted in the formation of biocompatible HA microspheres with a hollow core diameter equal to 0.6 the external diameter, high surface area (~100 m2/g), and a mesoporous shell wall (pore size ≈13 nm). After loading with a solution of BSA in phosphate-buffered saline (PBS) (5 mg BSA/ml), the release kinetics of BSA from the HA microspheres into a PBS medium were measured using a micro bicinchoninic acid (BCA) protein assay. Release of BSA initially increased linearly with time, but almost ceased after 24–48 h. Modification of the BSA release kinetics was achieved by modifying the microstructure of the as-prepared HA microspheres using a controlled heat treatment (1–24 h at 600–900°C). Sustained release of BSA was achieved over 7–14 days from HA microspheres heated for 5 h at 600°C. The amount of BSA released at a given time was dependent on the concentration of BSA initially loaded into the HA microspheres. These hollow HA microspheres could provide a novel inorganic device for controlled local delivery of proteins and drugs. PMID:21290170

  10. Polarization properties of fluorescent BSA protected Au25 nanoclusters

    NASA Astrophysics Data System (ADS)

    Raut, Sangram; Chib, Rahul; Rich, Ryan; Shumilov, Dmytro; Gryczynski, Zygmunt; Gryczynski, Ignacy

    2013-03-01

    BSA protected gold nanoclusters (Au25) are attracting a great deal of attention due to their unique spectroscopic properties and possible use in biophysical applications. Although there are reports on synthetic strategies, spectroscopy and applications, little is known about their polarization behavior. In this study, we synthesized the BSA protected Au25 nanoclusters and studied their steady state and time resolved fluorescence properties including polarization behavior in different solvents: glycerol, propylene glycol and water. We demonstrated that the nanocluster absorption spectrum can be separated from the extinction spectrum by subtraction of Rayleigh scattering. The nanocluster absorption spectrum is well approximated by three Gaussian components. By a comparison of the emissions from BSA Au25 clusters and rhodamine B in water, we estimated the quantum yield of nanoclusters to be higher than 0.06. The fluorescence lifetime of BSA Au25 clusters is long and heterogeneous with an average value of 1.84 μs. In glycerol at -20 °C the anisotropy is high, reaching a value of 0.35. However, the excitation anisotropy strongly depends on the excitation wavelengths indicating a significant overlap of the different transition moments. The anisotropy decay in water reveals a correlation time below 0.2 μs. In propylene glycol the measured correlation time is longer and the initial anisotropy depends on the excitation wavelength. BSA Au25 clusters, due to long lifetime and high polarization, can potentially be used in studying large macromolecules such as protein complexes with large molecular weight.BSA protected gold nanoclusters (Au25) are attracting a great deal of attention due to their unique spectroscopic properties and possible use in biophysical applications. Although there are reports on synthetic strategies, spectroscopy and applications, little is known about their polarization behavior. In this study, we synthesized the BSA protected Au25 nanoclusters and

  11. Endocytic uptake of nonenzymatically glycosylated proteins is mediated by a scavenger receptor for aldehyde-modified proteins.

    PubMed

    Takata, K; Horiuchi, S; Araki, N; Shiga, M; Saitoh, M; Morino, Y

    1988-10-15

    Long term incubation of proteins with glucose, named the Maillard reaction (Maillard, L. C. (1912) C. R. Acad. Sci. (Paris) 154, 66-68), gives rise to advanced glycosylation end product (AGE) with fluorescence, color, as well as cross-linked properties. The receptor-mediated endocytosis of AGE-proteins by macrophages was reported (Vlassara, H., Brownlee, M., and Cerami, A. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 5588-5592). The present study on the binding of AGE-bovine serum albumin (BSA) to rat peritoneal macrophages and sinusoidal liver cells demonstrated the presence of a saturable, high affinity receptor for AGE-BSA with Kd = 2.4 x 10(-7) M (macrophages) and 2.1 x 10(-7) M (sinusoidal cells). The cellular binding of AGE-BSA and its endocytic uptake by these cells were competitively inhibited by BSA preparations modified with aliphatic aldehydes such as formaldehyde or glycolaldehyde, ligands known to be specific for a scavenger receptor for aldehyde-modified proteins (Horiuchi, S., Murakami, M., Takata, K., and Morino, Y. (1986). J. Biol. Chem. 261, 4962-4966). These ligands also had a profound in vivo effect on the plasma clearance of 125I-AGE-BSA as well as its hepatic uptake. Thus, endocytic uptake of AGE-proteins by macrophages appeared to be mediated by a scavenger receptor for aldehyde-modified proteins. This provides evidence for the biological importance of the scavenger receptor in eliminating senescent macromolecules from the circulation.

  12. Systematic investigation of the toxicity interaction of ZnSe@ZnS QDs on BSA by spectroscopic and microcalorimetry techniques.

    PubMed

    Ding, Ling; Zhou, Peijiang; Zhan, Hongju; Zhao, Xiaohu; Chen, Chi; He, Zhenyu

    2013-08-01

    The interaction of ZnSe@ZnS quantum dots (QDs) and bovine serum albumin (BSA) was investigated by means of fluorescence (FL) spectrometry, circular dichroism (CD) spectra, and isothermal titration calorimetry (ITC). The fluorescence intensity of BSA decreased regularly with the increasing of QDs concentration. The decrease of BSA fluorescence intensity was proved to be a kind of static quenching. CD results show the helicity of BSA decreased from 38.04% to 26.51% with the addition of QDs, which suggests a stronger structural change that is related to a low degree of surface coverage. And also, both ion strength and pH value could affect the interaction between BSA and QDs, suggesting that both the static electronic attraction and H-bond contribute to the interaction between BSA and QDs. The thermodynamics of interaction between BSA and QDs were calculated from ITC data. Both enthalpy and entropy changes were favorable for the interaction in Tris-buffer, while only enthalpy change was favorable for the interaction in NaCl or HCl solution.

  13. Cytotoxicity of BSA-Stabilized Gold Nanoclusters: In Vitro and In Vivo Study.

    PubMed

    Dong, Liyun; Li, Mulin; Zhang, Song; Li, Jun; Shen, Guanxin; Tu, Yating; Zhu, Jintao; Tao, Juan

    2015-06-01

    Gold nanoclusters (Au NCs) are one of the most promising fluorescent nanomaterials for bioimaging, targeting, and cancer therapy due to their tunable optical properties, yet their biocompatibility still remains unclear. Herein, the cytotoxicity of bovine serum albumin (BSA)-stabilized Au NCs is studied by using three tumor cell lines and two normal cell lines. The results indicate that Au NCs induce the decline of cell viabilities of different cell lines to varying degrees in a dose- and time-dependent manner, and umbilical vein endothelial cells which had a higher intake of Au NCs than melanoma cells show more toxicity. Addition of free BSA to BSA-Au NCs solutions can relieve the cytotoxicity, implying that BSA can prevent cell damage. Moreover, Au NCs increase intracellular reactive oxygen species (ROS) production, further causing cell apoptosis. Furthermore, N-acetylcysteine, a ROS scavenger, partially reverses Au NCs-induced cell apoptosis and cytotoxicity, indicating that ROS might be one of the primary reasons for the toxicity of BSA-Au NCs. Surprisingly, Au NCs with concentrations of 5 and 20 nM significantly inhibit tumor growth in the xenograft mice model of human liver cancer, which might provide a new avenue for the design of anti-cancer drug delivery vehicles.

  14. Protein encapsulated magnetic carriers for micro/nanoscale drug delivery systems.

    SciTech Connect

    Xie, Y.; Kaminski, M. D.; Mertz, C. J.; Finck, M. R.; Guy, S. G.; Chen, H.; Rosengart, A. J.; Chemical Engineering; Univ. of Chicago, Pritzker School of Medicine

    2005-01-01

    Novel methods for drug delivery may be based on nanotechnology using non-invasive magnetic guidance of drug loaded magnetic carriers to the targeted site and thereafter released by external ultrasound energy. The key building block of this system is to successfully synthesize biodegradable, magnetic drug carriers. Magnetic carriers using poly(D,L-lactide-co-glycolide) (PLGA) or poly(lactic acid)-poly(ethylene glycol) (PLA-PEG) as matrix materials were loaded with bovine serum albumin (BSA) by a double-emulsion technique. BSA-loaded magnetic microspheres were characterized for size, morphology, surface charge, and magnetization. The BSA encapsulation efficiency was determined by recovering albumin from the microspheres using dimethyl sulfoxide and 0.05N NaOH/0.5% SDS then quantifying with the Micro-BCA protein assay. BSA release profiles were also determined by the Micro-BCA protein assay. The microspheres had drug encapsulation efficiencies up to 90% depending on synthesis parameters. Particles were spherical with a smooth or porous surface having a size range less than 5 {mu}m. The surface charge (expressed as zeta potential) was near neutral, optimal for prolonged intravascular survival. The magnetization of these BSA loaded magnetic carriers was 2 to 6 emu/g, depending on the specific magnetic materials used during synthesis.

  15. Intermolecular forces in bovine serum albumin solutions exhibiting solidlike mechanical behaviors.

    PubMed

    Ikeda, S; Nishinari, K

    2000-01-01

    Mechanical properties of bovine serum albumin (BSA) solutions were analyzed to gain information on intermolecular forces that stabilize the system under normal physiological conditions. BSA solutions showed unexpectedly large zero shear viscosity values under steady shear flows but responded like solids to sinusoidal linear strains: the storage shear moduli were always larger than the loss shear moduli in the frequency range 1-100 rad/s. These results suggest that BSA solutions are so-called colloidal crystals in which colloidal particles are ordered in an array due to strong repulsive forces among particles. However, the pair potential between BSA molecules predicted based on the conventional Derjaguin-Landau-Verwey-Overbeek theory failed to explain these remarkable mechanical properties of BSA solutions. Additional repulsive forces other than electrostatic must be introduced to explain stability of BSA aqueous dispersions.

  16. Spectroscopic studies on the interaction between troxerutin and bovine serum albumin

    NASA Astrophysics Data System (ADS)

    Wang, Tianhu; Zhao, Zhimin; Zhang, Lin; Ji, Lei

    2009-11-01

    The interaction between troxerutin and bovine serum albumin (BSA) was investigated by fluorescence and absorption spectroscopy under simulative physiological conditions. Results show that troxerutin causes the fluorescence quenching of BSA through a static quenching procedure. The binding constant KA and number of binding sites n of troxerutin with BSA were obtained. Positive values of thermodynamic parameters enthalpy change (Δ H) and entropy change (Δ S) indicate that the interaction between troxerutin and BSA is driven mainly by hydrophobic forces. It seems that the binding is spontaneous at standard state for the change in standard Gibbs free energy (Δ G) value is negative. The binding distance between the donor (BSA) and the acceptor (troxerutin) was calculated to be about 4.21 nm based on the Förster theory. The effect of troxerutin on the conformation of BSA was also analyzed by using synchronous fluorescence spectroscopy.

  17. Spectrometric studies on the interaction of fluoroquinolones and bovine serum albumin

    NASA Astrophysics Data System (ADS)

    Ni, Yongnian; Su, Shaojing; Kokot, Serge

    2010-02-01

    The interaction between fluoroquinolones (FQs), ofloxacin and enrofloxacin, and bovine serum albumin (BSA) was investigated by fluorescence and UV-vis spectroscopy. It was demonstrated that the fluorescence quenching of BSA by FQ is a result of the formation of the FQ-BSA complex stabilized, in the main, by hydrogen bonds and van der Waals forces. The Stern-Volmer quenching constant, KSV, and the corresponding thermodynamic parameters, Δ H, Δ S and Δ G, were estimated. The distance, r, between the donor, BSA, and the acceptor, FQ, was estimated from fluorescence resonance energy transfer (FRET). The effect of FQ on the conformation of BSA was analyzed with the aid of UV-vis absorbance spectra and synchronous fluorescence spectroscopy. Spectral analysis showed that the two FQs affected the conformation of the BSA but in a different manner. Thus, with ofloxacin, the polarity around the tryptophan residues decreased and the hydrophobicity increased, while for enrofloxacin, the opposite effect was observed.

  18. Study on the interaction between NCP-(4-hydroxycoumarins) and bovine serum albumin by spectroscopic techniques.

    PubMed

    Yu, Xianyong; Lu, Shiyu; Yang, Ying; Li, Xiaofang; Yi, Pinggui

    2012-06-01

    The interaction between N-confused porphyrins-(4-hydroxycoumarins) diad (NCP-(4-hydroxycoumarins)) and bovine serum albumin (BSA) was studied using fluorescence and ultraviolet spectroscopy at different temperatures under imitated physiological conditions. The experimental results showed that the fluorescence of BSA was quenched by NCP-(4-hydroxycoumarins) through a combined quenching procedure. The binding constants, binding sites and corresponding thermodynamic parameters between NCP-(4-hydroxycoumarins) and BSA at different temperatures were obtained. According to Förster non-radiation energy transfer theory, the binding distance between BSA and NCP-(4-hydroxycoumarins) was calculated to be about 2.1 nm. The effect of NCP-(4-hydroxycoumarins) on the conformation of BSA was analyzed using synchronous fluorescence spectroscopy. In addition, the effect of some metal ions Cu(2+), Ca(2+), Mg(2+), and Ni(2+) on the binding constant between NCP-(4-hydroxycoumarins) and BSA was examined.

  19. Study on the interaction of food colourant quinoline yellow with bovine serum albumin by spectroscopic techniques.

    PubMed

    Shahabadi, Nahid; Maghsudi, Maryam; Rouhani, Shohre

    2012-12-01

    The interaction of a food colourant, quinoline yellow (Qy), and bovine serum albumin (BSA) was investigated by spectrophotometry, spectrofluorometry, FT-IR and circular dichroism (CD) techniques. The experimental results indicated that the quenching mechanism of BSA by the dye was a static procedure. Various binding parameters were evaluated. The negative value of ΔH, negative value of ΔS and the negative value of ΔG indicated that van der Waals force and hydrogen bonding play major roles in the binding of Qy and BSA. Based on Forster's theory of non-radiation energy transfer, the binding distance, r, between the donor (BSA) and acceptor (Qy) was evaluated. The results of CD and UV-vis spectroscopy showed that this dye could bind to BSA and the conformation of BSA changed.

  20. Allosteric Ligand Binding and Anisotropic Energy Flow in Albumin

    NASA Astrophysics Data System (ADS)

    Dyer, Brian

    2014-03-01

    Protein allostery usually involves propagation of local structural changes through the protein to a remote site. Coupling of structural changes at remote sites is thought to occur through anisotropic energy transport, but the nature of this process is poorly understood. We have studied the relationship between allosteric interactions of remote ligand binding sites of the protein and energy flow through the structure of bovine serum albumin (BSA). We applied ultrafast infrared spectroscopy to probe the flow of energy through the protein backbone following excitation of a heater dye, a metalloporphyrin or malachite green, bound to different binding sites in the protein. We observe ballistic flow through the protein structure following input of thermal energy into the flexible ligand binding sites. We also observe anisotropic heat flow through the structure, without local heating of the rigid helix bundles that connect these sites. We will discuss the implications of this efficient energy transport mechanism with regard to the allosteric propagation of binding energy through the connecting helix structures.

  1. Suppressing the cytotoxicity of CuO nanoparticles by uptake of curcumin/BSA particles.

    PubMed

    Zhang, Wenjing; Jiang, Pengfei; Chen, Ying; Luo, Peihua; Li, Guanqun; Zheng, Botuo; Chen, Wei; Mao, Zhengwei; Gao, Changyou

    2016-05-01

    The adverse effects of metal-based nanoparticles on human beings and the environment have received extensive attention recently. It is urgently required to develop a simple and effective method to suppress the toxicity of metal-based nanomaterials. In this study, a hydrophobic antioxidant and a chelation agent curcumin (CUR) were encapsulated into bovine serum albumin (BSA) particles by a simple co-precipitation method, and followed by glutaraldehyde cross-linking. The CUR/BSA particles had an average size of 300 nm in diameter with a negatively charged surface and sustained curcumin release properties. The cellular uptake and cytotoxicity of CUR/BSA particles were followed on A549 cells, HepG2 cells and RAW264.7 cells. The CUR/BSA particles had higher intracellular accumul