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Sample records for serum protein binding

  1. Erythropoietin binding protein from mammalian serum

    DOEpatents

    Clemons, G.K.

    1997-04-29

    Purified mammalian erythropoietin binding-protein is disclosed, and its isolation, identification, characterization, purification, and immunoassay are described. The erythropoietin binding protein can be used for regulation of erythropoiesis by regulating levels and half-life of erythropoietin. A diagnostic kit for determination of level of erythropoietin binding protein is also described. 11 figs.

  2. Erythropoietin binding protein from mammalian serum

    DOEpatents

    Clemons, Gisela K.

    1997-01-01

    Purified mammalian erythropoietin binding-protein is disclosed, and its isolation, identification, characterization, purification, and immunoassay are described. The erythropoietin binding protein can be used for regulation of erythropoiesis by regulating levels and half-life of erythropoietin. A diagnostic kit for determination of level of erythropoietin binding protein is also described.

  3. Grizzly bear corticosteroid binding globulin: Cloning and serum protein expression.

    PubMed

    Chow, Brian A; Hamilton, Jason; Alsop, Derek; Cattet, Marc R L; Stenhouse, Gordon; Vijayan, Mathilakath M

    2010-06-01

    Serum corticosteroid levels are routinely measured as markers of stress in wild animals. However, corticosteroid levels rise rapidly in response to the acute stress of capture and restraint for sampling, limiting its use as an indicator of chronic stress. We hypothesized that serum corticosteroid binding globulin (CBG), the primary transport protein for corticosteroids in circulation, may be a better marker of the stress status prior to capture in grizzly bears (Ursus arctos). To test this, a full-length CBG cDNA was cloned and sequenced from grizzly bear testis and polyclonal antibodies were generated for detection of this protein in bear sera. The deduced nucleotide and protein sequences were 1218 bp and 405 amino acids, respectively. Multiple sequence alignments showed that grizzly bear CBG (gbCBG) was 90% and 83% identical to the dog CBG nucleotide and amino acid sequences, respectively. The affinity purified rabbit gbCBG antiserum detected grizzly bear but not human CBG. There were no sex differences in serum total cortisol concentration, while CBG expression was significantly higher in adult females compared to males. Serum cortisol levels were significantly higher in bears captured by leg-hold snare compared to those captured by remote drug delivery from helicopter. However, serum CBG expression between these two groups did not differ significantly. Overall, serum CBG levels may be a better marker of chronic stress, especially because this protein is not modulated by the stress of capture and restraint in grizzly bears. PMID:20347821

  4. 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.

  5. Differences in the serum binding determinants of isradipine and darodipine--consequences for serum protein binding in various diseases.

    PubMed Central

    Pinquier, J L; Urien, S; Chaumet-Riffaud, P; Tillement, J P

    1989-01-01

    1. Serum protein binding of isradipine and darodipine, and serum concentrations of alpha 1-acid glycoprotein (AAG), albumin (HSA) and non-esterified fatty acids (NEFA) were measured in three groups of patients, I: healthy subjects (n = 20); II: patients with inflammatory disorders (n = 15) and III: patients with hepatic insufficiency (n = 17). 2. AAG was increased significantly in group II patients (P less than 0.001) and decreased in group III patients (P less than 0.001); HSA was decreased significantly in group II and group III patients (P less than 0.001). 3. The free percentage of isradipine was decreased significantly in group II patients (P less than 0.05) and increased in group III patients (P less than 0.05) and multivariate analysis showed that these variations were inversely related to changes in AAG concentration. 4. The free percentage of darodipine was increased significantly in group II and III patients (P less than 0.05) due to a decrease in HSA concentration, as shown by multivariate analysis. 5. The changes in free serum percentages of isradipine and darodipine were inversely related to concomitant changes in the concentration of the serum protein for which they showed the highest affinity, AAG for isradipine and HSA for darodipine, respectively. 6. The unexplained variability in the binding data was greater when AAG was the major determinant of binding (isradipine). PMID:2531607

  6. Absence of serum growth hormone binding protein in patients with growth hormone receptor deficiency (Laron dwarfism)

    SciTech Connect

    Daughaday, W.H.; Trivedi, B.

    1987-07-01

    It has recently been recognized that human serum contains a protein that specifically binds human growth hormone (hGH). This protein has the same restricted specificity for hGH as the membrane-bound GH receptor. To determine whether the GH-binding protein is a derivative of, or otherwise related to, the GH receptor, the authors have examined the serum of three patients with Laron-type dwarfism, a condition in which GH refractoriness has been attributed to a defect in the GH receptor. The binding of /sup 125/I-labeled hGH incubated with serum has been measured after gel filtration of the serum through an Ultrogel AcA 44 minicolumn. Results are expressed as percent of specifically bound /sup 125/I-hGH and as specific binding relative to that of a reference serum after correction is made for endogenous GH. The mean +/- SEM of specific binding of sera from eight normal adults (26-46 years of age) was 21.6 +/- 0.45%, and the relative specific binding was 101.1 +/- 8.6%. Sera from 11 normal children had lower specific binding of 12.5 +/- 1.95% and relative specific binding of 56.6 +/- 9.1%. Sera from three children with Laron-type dwarfism lacked any demonstrable GH binding, whereas sera from 10 other children with other types of nonpituitary short stature had normal relative specific binding. They suggest that the serum GH-binding protein is a soluble derivative of the GH receptor. Measurement of the serum GH-binding protein may permit recognition of other abnormalities of the GH receptor.

  7. Characterization of the comparative drug binding to intra- (liver fatty acid binding protein) and extra- (human serum albumin) cellular proteins.

    PubMed

    Rowland, Andrew; Hallifax, David; Nussio, Matthew R; Shapter, Joseph G; Mackenzie, Peter I; Brian Houston, J; Knights, Kathleen M; Miners, John O

    2015-01-01

    1. This study compared the extent, affinity, and kinetics of drug binding to human serum albumin (HSA) and liver fatty acid binding protein (LFABP) using ultrafiltration and surface plasmon resonance (SPR). 2. Binding of basic and neutral drugs to both HSA and LFABP was typically negligible. Binding of acidic drugs ranged from minor (fu > 0.8) to extensive (fu < 0.1). Of the compounds screened, the highest binding to both HSA and LFABP was observed for the acidic drugs torsemide and sulfinpyrazone, and for β-estradiol (a polar, neutral compound). 3. The extent of binding of acidic drugs to HSA was up to 40% greater than binding to LFABP. SPR experiments demonstrated comparable kinetics and affinity for the binding of representative acidic drugs (naproxen, sulfinpyrazone, and torsemide) to HSA and LFABP. 4. Simulations based on in vitro kinetic constants derived from SPR experiments and a rapid equilibrium model were undertaken to examine the impact of binding characteristics on compartmental drug distribution. Simulations provided mechanistic confirmation that equilibration of intracellular unbound drug with the extracellular unbound drug is attained rapidly in the absence of active transport mechanisms for drugs bound moderately or extensively to HSA and LFABP. PMID:25801059

  8. THE INFLUENCE OF SERUM BINDING PROTEINS AND CLEARANCE ON THE COMPARATIVE RECEPTOR BINDING POTENCY OF ENDOCRINE ACTIVE COMPOUNDS

    EPA Science Inventory

    THE INFLUENCE OF SERUM BINDING PROTEINS AND CLEARANCE ON THE COMPARATIVE RECEPTOR BINDING POTENCY OF ENDOCRINE ACTIVE COMPOUNDS. JG Teeguarden1 and HA Barton2. 1ENVIRON International, Ruston LA; 2US EPA, ORD, NHEERL, ETD, Pharmacokinetics Branch, RTP, NC.

    One measure of th...

  9. Determination of protein-ligand binding affinity by NMR: observations from serum albumin model systems.

    PubMed

    Fielding, Lee; Rutherford, Samantha; Fletcher, Dan

    2005-06-01

    The usefulness of bovine serum albumin (BSA) as a model protein for testing NMR methods for the study of protein-ligand interactions is discussed. Isothermal titration calorimetry established the binding affinity and stoichiometry of the specific binding site for L-tryptophan, D-tryptophan, naproxen, ibuprofen, salicylic acid and warfarin. The binding affinities of the same ligands determined by NMR methods are universally weaker (larger KD). This is because the NMR methods are susceptible to interference from additional non-specific binding. The L-tryptophan-BSA and naproxen-BSA systems were the best behaved model systems. PMID:15816062

  10. 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

  11. Binding of labeled thyroxin analog to serum proteins evaluated after radioimmunoassay of free thyroxin

    SciTech Connect

    Arevalo, G.

    1989-03-01

    In ambulatory patients, assay of free thyroxin (FT4) in serum correlates well with thyroid status and with results obtained by equilibrium dialysis. The validity of FT4 results has been questioned mainly in euthyroid patients with altered concentrations of thyroid hormone-binding proteins, as in nonthyroidal illness, hereditary analbuminemia, familial dysalbuminemic hyperthyroxinemia (FDH), and the presence of iodothyronine-binding antibodies. I present here a study of the binding of (/sup 125/I)T4-derivative to serum proteins in the supernate, which is ordinarily discarded after determination of FT4 by one-step radioimmunoassay with dextran-coated charcoal used to separate the free and bound fractions. The results are expressed as a ratio, with results for a normal serum pool as reference. The average ratio was high in hyperthyroid subjects, 1.26 (SD 0.12, n = 25), and in hypoalbuminemia, 1.20 (SD 0.10, n = 15), and low in FDH, 0.62 (SD 0.11, n = 9), and hypothyroid subjects, 0.90 (SD 0.06, n = 20). In normal individuals it was 0.98 (SD 0.05, n = 30). Determination of the analog-binding rate complements the FT4 result and allows for the recognition of cases with abnormal binding by serum proteins, without recourse to other tests recommended for thyroid-function studies.

  12. Does binding of complement factor H to the meningococcal vaccine antigen, factor H binding protein, decrease protective serum antibody responses?

    PubMed

    Granoff, Dan M; Ram, Sanjay; Beernink, Peter T

    2013-08-01

    Factor H binding protein (fHbp) is a principal antigen in a multicomponent meningococcal vaccine recently licensed in Europe for prevention of serogroup B diseases. The protein recruits the complement downregulator, factor H (fH), to the bacterial surface, which enables the organism to resist complement-mediated bacteriolysis. Binding is specific for human fH. In preclinical studies, mice and rabbits immunized with fHbp vaccines developed serum bactericidal antibody responses, which in humans predict protection against developing meningococcal disease. These studies, however, were in animals whose fH did not bind to the vaccine antigen. Here we review the immunogenicity of fHbp vaccines in human fH transgenic mice. The data suggest that animals with high serum human fH concentrations have impaired protective antibody responses. Further, mutant fHbp vaccines with single amino acid substitutions that decrease fH binding are superior immunogens, possibly by unmasking epitopes in the fH binding site that are important for eliciting serum bactericidal antibody responses. Humans immunized with fHbp vaccines develop serum bactericidal antibody, but achieving broad coverage in infants required incorporation of additional antigens, including outer membrane vesicles, which increased rates of fever and local reactions at the injection site. The experimental results in transgenic mice predict that fHbp immunogenicity can be improved in humans by using mutant fHbp vaccines with decreased fH binding. These results have important public health implications for developing improved fHbp vaccines for control of serogroup B meningococcal disease and for development of vaccines against other microbes that bind host molecules. PMID:23740919

  13. Pharmacokinetics of warfarin in rats: role of serum protein binding and tissue distribution

    SciTech Connect

    Cheung, W.K.

    1985-01-01

    The purpose of this study was to explore the role of serum protein binding and tissue distribution in the non-linear pharmacokinetics of warfarin in rats. The first phase of the research was an attempt to elucidate the causes of intersubject differences in serum protein binding of warfarin in rats. It was found that the distribution of S-warfarin between blood and liver, kidneys, muscle, or fatty tissue was non-linear. Based on the tissue distribution data obtained, a physiologically-based pharmacokinetic model was developed to describe the time course of S-warfarin concentrations in the serum and tissues of rats. The proposed model was able to display the dose-dependent pharmacokinetics of warfarin in rats. Namely a lower clearance and a smaller apparent volume of distribution with increasing dose, which appear to be due to the presence of capacity-limited, high-affinity binding sites for warfarin in various tissues. To determine if the binding of warfarin to the high-affinity binding sites in the liver of rats is reversible, concentrations of S-warfarin in the liver and serum of rats were monitored for a very long time after an intravenous injection of a 1 mg/kg dose. In another study in rats, non-radioactive warfarin was found to be able to displace tissue-bound C/sup 14/-warfarin which was administered about 200 hours before the i.v. injection of the non-radioactive warfarin, showing that the binding of warfarin to the high-affinity binding sites in the body is persistent and reversible.

  14. Association of androgen with gender difference in serum adipocyte fatty acid binding protein levels

    PubMed Central

    Hu, Xiang; Ma, Xiaojing; Pan, Xiaoping; Luo, Yuqi; Xu, Yiting; Xiong, Qin; Bao, Yuqian; Jia, Weiping

    2016-01-01

    Clinical investigations have indicated women have higher levels of adipocyte fatty acid binding protein (A-FABP) than men. The present study aimed to identify factors related to gender difference in serum A-FABP levels. A total of 507 participants (194 men, 132 premenopausal women, and 181 postmenopausal women) were enrolled in the present study. Serum A-FABP levels increased in the order from men to premenopausal women to postmenopausal women in both body mass index categories (<25.0 and ≥25.0 kg/m2; all P < 0.05). Multiple stepwise regression analyses showed that after adjustment for factors related to serum A-FABP levels, the trunk fat mass was an independent and positive factor of serum A-FABP levels. For men, total testosterone was associated independently and inversely with serum A-FABP levels. For pre- and postmenopausal women, bioavailable testosterone and total testosterone were independent and positive factors associated with serum A-FABP levels, respectively. The present study demonstrated that the androgen was correlated with the serum A-FABP levels negatively in men, but positively in women. With these effects on the fat content, especially trunk fat, androgen might contribute to the gender difference in serum A-FABP levels. PMID:27270834

  15. Association of androgen with gender difference in serum adipocyte fatty acid binding protein levels.

    PubMed

    Hu, Xiang; Ma, Xiaojing; Pan, Xiaoping; Luo, Yuqi; Xu, Yiting; Xiong, Qin; Bao, Yuqian; Jia, Weiping

    2016-01-01

    Clinical investigations have indicated women have higher levels of adipocyte fatty acid binding protein (A-FABP) than men. The present study aimed to identify factors related to gender difference in serum A-FABP levels. A total of 507 participants (194 men, 132 premenopausal women, and 181 postmenopausal women) were enrolled in the present study. Serum A-FABP levels increased in the order from men to premenopausal women to postmenopausal women in both body mass index categories (<25.0 and ≥25.0 kg/m(2); all P < 0.05). Multiple stepwise regression analyses showed that after adjustment for factors related to serum A-FABP levels, the trunk fat mass was an independent and positive factor of serum A-FABP levels. For men, total testosterone was associated independently and inversely with serum A-FABP levels. For pre- and postmenopausal women, bioavailable testosterone and total testosterone were independent and positive factors associated with serum A-FABP levels, respectively. The present study demonstrated that the androgen was correlated with the serum A-FABP levels negatively in men, but positively in women. With these effects on the fat content, especially trunk fat, androgen might contribute to the gender difference in serum A-FABP levels. PMID:27270834

  16. Vitamin D binding protein as a serum biomarker of Alzheimer's disease.

    PubMed

    Bishnoi, Ram J; Palmer, Raymond F; Royall, Donald R

    2015-01-01

    Vitamin D binding protein (VDBP), a multifunctional protein, has been found to be elevated in the cerebrospinal fluid (CSF) of neurodegenerative disorder cases, implicating it in the pathogenesis of Alzheimer's disease (AD). However, the contribution of VDBP to AD has not been fully explored. We used a Multiple Indicators Multiple Causes (MIMIC) approach to examine the relationship between serum VDBP levels and cognitive performance in a well characterized AD cohort, the Texas Alzheimer's Research and Care Consortium (TARCC). Instead of categorical diagnoses, we used a latent dementia phenotype (d), which has been validated in several prior studies using this dataset. We found that serum VDBP levels are significantly positively associated with d scores, which in turn are inversely related to cognitive performance. This suggests that d mediates the adverse effects of serum VDB on cognition and therefore that its effects are specifically dementing. d scores are also specifically related to default mode network (DMN) structure. VDBP acts as an amyloid-β (Aβ) scavenger, and Aβ deposition in the DMN is seen in the pre-clinical stages of AD. We speculate then that serum effects of VDBP are mediated through changes in DMN structure or function, most probably via Aβ. Aβ affects the DMN early in the course of AD. Therefore, raised serum VDBP levels may be a useful indicator of future dementia and/or dementia conversion. This might be confirmed through longitudinal analysis of TARCC data. PMID:25079796

  17. Serum amyloid A is a retinol binding protein that transports retinol during bacterial infection

    PubMed Central

    Derebe, Mehabaw G; Zlatkov, Clare M; Gattu, Sureka; Ruhn, Kelly A; Vaishnava, Shipra; Diehl, Gretchen E; MacMillan, John B; Williams, Noelle S; Hooper, Lora V

    2014-01-01

    Retinol plays a vital role in the immune response to infection, yet proteins that mediate retinol transport during infection have not been identified. Serum amyloid A (SAA) proteins are strongly induced in the liver by systemic infection and in the intestine by bacterial colonization, but their exact functions remain unclear. Here we show that mouse and human SAAs are retinol binding proteins. Mouse and human SAAs bound retinol with nanomolar affinity, were associated with retinol in vivo, and limited the bacterial burden in tissues after acute infection. We determined the crystal structure of mouse SAA3 at a resolution of 2 Å, finding that it forms a tetramer with a hydrophobic binding pocket that can accommodate retinol. Our results thus identify SAAs as a family of microbe-inducible retinol binding proteins, reveal a unique protein architecture involved in retinol binding, and suggest how retinol is circulated during infection. DOI: http://dx.doi.org/10.7554/eLife.03206.001 PMID:25073702

  18. Serum Mac-2 binding protein is a novel biomarker for chronic pancreatitis

    PubMed Central

    Maekawa, Tomohiro; Kamada, Yoshihiro; Ebisutani, Yusuke; Ueda, Makiko; Hata, Tomoki; Kawamoto, Koichi; Takamatsu, Shinji; Mizutani, Kayo; Shimomura, Mayuka; Sobajima, Tomoaki; Fujii, Hironobu; Nakayama, Kotarosumitomo; Nishino, Kimihiro; Yamada, Makoto; Kumada, Takashi; Ito, Toshifumi; Eguchi, Hidetoshi; Nagano, Hiroaki; Miyoshi, Eiji

    2016-01-01

    AIM: To determine the efficacy of Mac-2 binding protein (Mac-2bp) for diagnosis of chronic pancreatitis. METHODS: Fifty-nine healthy volunteers (HV), 162 patients with chronic pancreatitis (CP), and 94 patients with pancreatic ductal adenocarcinoma (PDAC) were enrolled in this study. We measured serum Mac-2bp using our developed enzyme-linked immunosorbent assay kit. Additional biochemical variables were measured using an automated analyzer (including aminotransferase, alanine aminotransferase, γ-glutamyltransferase, alkaline phosphatase, triglyceride, C-reactive protein, and amylase levels) or chemiluminescent enzyme immunoassay (carbohydrate antigen 19-9 and carcinoembryonic antigen). The ability of Mac-2bp to predict CP diagnosis accurately was assessed using receiver operating characteristic (ROC) analyses. RESULTS: Serum Mac-2bp levels were significantly increased in CP patients compared to HV (P < 0.0001) and PDAC patients (P < 0.0001). Area under the ROC curve values of Mac-2bp for the discrimination of CP from HV and PDAC were 0.727 and 0.784, respectively. Multivariate analyses demonstrated that serum Mac-2bp levels were independent determinants for CP diagnosis from HV and PDAC patients. Immunohistological staining showed that Mac-2bp was expressed faintly in the pancreas tissues of both CP and PDAC patients. Serum aspartate aminotransferase, alanine aminotransferase, γ-glutamyltransferase, alkaline phosphatase, and triglyceride levels were significantly higher in patients with CP or PDAC. Serum Mac-2bp levels were highly correlated with protein levels of alanine aminotransferase, γ-glutamyltransferase, and C-reactive protein, but not amylase, suggesting that the damaged liver produces Mac-2bp. CONCLUSION: Measurement of serum Mac-2bp may be a novel and useful biomarker for CP diagnosis as well as liver fibrosis in the general population. PMID:27158210

  19. Hypophysectomy eliminates and growth hormone (GH) maintains the midpregnancy elevation in GH receptor and serum binding protein in the mouse

    SciTech Connect

    Sanchez-Jimenez, F.; Fielder, P.J.; Martinez, R.R.; Smith, W.C.; Talamantes, F. )

    1990-02-01

    ({sup 125}I)Iodomouse GH (({sup 125}I)iodo-mGH) binding to samples of serum and hepatic microsomal membranes was measured in hypophysectomized pregnant, sham-operated pregnant, intact pregnant, and intact adult virgin mice. Surgeries were carried out on day 11 of pregnancy, and the animals were killed on day 14. The binding of mGH to both serum and hepatic microsomal membranes of intact virgin mice was much lower than to those of intact pregnant mice. In hypophysectomized mice, the mGH-binding capacity of both serum and hepatic microsomes decreased to values similar to those of nonpregnant mice. No significant differences were observed between intact and sham-operated pregnant animals in the maternal serum mGH concentration, the serum GH-binding protein concentration, or the hepatic GH receptor concentration. GH receptor and binding protein-encoding mRNAs were also higher in intact and sham-operated pregnant mice than in virgin and hypophysectomized mice. Hypophysectomized mice were treated with 200 micrograms/day bovine GH, administered by osmotic minipump; after 3 days of treatment, a significant elevation of hepatic GH receptor and serum GH-binding protein levels was observed. These results demonstrate an up-regulation of hepatic GH receptors and serum GH-binding protein by GH during pregnancy in the mouse.

  20. Marsupial and monotreme serum immunoglobulin binding by proteins A, G and L and anti-kangaroo antibody.

    PubMed

    Vaz, Paola K; Hartley, Carol A; Browning, Glenn F; Devlin, Joanne M

    2015-12-01

    Serological studies are often conducted to examine exposure to infectious agents in wildlife populations. However, specific immunological reagents for wildlife species are seldom available and can limit the study of infectious diseases in these animals. This study examined the ability of four commercially available immunoglobulin-binding reagents to bind serum immunoglobulins from 17 species within the Marsupialia and Monotremata. Serum samples were assessed for binding, using immunoblots and ELISAs (Enzyme-linked immunosorbent assays), to three microbially-derived proteins - staphylococcal protein A, streptococcal protein G and peptostreptococcal protein L. Additionally, an anti-kangaroo antibody was included for comparison. The inter- and intra-familial binding patterns of the reagents to serum immunoglobulins varied and evolutionary distance between animal species was not an accurate predictor of the ability of reagents to bind immunoglobulins. Results from this study can be used to inform the selection of appropriate immunological reagents in future serological studies in these clades. PMID:26523413

  1. Serum complements and heart fatty acid binding protein in Bangladeshi patients with acute myocardial infarction

    PubMed Central

    Akhtar, Nayareen; Taher, Abu; Rahman, Rezwanur; Chowdhury, Ashesh Kumar

    2012-01-01

    The complement system is activated following acute myocardial infarction (AMI). Heart fatty acid binding protein (H-FABP) is a sensitive early biomarker of myocardial necrosis that can be used to confirm or exclude a diagnosis of AMI and to monitor recurrent infarction. This study was designed to detect changes in C3, C4 and H-FABP after AMI. Forty patients with AMI and a control group of 40 apparently healthy people were included. Selections were based on inclusion and exclusion criteria. The baseline characteristics were not significantly different between the groups. Patients’ blood samples were collected within 12 h of admission. Significant increases in C3 (AMI group 1.4260+0.04, healthy group 1.26040+0.04; p<0.05), C4 (AMI group 0.29305±0.013, healthy group 0.20860±0.012; p<0.05) and H-FABP (AMI group 12.3±1.69, healthy group 0.16±0.057; p<0.001) were seen in patients with AMI. The correlation between serum C3 and body mass index (BMI, r=0.33; p<0.05), serum C4 and BMI(r=0.313; p<0.05), serum C3 and total cholesterol high density lipoprotein (HDL, r=0.32; p<0.05), serum C4 and HbA1C (r=0.335; p<0.05) and serum C3 and troponin I (r= 0.325p<0.05) was found to be significant. But the correlation between serum C3 and waist:hip ratio (p=0.56), serum C4 and waist:hip ratio (p=0.83), serum C4 and total cholesterol HDL (p=0.993), serum C3 and HbA1C (p=0.440), serum C3 and random blood sugar (p=0.563), serum C4 and random blood sugar (p=0.828) and serum C4 and troponin I (p=0.373) was not significant. The significant complement activation detected in the plasma of patients with AMI indicated that complement plays a part in the pathogenesis of myocardial infarction. A significant increase of H-FABP improves the diagnosis of AMI.

  2. Purification, properties and identification of a serum DNA binding protein (64DP) and its microheterogeneity.

    PubMed

    Tsuda, M; Ohkubo, T; Kamiguchi, H; Suzuki, K; Nakasaki, H; Mitomi, T; Katsunuma, T

    1982-03-01

    A DNA binding protein with a molecular weight of 64,000, designated 64DP, has been purified and characterized. This protein was isolated from adult human pooled serum by DEAE Sephadex column Chromatography, DNA cellulose affinity column chromatography, ammonium sulfate fractionation and Sephadex G-150 gel filtration. The final preparation of 64DP was homogeneous, as judged from polyacrylamide gel electrophoresis with or without sodium dodecyl sulfate and sedimentation experiments. Physicochemical and immunochemical properties of this protein were very similar or identical to those of alpha-1-antichymotrypsin with some differences in electric mobility and th pattern of isoelectric focusing. Furthermore, the general properties of 64DP from various sera were practically similar with the exception that isolectric focusing analysis showed microheterogeneity among 64DP purified from various sera. PMID:6808710

  3. Structural modification of serum vitamin D3-binding protein and immunosuppression in AIDS patients.

    PubMed

    Yamamoto, N; Naraparaju, V R; Srinivasula, S M

    1995-11-01

    A serum glycoprotein, vitamin D3-binding protein (Gc protein), can be converted by beta-galactosidase of stimulated B lymphocytes and sialidase of T lymphocytes to a potent macrophage-activating factor (MAF), a protein with N-acetylgalactosamine as the remaining sugar moiety. Thus, Gc protein is a precursor for MAF. Treatment of purified Gc protein with immobilized beta-galactosidase and sialidase generates an extremely high-titered MAF (GcMAF). When peripheral blood monocytes/macrophages of 46 HIV-infected patients were treated with GcMAF (100 pg/ml), the monocytes/macrophages of all patients were efficiently activated. However, the MAF precursor activity of plasma Gc protein was low in 16 (35%) of of these patients. Loss of the MAF precursor activity appeared to be due to deglycosylation of plasma Gc protein by alpha-N-acetylgalactosaminidase found in the patient blood stream. Levels of plasma alpha-N-acetylgalactosaminidase activity in individual patients had an inverse correlation with the MAF precursor activity of their plasma Gc protein. Thus, precursor activity of Gc protein and alpha-N-acetylgalactosaminidase activity in patient blood can serve as diagnostic and prognostic indices. PMID:8573395

  4. Deglycosylation of serum vitamin D3-binding protein leads to immunosuppression in cancer patients.

    PubMed

    Yamamoto, N; Naraparaju, V R; Asbell, S O

    1996-06-15

    Serum vitamin D3-binding protein (Gc protein) can be converted by beta-galactosidase of B cells and sialidase of T cells to a potent macrophage activating factor, a protein with N-acetylgalactosamine as the remaining sugar moiety. Thus, Gc protein is the precursor of the macrophage activating factor (MAF). Treatment of Gc protein with immobilized beta-galactosidase and sialidase generates an extremely high titered MAF, Gc-MAF. When peripheral blood monocytes/macrophages of 52 patients bearing various types of cancer were incubated with 100 pg/ml of GcMAF, the monocytes/macrophages of all patients were efficiently activated. However, the MAF precursor activity of patient plasma Gc protein was found to be severely reduced in about 25% of this patient population. About 45% of the patients had moderately reduced MAF precursor activities. Loss of the precursor activity was found to be due to deglycosylation of plasma Gc protein by alpha-N-acetylgalactosaminidase detected in the patient's bloodstream. The source of the enzyme appeared to be cancerous cells. Radiation therapy decreased plasma alpha-N-acetylgalactosaminidase activity with concomitant increase of precursor activity. This implies that radiation therapy decreases the number of cancerous cells capable of secreting alpha-N-acetylgalactosaminidase. Both alpha-N-acetylgalactosaminidase activity and MAF precursor activity of Gc protein in patient bloodstream can serve as diagnostic and prognostic indices. PMID:8665521

  5. Association Between Serum Levels of Adipocyte Fatty Acid-binding Protein and Free Thyroxine

    PubMed Central

    Tseng, Fen-Yu; Chen, Pei-Lung; Chen, Yen-Ting; Chi, Yu-Chao; Shih, Shyang-Ron; Wang, Chih-Yuan; Chen, Chi-Ling; Yang, Wei-Shiung

    2015-01-01

    Abstract Adipocyte fatty acid-binding protein (AFABP) has been shown to be a biomarker of body weight change and atherosclerosis. Changes in thyroid function are associated with changes in body weight and risks of cardiovascular diseases. The association between AFABP and thyroid function status has been seldom evaluated. The aim of this study was to compare the serum AFABP concentrations in hyperthyroid patients and those in euthyroid individuals, and to evaluate the associations between serum AFABP and free thyroxine (fT4) levels. For this study, 30 hyperthyroid patients and 30 euthyroid individuals at a referral medical center were recruited. The patients with hyperthyroidism were treated with antithyroid regimens as clinically indicated. No medication was given to the euthyroid individuals. The body weight, body mass index, thyroid function, serum levels of AFABP, and biochemical data of both groups at baseline and at the 6th month were compared. Associations between AFABP and fT4 levels were also analyzed. At the baseline, the hyperthyroid patients had significantly higher serum AFABP levels than the euthyroid individuals (median [Q1, Q3]: 22.8 [19.4, 30.6] ng/mL vs 18.6 [15.3, 23.2] ng/mL; P = 0.038). With the antithyroid regimens, the AFABP serum levels of the hyperthyroid patients decreased to 16.6 (15.0, 23.9) ng/mL at the 6th month. No difference in the AFABP level was found between the hyperthyroid and the euthyroid groups at the 6th month. At baseline, sex (female vs male, ß = 7.65, P = 0.022) and fT4 level (ß = 2.51, P = 0.018) were significantly associated with AFABP levels in the univariate regression analysis. At the 6th month, sex and fT4 level (ß = 8.09, P < 0.001 and ß = 3.61, P = 0.005, respectively) were also significantly associated with AFABP levels. The associations between sex and fT4 level with AFABP levels remained significant in the stepwise multivariate regression analysis, both at baseline and at

  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. Liver Retinol Transporter and Receptor for Serum Retinol-binding Protein (RBP4)*

    PubMed Central

    Alapatt, Philomena; Guo, Fangjian; Komanetsky, Susan M.; Wang, Shuping; Cai, Jinjin; Sargsyan, Ashot; Rodríguez Díaz, Eduardo; Bacon, Brandon T.; Aryal, Pratik; Graham, Timothy E.

    2013-01-01

    Vitamin A (retinol) is absorbed in the small intestine, stored in liver, and secreted into circulation bound to serum retinol-binding protein (RBP4). Circulating retinol may be taken up by extrahepatic tissues or recycled back to liver multiple times before it is finally metabolized or degraded. Liver exhibits high affinity binding sites for RBP4, but specific receptors have not been identified. The only known high affinity receptor for RBP4, Stra6, is not expressed in the liver. Here we report discovery of RBP4 receptor-2 (RBPR2), a novel retinol transporter expressed primarily in liver and intestine and induced in adipose tissue of obese mice. RBPR2 is structurally related to Stra6 and highly conserved in vertebrates, including humans. Expression of RBPR2 in cultured cells confers high affinity RBP4 binding and retinol transport, and RBPR2 knockdown reduces RBP4 binding/retinol transport. RBPR2 expression is suppressed by retinol and retinoic acid and correlates inversely with liver retinol stores in vivo. We conclude that RBPR2 is a novel retinol transporter that potentially regulates retinol homeostasis in liver and other tissues. In addition, expression of RBPR2 in liver and fat suggests a possible role in mediating established metabolic actions of RBP4 in those tissues. PMID:23105095

  8. Serum vitamin D, vitamin D binding protein, and lung cancer survival

    PubMed Central

    Anic, Gabriella M.; Weinstein, Stephanie J.; Mondul, Alison M.; Männistö, Satu; Albanes, Demetrius

    2014-01-01

    Objectives Vitamin D may prolong cancer survival by inhibiting tumor progression and metastasis, however, there are limited epidemiologic studies regarding the association between circulating 25-hydroxyvitamin D (25(OH)D) and lung cancer survival. The aim of this study was to examine the relationship between serum 25(OH)D and lung cancer specific survival and to evaluate whether vitamin D binding protein (DBP) concentration modified this association. Materials and Methods 25(OH)D and DBP were measured in fasting serum samples from 500 male lung cancer cases in the Alpha-Tocopherol, Beta-Carotene Cancer Prevention Study. Cox proportional hazards regression was used to estimate hazard ratios (HRs) and 95% confidence intervals (CI) for lung cancer related death according to quartiles of season-specific 25(OH)D, DBP, and the molar ratio of 25(OH)D:DBP, a proxy for free circulating 25(OH)D. Results Comparing highest to lowest quartiles, serum 25(OH)D (HR=1.18; 95% CI: 0.89–1.56) and DBP (HR=0.95; 95% CI: 0.71–1.26) were not associated with lung cancer survival and DBP concentration did not modify the association with 25(OH)D (p for interaction=0.56). There was suggestion of an association between higher serum 25(OH)D and better survival from adenocarcinoma (HR=0.64; 95% CI: 0.17–2.45) and small cell carcinoma (HR=0.55; 95% CI: 0.21–1.45), but these estimates were based on a relatively small number of cases. Conclusion Serum 25(OH)D was not associated with overall lung cancer survival regardless of DBP concentration, however, these findings should be examined in other studies that include women and subjects with higher 25(OH)D levels. PMID:25456734

  9. Serum Galectin-9 and Galectin-3-Binding Protein in Acute Dengue Virus Infection

    PubMed Central

    Liu, Kuan-Ting; Liu, Yao-Hua; Chen, Yen-Hsu; Lin, Chun-Yu; Huang, Chung-Hao; Yen, Meng-Chi; Kuo, Po-Lin

    2016-01-01

    Dengue fever is a serious threat for public health and induces various inflammatory cytokines and mediators, including galectins and glycoproteins. Diverse immune responses and immunological pathways are induced in different phases of dengue fever progression. However, the status of serum galectins and glycoproteins is not fully determined. The aim of this study was to investigate the serum concentration and potential interaction of soluble galectin-1, galectin-3, galectin-9, galectin-3 binding protein (galectin-3BP), glycoprotein 130 (gp130), and E-, L-, and P-selectin in patients with dengue fever in acute febrile phase. In this study, 317 febrile patients (187 dengue patients, 150 non-dengue patients that included 48 patients with bacterial infection and 102 patients with other febrile illness) who presented to the emergency department and 20 healthy controls were enrolled. Our results showed the levels of galectin-9 and galectin-3BP were significantly higher in dengue patients than those in healthy controls. Lower serum levels of galectin-1, galectin-3, and E-, L-, and P-selectin in dengue patients were detected compared to bacteria-infected patients, but not to healthy controls. In addition, strong correlation between galectin-9 and galectin-3BP was observed in dengue patients. In summary, our study suggested galectin-9 and galectin-3BP might be critical inflammatory mediators in acute dengue virus infection. PMID:27240351

  10. Serum Galectin-9 and Galectin-3-Binding Protein in Acute Dengue Virus Infection.

    PubMed

    Liu, Kuan-Ting; Liu, Yao-Hua; Chen, Yen-Hsu; Lin, Chun-Yu; Huang, Chung-Hao; Yen, Meng-Chi; Kuo, Po-Lin

    2016-01-01

    Dengue fever is a serious threat for public health and induces various inflammatory cytokines and mediators, including galectins and glycoproteins. Diverse immune responses and immunological pathways are induced in different phases of dengue fever progression. However, the status of serum galectins and glycoproteins is not fully determined. The aim of this study was to investigate the serum concentration and potential interaction of soluble galectin-1, galectin-3, galectin-9, galectin-3 binding protein (galectin-3BP), glycoprotein 130 (gp130), and E-, L-, and P-selectin in patients with dengue fever in acute febrile phase. In this study, 317 febrile patients (187 dengue patients, 150 non-dengue patients that included 48 patients with bacterial infection and 102 patients with other febrile illness) who presented to the emergency department and 20 healthy controls were enrolled. Our results showed the levels of galectin-9 and galectin-3BP were significantly higher in dengue patients than those in healthy controls. Lower serum levels of galectin-1, galectin-3, and E-, L-, and P-selectin in dengue patients were detected compared to bacteria-infected patients, but not to healthy controls. In addition, strong correlation between galectin-9 and galectin-3BP was observed in dengue patients. In summary, our study suggested galectin-9 and galectin-3BP might be critical inflammatory mediators in acute dengue virus infection. PMID:27240351

  11. Identification of a protein-binding site that mediates transcriptional response of the c-fos gene to serum factors.

    PubMed

    Treisman, R

    1986-08-15

    Transient transcriptional activation of the c-fos gene following serum stimulation of susceptible cells requires a conserved DNA element located 300 bp 5' to the mRNA cap site. A DNA-binding gel electrophoresis assay was used to detect a protein(s) in HeLa cell nuclear extracts that specifically binds to the 5' activating element. The protein recognizes a region of dyad symmetry within the 5' activating element, defined by binding competition, dimethylsulphate (DMS) interference and DNAase I and DMS protection studies. A single 22 bp synthetic copy of the dyad symmetry element will both compete efficiently for protein binding and restore serum regulation to c-fosH genes that lack the 5' activating element. PMID:3524858

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

    PubMed

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

    2016-08-14

    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. PMID:27241081

  13. Binding of radioiodinated human. beta. -endorphin to serum proteins from rats and humans, determined by several methods

    SciTech Connect

    Sato, H.; Sugiyama, Y.; Sawada, Y.; Iga, T.; Hanano, M.

    1985-10-07

    Binding of immunoreactive radioiodinated human ..beta..-endorphin (/sup 125/I-..beta..-EP) to rat serum was demonstrated by gel filtration of /sup 125/I-..beta..-EP in pooled rat serum on Sephadex G-200. Two radioactive peaks associated with proteins eluted from the column. The first peak eluted at the void volume containing lipoproteins, ..cap alpha../sub 2/- and ..beta../sub 2/-macroglobulins, and the second peak at the fraction of albumin. Binding of /sup 125/I-..beta..-EP to albumin was directly proved by gel filtration of /sup 125/I-..beta..-EP in buffer containing 4% human serum albumin on Sephadex G-200. Equilibrium dialysis was not applicable to investigating the interaction of /sup 125/I-..beta..-EP with serum proteins, because of the intense nonspecific adsorption to the semi-permeable membrane and the degradation of the peptide during dialysis. Therefore, in order to quantitatively evaluate the binding of /sup 125/I-..beta..-EP in sera from rats and humans, the authors utilized four other methods (ultrafiltration, charcoal adsorption, polyethylene glycol precipitation and equilibrium gel filtration). These methods corresponded well with each other and indicated 35-44% binding of /sup 125/I-..beta..-EP in rat serum. Binding of /sup 125/I-..beta..-EP in normal human serum was 36%, determined by ultrafiltration. Serum protein binding of /sup 125/I-..beta..-EP was concentration independent over the concentration range studied (1-1000 nM). 23 references, 4 figures, 1 table.

  14. The vitamin E-binding protein afamin increases in maternal serum during pregnancy

    PubMed Central

    Hubalek, Michael; Buchner, Hannes; Mörtl, Manfred G.; Schlembach, Dietmar; Huppertz, Berthold; Firulovic, Branka; Köhler, Wolfgang; Hafner, Erich; Dieplinger, Benjamin; Wildt, Ludwig; Dieplinger, Hans

    2014-01-01

    Background Afamin is a liver-derived plasma glycoprotein with vitamin E-binding properties and a putative function in fertility. This study evaluated serum afamin concentrations during and postpartum to uncomplicated pregnancies and investigated a potential association between afamin concentrations and pregnancy outcome. Methods Afamin serum concentrations were measured in women with uncomplicated pregnancies in a retrospective cohort (n = 466) at different gestational ages and a prospective observational study (n = 76) in the first, second and third trimester. Furthermore, afamin was determined in the first trimester in a cross-sectional pilot study including women with preeclampsia (PE), pregnancy-induced hypertension (PIH) and women without pregnancy complications (n = 13 each). Finally, expression of afamin was investigated in human placental tissue by RT-PCR and immunohistochemistry. Results Afamin concentrations increased linearly almost two-fold during pregnancy in both retrospective and prospective studies in women without pregnancy complications with median afamin serum concentrations of 61.9 mg/l, 79.6 mg/l, and 98.6 mg/l in the first, second, and third trimester, respectively. After delivery, median afamin concentrations decreased to baseline values of 54.6 mg/l. In the pilot study with pregnancy complications, women with PE displayed significantly higher median afamin concentrations than did women with uncomplicated pregnancy (70.0 mg/l vs. 55.4 mg/l, P = 0.007). Expression analyses revealed no placental afamin expression at either mRNA or protein level in uncomplicated pregnancy. Conclusion A linear increase in the maternally expressed glycoprotein afamin during pregnancy may serve as basic reference for subsequent investigations of afamin in pregnancy-related disorders. PMID:24768783

  15. Intestinal Dysbiosis and Lowered Serum Lipopolysaccharide-Binding Protein in Parkinson’s Disease

    PubMed Central

    Hasegawa, Satoru; Goto, Sae; Tsuji, Hirokazu; Okuno, Tatsuya; Asahara, Takashi; Nomoto, Koji; Shibata, Akihide; Fujisawa, Yoshiro; Minato, Tomomi; Okamoto, Akira; Ohno, Kinji; Hirayama, Masaaki

    2015-01-01

    Background The intestine is one of the first affected organs in Parkinson’s disease (PD). PD subjects show abnormal staining for Escherichia coli and α-synuclein in the colon. Methods We recruited 52 PD patients and 36 healthy cohabitants. We measured serum markers and quantified the numbers of 19 fecal bacterial groups/genera/species by quantitative RT-PCR of 16S or 23S rRNA. Although the six most predominant bacterial groups/genera/species covered on average 71.3% of total intestinal bacteria, our analysis was not comprehensive compared to metagenome analysis or 16S rRNA amplicon sequencing. Results In PD, the number of Lactobacillus was higher, while the sum of analyzed bacteria, Clostridium coccoides group, and Bacteroides fragilis group were lower than controls. Additionally, the sum of putative hydrogen-producing bacteria was lower in PD. A linear regression model to predict disease durations demonstrated that C. coccoides group and Lactobacillus gasseri subgroup had the largest negative and positive coefficients, respectively. As a linear regression model to predict stool frequencies showed that these bacteria were not associated with constipation, changes in these bacteria were unlikely to represent worsening of constipation in the course of progression of PD. In PD, the serum lipopolysaccharide (LPS)-binding protein levels were lower than controls, while the levels of serum diamine oxidase, a marker for intestinal mucosal integrity, remained unchanged in PD. Conclusions The permeability to LPS is likely to be increased without compromising the integrity of intestinal mucosa in PD. The increased intestinal permeability in PD may make the patients susceptible to intestinal dysbiosis. Conversely, intestinal dysbiosis may lead to the increased intestinal permeability. One or both of the two mechanisms may be operational in development and progression of PD. PMID:26539989

  16. 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

  17. Inclusion of a non-immunoglobulin binding protein in two-site ELISA for quantification of human serum proteins without interference by heterophilic serum antibodies.

    PubMed

    Andersson, Mårten; Rönnmark, Jenny; Areström, Iréne; Nygren, Per Ake; Ahlborg, Niklas

    2003-12-01

    Measurement of human serum molecules with two-site ELISA can be biased by the presence of human heterophilic anti-animal immunoglobulin antibodies (HAIA) that cause false-positive signals by cross-linking the monoclonal (mAb) and/or polyclonal antibodies (pAb) used for the pre- (capture) and post-analyte steps (detection). To evaluate a novel ELISA format designed to avoid interference by HAIA, a target-specific non-immunoglobulin (Ig) affinity protein (affibody) was used to replace one of the antibodies. First, a human IgA-binding affibody (Z(IgA)) selected by phage display technology from a combinatorial library of a single Staphylococcus aureus protein A domain was used. The detection range of IgA standard using an ELISA based on Z(IgA) for capture and goat pAb against IgA (pAb(IgA)) for detection was comparable with that of using pAb(IgA) for both capture and detection. Secondly, another affibody (Z(Apo)) was combined with mAb and used to detect recombinant human apolipoprotein A-1. The affibody/antibody ELISAs were also used to quantify human serum levels of IgA and apolipoprotein A1. To verify that human serum did not cause false-positive signals in the affibody/antibody ELISA format, the ability of human serum to cross-link affibodies, mAb (mouse or rat) and/or pAb (goat) displaying non-matched specificities was assessed; affibodies and antibodies were not cross-linked whereas all combinations of mAb and/or pAb were cross-linked. The combination of affibodies and antibodies for analysis of human serum molecules represents a novel two-site ELISA format which precludes false-positive signals caused by HAIA. PMID:14659914

  18. Serum and testicular testosterone and androgen binding protein profiles following subchronic treatment with carbendazim.

    PubMed

    Rehnberg, G L; Cooper, R L; Goldman, J M; Gray, L E; Hein, J F; McElroy, W K

    1989-10-01

    While the general toxicity of the benzimidazole pesticides for mammals is low, one of these compounds, carbendazim (MBC), causes degeneration of testicular tissue and decreases spermatogenic activity at doses well below the LD50 value. A study conducted by S. D. Carter, R. A. Hess, and J. W. Laskey (1987, Biol. Reprod. 37, 709-717) showed that treatment with 400 mg/kg/day MBC resulted in severe seminiferous tubular atrophy and infertility. Since spermatogenesis is an androgen-dependent process, we characterized the effects of MBC (0-400 mg/kg/day) on the endocrine function of the rat testes. Following subchronic (85 day) exposure, serum hormones (TSH, LH, FSH, and Prl) were measured as were androgen binding protein (ABP) and testosterone in testicular fluids (interstitial fluid and seminiferous tubule fluid). In addition, the functional capacity of the Leydig cell to secrete testosterone was assessed in vitro following an hCG challenge. Subchronic treatment with MBC at doses of 50-100 mg/kg/day had no effect on pituitary or testicular hormone concentrations: 200 mg/kg/day elevated the testosterone concentration in the seminiferous tubule fluid and the ABP concentration in both the interstitial fluid and the seminiferous tubule fluid without affecting serum testosterone or ABP concentrations. The 400 mg/kg/day dose resulted in increased concentration of both testosterone and ABP in the interstitial fluid and seminiferous tubule fluid and elevated serum ABP, with no change in serum testosterone. This endocrine profile is consistent with the testicular atrophy and "Sertoli cell-only" syndrome seen in these animals as reported by Gray et al. (1987, Toxicologist 7, 717). We conclude that seminiferous tubule fluid testosterone may be a result of two factors: (1) increased interstitial fluid testosterone concentrations and (2) decreased testosterone outflow from the testis to the general circulation. Also, increased ABP in the interstitial fluid may reflect a change in

  19. Integration of binding peptide selection and multifunctional particles as tool-box for capture of soluble proteins in serum.

    PubMed

    Cusano, Angela Maria; Causa, Filippo; Moglie, Raffaella Della; Falco, Nunzia; Scognamiglio, Pasqualina Liana; Aliberti, Anna; Vecchione, Raffaele; Battista, Edmondo; Marasco, Daniela; Savarese, Marika; Raucci, Umberto; Rega, Nadia; Netti, Paolo Antonio

    2014-10-01

    In this paper, we report on a general approach for the detection of a specific tumoural biomarker directly in serum. Such detection is made possible using a protein-binding peptide selected through an improved phage display technique and then conjugated to engineered microparticles (MPs). Protein biomarkers represent an unlimited source of information for non-invasive diagnostic and prognostic tests; MP-based assays are becoming largely used in manipulation of soluble biomarkers, but their direct use in serum is hampered by the complex biomolecular environment. Our technique overcomes the current limitations as it produces a selective MP--engineered with an antifouling layer--that 'captures' the relevant protein staying impervious to the background. Our system succeeds in fishing-out the human tumour necrosis factor alpha directly in serum with a high selectivity degree. Our method could have great impact in soluble protein manipulation and detection for a wide variety of diagnostic applications. PMID:25100324

  20. Integration of binding peptide selection and multifunctional particles as tool-box for capture of soluble proteins in serum

    PubMed Central

    Cusano, Angela Maria; Causa, Filippo; Moglie, Raffaella Della; Falco, Nunzia; Scognamiglio, Pasqualina Liana; Aliberti, Anna; Vecchione, Raffaele; Battista, Edmondo; Marasco, Daniela; Savarese, Marika; Raucci, Umberto; Rega, Nadia; Netti, Paolo Antonio

    2014-01-01

    In this paper, we report on a general approach for the detection of a specific tumoural biomarker directly in serum. Such detection is made possible using a protein-binding peptide selected through an improved phage display technique and then conjugated to engineered microparticles (MPs). Protein biomarkers represent an unlimited source of information for non-invasive diagnostic and prognostic tests; MP-based assays are becoming largely used in manipulation of soluble biomarkers, but their direct use in serum is hampered by the complex biomolecular environment. Our technique overcomes the current limitations as it produces a selective MP—engineered with an antifouling layer—that ‘captures’ the relevant protein staying impervious to the background. Our system succeeds in fishing-out the human tumour necrosis factor alpha directly in serum with a high selectivity degree. Our method could have great impact in soluble protein manipulation and detection for a wide variety of diagnostic applications. PMID:25100324

  1. Identification and Characterization of a Non-retinoid Ligand for Retinol-binding Protein 4 Which Lowers Serum Retinol-binding Protein 4 Levels in Vivo*

    PubMed Central

    Motani, Alykhan; Wang, Zhulun; Conn, Marion; Siegler, Karen; Zhang, Ying; Liu, Qingxiang; Johnstone, Sheree; Xu, Haoda; Thibault, Steve; Wang, Yingcai; Fan, Pingchen; Connors, Richard; Le, Hoa; Xu, Guifen; Walker, Nigel; Shan, Bei; Coward, Peter

    2009-01-01

    Retinol-binding protein 4 (RBP4) transports retinol from the liver to extrahepatic tissues, and RBP4 lowering is reported to improve insulin sensitivity in mice. We have identified A1120, a high affinity (Ki = 8.3 nm) non-retinoid ligand for RBP4, which disrupts the interaction between RBP4 and its binding partner transthyretin. Analysis of the RBP4-A1120 co-crystal structure reveals that A1120 induces critical conformational changes at the RBP4-transthyretin interface. Administration of A1120 to mice lowers serum RBP4 and retinol levels but, unexpectedly, does not improve insulin sensitivity. In addition, we show that Rpb4-/- mice display normal insulin sensitivity and are not protected from high fat diet-induced insulin resistance. We conclude that lowering RBP4 levels does not improve insulin sensitivity in mice. Therefore, RBP4 lowering may not be an effective strategy for treating diabetes. PMID:19147488

  2. A fluorescence-based high throughput assay for the determination of small molecule–human serum albumin protein binding

    PubMed Central

    McCallum, Megan M.; Pawlak, Alan J.; Shadrick, William R.; Simeonov, Anton; Jadhav, Ajit; Yasgar, Adam; Maloney, David J.; Arnold, Leggy A.

    2014-01-01

    Herein, we describe the development of a fluorescence-based high throughput assay to determine the small molecule binding towards human serum albumin (HSA). This innovative competition assay is based on the use of a novel fluorescent small molecule Red Mega 500 with unique spectroscopic and binding properties. The commercially available probe displays a large fluorescence intensity difference between the protein-bound and protein-unbound state. The competition of small molecules for HSA binding in the presence of probe resulted in low fluorescence intensities. The assay was evaluated with the LOPAC small molecule library of 1280 compounds identifying known high protein binders. The small molecule competition of HSA–Red Mega 500 binding was saturable at higher compound concentrations and exhibited IC50 values between 3–24 μM. The compound affinity towards HSA was confirmed by isothermal titration calorimetry indicating that the new protein binding assay is a valid high throughput assay to determine plasma protein binding. PMID:24390461

  3. Serum Vitamin D, Vitamin D Binding Protein, and Risk of Colorectal Cancer

    PubMed Central

    Anic, Gabriella M.; Weinstein, Stephanie J.; Mondul, Alison M.; Männistö, Satu; Albanes, Demetrius

    2014-01-01

    Background We previously reported a positive association between serum 25-hydroxyvitamin D (25(OH)D) and colorectal cancer risk. To further elucidate this association, we examined the molar ratio of 25(OH)D to vitamin D binding protein (DBP), the primary 25(OH)D transport protein, and whether DBP modified the association between 25(OH)D and colorectal cancer risk. Methods In a nested case-control study within the Alpha-Tocopherol, Beta-Carotene Cancer Prevention Study, controls were 1∶1 matched to 416 colorectal cancer cases based on age and date of blood collection. Logistic regression was used to estimate odds ratios (ORs) and 95% confidence intervals (CI) for quartiles of 25(OH)D, DBP, and the molar ratio of 25(OH)D:DBP, a proxy for free, unbound circulating 25(OH)D. Results Comparing highest to lowest quartiles, DBP was not associated with colorectal cancer risk (OR = 0.91; 95% CI: 0.58, 1.42, p for trend  = 0.58); however, a positive risk association was observed for the molar ratio of 25(OH)D:DBP (OR = 1.44; 95% CI: 0.92, 2.26, p for trend  = 0.04). In stratified analyses, the positive association between 25(OH)D and colorectal cancer was stronger among men with DBP levels above the median (OR = 1.89; 95% CI: 1.07, 3.36, p for trend  = 0.01) than below the median (OR = 1.20; 95% CI: 0.68, 2.12, p for trend  = 0.87), although the interaction was not statistically significant (p for interaction  = 0.24). Conclusion Circulating DBP may influence the association between 25(OH)D and colorectal cancer in male smokers, with the suggestion of a stronger positive association in men with higher DBP concentrations. This finding should be examined in other populations, especially those that include women and non-smokers. PMID:25036524

  4. Interaction study on bovine serum albumin physically binding to silver nanoparticles: Evolution from discrete conjugates to protein coronas

    NASA Astrophysics Data System (ADS)

    Guo, Jun; Zhong, Ruibo; Li, Wanrong; Liu, Yushuang; Bai, Zhijun; Yin, Jun; Liu, Jingran; Gong, Pei; Zhao, Xinmin; Zhang, Feng

    2015-12-01

    The nanostructures formed by inorganic nanoparticles together with organic molecules especially biomolecules have attracted increasing attention from both industries and researching fields due to their unique hybrid properties. In this paper, we systemically studied the interactions between amphiphilic polymer coated silver nanoparticles and bovine serum albumins by employing the fluorescence quenching approach in combination with the Stern-Volmer and Hill equations. The binding affinity was determined to 1.30 × 107 M-1 and the interaction was spontaneously driven by mainly the van der Waals force and hydrogen-bond mediated interactions, and negatively cooperative from the point of view of thermodynamics. With the non-uniform coating of amphiphilic polymer, the silver nanoparticles can form protein coronas which can become discrete protein-nanoparticle conjugates when controlling their molar ratios of mixing. The protein's conformational changes upon binding nanoparticles was also studied by using the three-dimensional fluorescence spectroscopy.

  5. Quantitative Proteomics Identifies Serum Response Factor Binding Protein 1 as a Host Factor for Hepatitis C Virus Entry.

    PubMed

    Gerold, Gisa; Meissner, Felix; Bruening, Janina; Welsch, Kathrin; Perin, Paula M; Baumert, Thomas F; Vondran, Florian W; Kaderali, Lars; Marcotrigiano, Joseph; Khan, Abdul G; Mann, Matthias; Rice, Charles M; Pietschmann, Thomas

    2015-08-01

    Hepatitis C virus (HCV) enters human hepatocytes through a multistep mechanism involving, among other host proteins, the virus receptor CD81. How CD81 governs HCV entry is poorly characterized, and CD81 protein interactions after virus binding remain elusive. We have developed a quantitative proteomics protocol to identify HCV-triggered CD81 interactions and found 26 dynamic binding partners. At least six of these proteins promote HCV infection, as indicated by RNAi. We further characterized serum response factor binding protein 1 (SRFBP1), which is recruited to CD81 during HCV uptake and supports HCV infection in hepatoma cells and primary human hepatocytes. SRFBP1 facilitates host cell penetration by all seven HCV genotypes, but not of vesicular stomatitis virus and human coronavirus. Thus, SRFBP1 is an HCV-specific, pan-genotypic host entry factor. These results demonstrate the use of quantitative proteomics to elucidate pathogen entry and underscore the importance of host protein-protein interactions during HCV invasion. PMID:26212323

  6. Human serum albumin-mimetic chromatography based hexadecyltrimethylammonium bromide as a novel direct probe for protein binding of acidic drugs.

    PubMed

    Salary, Mina; Hadjmohammadi, Mohammadreza

    2015-10-10

    Human serum albumin (HSA) is the most important drug carrier in humans mainly binding acidic drugs. Negatively charged compounds bind more strongly to HSA than it would be expected from their lipophilicity alone. With the development of new acidic drugs, there is a high need for rapid and simple protein binding screening technologies. Biopartitioning micellar chromatography (BMC) is a mode of micellar liquid chromatography, which can be used as an in vitro system to model the biopartitioning process of drugs when there are no active processes. In this study, a new kind of BMC using hexadecyltrimethylammonium bromide (CTAB) as micellar mobile phases was used for the prediction of protein binding of acidic drugs based on the similar property of CTAB micelles to HSA. The use of BMC is simple, reproducible and can provide key information about the pharmacological behavior of drugs such as protein binding properties of new compounds during the drug discovery process. The relationships between the MLC retention data of a heterogeneous set of 17 acidic and neutral drugs and their plasma protein binding parameter were studied and second-order polynomial models obtained in two different concentrations (0.07 and 0.09M) of CTAB. However, the developed models are only being able to distinguish between strongly and weakly binding drugs. Also, the developed models were characterized by both the descriptive and predictive ability (R(2)=0.885, RCV(2)=0.838 and R(2)=0.898, RCV(2)=0.859 for 0.07 and 0.09M CTAB, respectively). The application of the developed model to a prediction set demonstrated that the model was also reliable with good predictive accuracy. PMID:25988296

  7. Photoactivable analogs for labeling 25-hydroxyvitamin D3 serum binding protein and for 1,25-dihydroxyvitamin D3 intestinal receptor protein

    NASA Technical Reports Server (NTRS)

    Kutner, A.; Link, R. P.; Schnoes, H. K.; DeLuca, H. F.

    1986-01-01

    3-Azidobenzoates and 3-azidonitrobenzoates of 25-hydroxyvitamin D3 as well as 3-deoxy-3-azido-25-hydroxyvitamin D3 and 3-deoxy-3-azido-1,25-dihydroxyvitamin D3 were prepared as photoaffinity labels for vitamin D serum binding protein and 1,25-dihydroxyvitamin D3 intestinal receptor protein. The compounds prepared were easily activated by short- or long-wavelength uv light, as monitored by uv and ir spectrometry. The efficacy of the compounds to compete with 25-hydroxyvitamin D3 or 1,25-dihydroxyvitamin D3 for the binding site of serum binding protein and receptor, respectively, was studied to evaluate the vitamin D label with the highest affinity for the protein. The presence of an azidobenzoate or azidonitrobenzoate substituent at the C-3 position of 25-OH-D3 significantly decreased (10(4)- to 10(6)-fold) the binding activity. However, the labels containing the azido substituent attached directly to the vitamin D skeleton at the C-3 position showed a high affinity, only 20- to 150-fold lower than that of the parent compounds with their respective proteins. Therefore, 3-deoxy-3-azidovitamins present potential ligands for photolabeling of vitamin D proteins and for studying the structures of the protein active sites.

  8. Transgenic Mice Overexpressing Serum Retinol-Binding Protein Develop Progressive Retinal Degeneration through a Retinoid-Independent Mechanism

    PubMed Central

    Du, Mei; Otalora, Laura; Martin, Ashley A.; Moiseyev, Gennadiy; Vanlandingham, Phillip; Wang, Qilong; Farjo, Rafal; Yeganeh, Alexander; Quiambao, Alexander

    2015-01-01

    Serum retinol-binding protein 4 (RBP4) is the sole specific transport protein for retinol in the blood, but it is also an adipokine with retinol-independent, proinflammatory activity associated with obesity, insulin resistance, type 2 diabetes, and cardiovascular disease. Moreover, two separate studies reported that patients with proliferative diabetic retinopathy have increased serum RBP4 levels compared to patients with mild or no retinopathy, yet the effect of increased levels of RBP4 on the retina has not been studied. Here we show that transgenic mice overexpressing RBP4 (RBP4-Tg mice) develop progressive retinal degeneration, characterized by photoreceptor ribbon synapse deficiency and subsequent bipolar cell loss. Ocular retinoid and bisretinoid levels are normal in RBP4-Tg mice, demonstrating that a retinoid-independent mechanism underlies retinal degeneration. Increased expression of pro-interleukin-18 (pro-IL-18) mRNA and activated IL-18 protein and early-onset microglia activation in the retina suggest that retinal degeneration is driven by a proinflammatory mechanism. Neither chronic systemic metabolic disease nor other retinal insults are required for RBP4 elevation to promote retinal neurodegeneration, since RBP4-Tg mice do not have coincident retinal vascular pathology, obesity, dyslipidemia, or hyperglycemia. These findings suggest that elevation of serum RBP4 levels could be a risk factor for retinal damage and vision loss in nondiabetic as well as diabetic patients. PMID:26055327

  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. PMID:26821345

  10. Serum insulin-like growth factor-I, IGF binding protein-3 and IGFBP-3 protease activity after cranial irradiation.

    PubMed

    Tillmann, V; Shalet, S M; Price, D A; Wales, J K; Pennells, L; Soden, J; Gill, M S; Whatmore, A J; Clayton, P E

    1998-01-01

    The relationship between peak growth hormone (GH), insulin-like growth factor I (IGF-I), IGF-I binding protein 3 (IGFBP-3) and IGFBP-3 protease activity was studied in 28 children and adolescents undergoing investigation of pituitary function 0.4-14.2 years after cranial or craniospinal irradiation for the treatment of CNS tumours distant from the hypothalamic-pituitary axis (n = 16) or prophylaxis against CNS leukaemia (n = 12). Seven out of 15 patients with GH deficiency (GHD) (defined as a peak GH concentration <7.5 ng/ml in a stimulation test) had IGF-I <-2 standard deviation score (SDS). None of the 28 patients had serum IGFBP-3 concentrations measured by radioimmunoassay (RIA) <-1.5 SDS with no difference between those with and without GHD. IGFBP-3 concentrations measured by RIA were strongly correlated to IGFBP-3 band density on Western ligand blot (WLB) (r = 0.71; p < 0.0001). IGFBP-3 protease activity was negatively correlated to IGFBP-3 by RIA (r = -0.55; p < 0.01) and to IGFBP-3 by WLB (r = -0.51; p < 0.01). Twenty-two patients had normal IGFBP-3 protease activity (<30% of the activity in pregnancy serum) indicating that serum IGFBP-3 protease activity does not account for the normal levels of IGFBP-3 in RIA. Low serum IGF-I but normal IGFBP-3 concentrations and in the majority normal IGFBP-3 protease activity was found in patients in the years after CNS irradiation. Neither serum IGF-I nor IGFBP-3 can be used as a reliable index of the development of radiation-induced GHD. PMID:9701699

  11. Analysis of S100 calcium binding protein B serum levels in different types of traumatic intracranial lesions.

    PubMed

    Wolf, Harald; Frantal, Sophie; Pajenda, Gholam; Leitgeb, Johannes; Sarahrudi, Kambiz; Hajdu, Stefan

    2015-01-01

    The objective of this study was to determine whether the type of intracranial traumatic lesions, the number of simultaneous traumatic lesions, and the occurrence of skull and facial bone fractures have an influence on S100 calcium binding protein B (S100B) serum levels. Patients with blunt traumatic brain injury were prospectively enrolled into this cohort study over a period of 13 months. Venous blood samples were obtained prior to emergency cranial CT scan in all patients within 3 h after injury. The patients were then assigned into six groups: 1) concussion, 2) epidural hematoma, 3) subdural hematoma, 4) subarachnoid hemorrhage, 5) brain contusions, and 6) brain edema. The study included 1696 head trauma patients with a mean age of 57.7 ± 25.3 years, and 126 patients (8%) had 182 traumatic lesions on CT. Significant differences in S100B serum levels were found between cerebral edema and the other four bleeding groups: epidural p = 0.0002, subdural p < 0.0001, subarachnoid p = 0.0001, brain contusions p = 0.0003, and concussion p < 0.0001. Significant differences in S100B values between patients with one or two intracranial lesions (p = 0.014) or with three (p < 0.0001) simultaneous intracranial lesions were found. In patients with intracranial traumatic lesions, skull fractures, as well as skull and facial bone fractures occurring together, were identified as significant additional factors for the increase in serum S100B levels (p < 0.0001). Older age was also associated with elevated S100B serum levels (p < 0.0001). Our data show that peak S100B serum levels were found in patients with cerebral edema and brain contusions. PMID:25068442

  12. Predicting binding affinities of diverse pharmaceutical chemicals to human serum plasma proteins using QSPR modelling approaches.

    PubMed

    Basant, N; Gupta, S; Singh, K P

    2016-01-01

    The prediction of the plasma protein binding (PPB) affinity of chemicals is of paramount significance in the drug development process. In this study, ensemble machine learning-based QSPR models have been established for a four-category classification and PPB affinity prediction of diverse compounds using a large PPB dataset of 930 compounds and in accordance with the OECD guidelines. The structural diversity of the chemicals was tested by the Tanimoto similarity index. The external predictive power of the developed QSPR models was evaluated through internal and external validations. In the QSPR models, XLogP was the most important descriptor. In the test data, the classification QSPR models rendered an accuracy of >93%, while the regression QSPR models yielded r(2) of >0.920 between the measured and predicted PPB affinities, with the root mean squared error <9.77. Values of statistical coefficients derived for the test data were above their threshold limits, thus put a high confidence in this analysis. The QSPR models in this study performed better than any of the previous studies. The results suggest that the developed QSPR models are reliable for predicting the PPB affinity of structurally diverse chemicals. They can be useful for initial screening of candidate molecules in the drug development process. PMID:26854728

  13. Photoaffinity labeling of serum vitamin D binding protein by 3-deoxy-3-azido-25-hydroxyvitamin D3

    SciTech Connect

    Link, R.P.; Kutner, A.; Schnoes, H.K.; DeLuca, H.F.

    1987-06-30

    3-Deoxy-3-azido-25-hydroxyvitamin D3 was covalently incorporated in the 25-hydroxyvitamin D3 binding site of purified human plasma vitamin D binding protein. Competition experiments showed that 3-deoxy-3-azido-25-hydroxyvitamin D3 and 25-hydroxyvitamin D3 bind at the same site on the protein. Tritiated 3-deoxy-3-azido-25-hydroxyvitamin D3 was synthesized from tritiated 25-hydroxyvitamin D3, retaining the high specific activity of the parent compound. The tritiated azido label bound reversibly to human vitamin D binding protein in the dark and covalently to human vitamin D binding protein after exposure to ultraviolet light. Reversible binding of tritiated 3-deoxy-3-azido-25-hydroxyvitamin D3 was compared to tritiated 25-hydroxyvitamin D3 binding to human vitamin D binding protein. Scatchard analysis of the data indicated equivalent maximum density binding sites with a KD,app of 0.21 nM for 25-hydroxyvitamin D3 and a KD,app of 1.3 nM for the azido derivative. Covalent binding was observed only after exposure to ultraviolet irradiation, with an average of 3% of the reversibly bound label becoming covalently bound to vitamin D binding protein. The covalent binding was reduced 70-80% when 25-hydroxyvitamin D3 was present, indicating strong covalent binding at the vitamin D binding site of the protein. When tritiated 3-deoxy-3-azido-25-hydroxyvitamin D3 was incubated with human plasma in the absence and presence of 25-hydroxyvitamin D3, 12% of the azido derivative was reversibly bound to vitamin D binding protein. After ultraviolet irradiation, four plasma proteins covalently bound the azido label, but vitamin D binding protein was the only protein of the four that was unlabeled in the presence of 25-hydroxyvitamin D3.

  14. Photoaffinity labeling of serum vitamin D binding protein by 3-deoxy-3-azido-25-hydroxyvitamin D/sub 3/

    SciTech Connect

    Link, R.P.; Kutner, A.; Schnoes, H.K.; DeLuca, H.F.

    1986-05-01

    3-Deoxy-3-azido-25-hydroxyvitamin D/sub 3/ (3-Az-25-OH-D/sub 3/) was covalently incorporated into the 25-hydroxyvitamin D/sub 3/ (25-OH-D/sub 3/) binding site of purified human serum vitamin D binding protein (hDBP). Competition experiments showed that 3-Az-25-OH-D/sub 3/ and 25-OH-D/sub 3/ bound at the same site. A K/sub I/ of 80 nM was determined from 3-Az-25-OH-D/sub 33/ inhibition of 25-OH-D/sub 3/ binding to hDBP. 3-Az-25-OH-(26,27-/sup 3/H)D/sub 3/, prepared from 25-OH-(26,27-/sup 3/H)D/sub 3/, bound reversibly to hDBP in the dark. Scatchard analysis of the data indicated a single class of sites with a K/sub D. app/ of 0.71 nM and a B/sub max/ of 0.11 nM for 3-Az-25-OH-(26,27-/sup 3/H)D/sub 3/ as compared to a K/sub D. app/ of 0.37 nM and a B/sub max/ of 0.43 nM for 25-OH-(26,27-/sup 3/H)D/sub 3/. The stability of 25-OH-D/sub 3/ binding to hDBP was tested under the UV irradiation conditions required for photoactivation of 3-Az-25-OH-D/sub 3/. After 3 minutes of medium intensity, short wavelength (254 nm) UV irradiation, 40-50% of the previously bound 25-OH-(26,27-/sup 3/H)D/sub 3/ was retained by hDBP and 60% of the unliganded protein was capable of subsequently binding 25-OH-(26,27-/sup 3/H)D/sub 3/. Maximum specific covalent incorporation of 3-Az-25-OH-(26,27-/sup 3/H)D/sub 3/ by hDBP was after 3 to 5 minutes of irradiation. As much as 7.5% of the initially reversibly bound 3-Az-25-OH-(26,27-/sup 3/H)D/sub 3/ could be covalently bound to the 25-OH-D/sub 3/ binding site of hDBP.

  15. 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

  16. Importance of Inhibition of Binding of Complement Factor H for Serum Bactericidal Antibody Responses to Meningococcal Factor H-binding Protein Vaccines

    PubMed Central

    Konar, Monica; Granoff, Dan M.; Beernink, Peter T.

    2013-01-01

    Background. Factor H (fH) binding protein (fHbp) is part of vaccines developed for prevention of meningococcal serogroup B disease. More than 610 fHbp amino acid sequence variants have been identified, which can be classified into 2 subfamilies. The extent of cross-protection within a subfamily has been difficult to assess because of strain variation in fHbp expression. Methods. Using isogenic mutant strains, we compared cross-protective serum antibody responses of mice immunized with 7 divergent fHbp variants in subfamily B, including identification numbers (ID) 1 and 55, which were chosen for vaccine development. Results and Conclusions. In the presence of the human complement downregulator fH, the ability of the anti-fHbp antibodies to deposit sufficient complement C3b on the bacterial surface to elicit bactericidal activity required inhibition of binding of fH by the anti-fHbp antibodies. With less bound fH, the bacteria became more susceptible to complement-mediated bactericidal activity. Among the different fHbp sequence variants, those more central in a phylogenic network than ID 1 or 55 elicited anti-fHbp antibodies with broader inhibition of fH binding and broader bactericidal activity. Thus, the more central variants show promise of extending protection to strains with divergent fHbp sequences that are covered poorly by fHbp variants in clinical development. PMID:23715659

  17. Protein stability, conformational change and binding mechanism of human serum albumin upon binding of embelin and its role in disease control.

    PubMed

    Yeggoni, Daniel Pushparaju; Rachamallu, Aparna; Subramanyam, Rajagopal

    2016-07-01

    Here, we present the inclusive binding mode of phytochemical embelin, an anticancer drug with human serum albumin (HSA) established under physiological condition. Also, to understand the pharmacological role of embelin molecule, here, we have studied the anti-cancer activity of embelin on human cervical cancer cell line (HeLa cell line), which revealed that embelin showed dose dependent inhibition in the growth of cancer cells and also induces 26.3% of apoptosis at an IC50 value of 29μM. Further, embelin was titrated with HSA and the fluorescence emission quenching of HSA due to the formation of the HSA-embelin complex was observed. The binding constant of this complex is 5.9±.01×10(4)M(-1) and the number of bound embelin molecules is approximately 1.0. Consequently, molecular displacement and computational docking experiments show that the embelin is binding to subdomain IB to HSA. Further evidence from microTOF-Q mass spectrometry showed an increase in mass from 66,563Da to 66,857Da observed for free HSA and HSA+embelin complex, signifying that there is robust binding of embelin with HSA. In addition, the variations of HSA secondary structural elements in presence of embelin were confirmed by circular dichroism which indicates partial unfolding of protein. Furthermore, the transmission electron micrographs established that complex formation leads to aggregation of HSA plus embelin. Molecular dynamics simulations revealed that the stability of the HSA-embelin complexes and results suggests that at around 3500ps the complex reaches equilibration state which clearly contributes to the understanding of the stability of the HSA-embelin complexes. PMID:27130964

  18. Angiotensin II receptor blockers decrease serum concentration of fatty acid-binding protein 4 in patients with hypertension.

    PubMed

    Furuhashi, Masato; Mita, Tomohiro; Moniwa, Norihito; Hoshina, Kyoko; Ishimura, Shutaro; Fuseya, Takahiro; Watanabe, Yuki; Yoshida, Hideaki; Shimamoto, Kazuaki; Miura, Tetsuji

    2015-04-01

    Elevated circulating fatty acid-binding protein 4 (FABP4/A-FABP/aP2), an adipokine, is associated with obesity, insulin resistance, hypertension and cardiovascular events. However, how circulating FABP4 level is modified by pharmacological agents remains unclear. We here examined the effects of angiotensin II receptor blockers (ARBs) on serum FABP4 level. First, essential hypertensives were treated with ARBs: candesartan (8 mg day(-1); n=7) for 2 weeks, olmesartan (20 mg day(-1); n=9) for 12 weeks, and valsartan (80 mg day(-1); n=94) or telmisartan (40 mg day(-1); n=91) for 8 weeks added to amlodipine (5 mg day(-1)). Treatment with ARBs significantly decreased blood pressure and serum FABP4 concentrations by 8-20% without significant changes in adiposity or lipid variables, though the M value determined by hyperinsulinemic-euglycemic glucose clamp, a sensitive index of insulin sensitivity, was significantly increased by candesartan. Next, alterations in FABP4 secretion from 3T3-L1 adipocytes were examined under several agents. Lipolytic stimulation of the β-adrenoceptor in 3T3-L1 adipocytes by isoproterenol increased FABP4 secretion, and conversely, insulin suppressed FABP4 secretion. However, treatment of 3T3-L1 adipocytes with angiotensin II or ARBs for 2 h had no effect on gene expression or secretion of FABP4 regardless of β-adrenoceptor stimulation. In conclusion, treatment with structurally different ARBs similarly decreases circulating FABP4 concentrations in hypertensive patients as a class effect of ARBs, which is not attributable to blockade of the angiotensin II receptor in adipocytes. Reduction of FABP4 levels by ARBs might be involved in suppression of cardiovascular events. PMID:25672659

  19. Characterization of the binding of metoprolol tartrate and guaifenesin drugs to human serum albumin and human hemoglobin proteins by fluorescence and circular dichroism spectroscopy.

    PubMed

    Duman, Osman; Tunç, Sibel; Kancı Bozoğlan, Bahar

    2013-07-01

    The interactions of metoprolol tartrate (MPT) and guaifenesin (GF) drugs with human serum albumin (HSA) and human hemoglobin (HMG) proteins at pH 7.4 were studied by fluorescence and circular dichroism (CD) spectroscopy. Drugs quenched the fluorescence spectra of HSA and HMG proteins through a static quenching mechanism. For each protein-drug system, the values of Stern-Volmer quenching constant, bimolecular quenching constant, binding constant and number of binding site on the protein molecules were determined at 288.15, 298.15, 310.15 and 318.15 K. It was found that the binding constants of HSA-MPT and HSA-GF systems were smaller than those of HMG-MPT and HMG-GF systems. For both drugs, the affinity of HMG was much higher than that of HSA. An increase in temperature caused a negative effect on the binding reactions. The number of binding site on blood proteins for MPT and GF drugs was approximately one. Thermodynamic parameters showed that MPT interacted with HSA through electrostatic attraction forces. However, hydrogen bonds and van der Waals forces were the main interaction forces in the formation of HSA-GF, HMG-MPT and HMG-GF complexes. The binding processes between protein and drug molecules were exothermic and spontaneous owing to negative ∆H and ∆G values, respectively. The values of binding distance between protein and drug molecules were calculated from Förster resonance energy transfer theory. It was found from CD analysis that the bindings of MPT and GF drugs to HSA and HMG proteins altered the secondary structure of HSA and HMG proteins. PMID:23471625

  20. Serum Fatty Acid-Binding Protein 4 Is a Predictor of Cardiovascular Events in End-Stage Renal Disease

    PubMed Central

    Furuhashi, Masato; Ishimura, Shutaro; Ota, Hideki; Hayashi, Manabu; Nishitani, Takahiro; Tanaka, Marenao; Yoshida, Hideaki; Shimamoto, Kazuaki; Hotamisligil, Gökhan S.; Miura, Tetsuji

    2011-01-01

    Background Fatty acid-binding protein 4 (FABP4/A-FABP/aP2), a lipid chaperone, is expressed in both adipocytes and macrophages. Recent studies have shown that FABP4 is secreted from adipocytes and that FABP4 level is associated with obesity, insulin resistance, and atherosclerosis. However, little is known about the impact of FABP4 concentrations on prognosis. We tested the hypothesis that FABP4 level predicts prognosis of patients with end-stage renal disease (ESRD), a group at high risk for atherosclerosis-associated morbidity and mortality. Methods and Results Biochemical markers including FABP4 were determined in 61 ESRD patients on chronic hemodialysis (HD). Serum FABP4 level in females (404.2±30.5 ng/ml) was significantly higher than that in males (315.8±30.0 ng/ml), and the levels in ESRD patients were about 20-times higher than those in age-, gender- and body mass index (BMI)-matched control subjects with normal renal function. FABP4 level was decreased by 57.2% after HD and was positively correlated with blood pressure, BMI, and levels of lipids and insulin. Multiple regression analysis indicated that HD duration, BMI, and triglycerides level were independent determinants for FABP4 level. ESRD patients with high FABP4 levels had higher cardiovascular mortality during the 7-year follow-up period. Cox proportional hazard regression analysis showed that logarithmically transformed FABP4 level was an independent predictor of cardiovascular death adjusted for age, gender, HD duration, BMI, and triglycerides level (hazard ratio, 7.75; 95% CI, 1.05–25.31). Conclusion These findings suggest that FABP4 level, being related to adiposity and metabolic disorders, is a novel predictor of cardiovascular mortality in ESRD. PMID:22102888

  1. Analysis of drug-protein binding using on-line immunoextraction and high-performance affinity microcolumns: Studies with normal and glycated human serum albumin.

    PubMed

    Matsuda, Ryan; Jobe, Donald; Beyersdorf, Jared; Hage, David S

    2015-10-16

    A method combining on-line immunoextraction microcolumns with high-performance affinity chromatography (HPAC) was developed and tested for use in examining drug-protein interactions with normal or modified proteins. Normal human serum albumin (HSA) and glycated HSA were used as model proteins for this work. High-performance immunoextraction microcolumns with sizes of 1.0-2.0 cm × 2.1mm i.d. and containing anti-HSA polyclonal antibodies were developed and tested for their ability to bind normal HSA or glycated HSA. These microcolumns were able to extract up to 82-93% for either type of protein at 0.05-0.10 mL/min and had a binding capacity of 0.34-0.42 nmol HSA for a 1.0 cm × 2.1mm i.d. microcolumn. The immunoextraction microcolumns and their adsorbed proteins were tested for use in various approaches for drug binding studies. Frontal analysis was used with the adsorbed HSA/glycated HSA to measure the overall affinities of these proteins for the drugs warfarin and gliclazide, giving comparable values to those obtained previously using similar protein preparations that had been covalently immobilized within HPAC columns. Zonal elution competition studies with gliclazide were next performed to examine the specific interactions of this drug at Sudlow sites I and II of the adsorbed proteins. These results were also comparable to those noted in prior work with covalently immobilized samples of normal HSA or glycated HSA. These experiments indicated that drug-protein binding studies can be carried out by using on-line immunoextraction microcolumns with HPAC. The same method could be used in the future with clinical samples and other drugs or proteins of interest in pharmaceutical studies or biomedical research. PMID:26381571

  2. Recognition of insulin-like growth factor (IGF) serum binding proteins by an antibody raised against a specific IGF-inhibitor

    SciTech Connect

    Ooi, G.T.; Herington, A.C.

    1988-10-31

    Evidence suggests that a specific inhibitor of the insulin-like growth factors (IGF) which acts by binding to IGF may be structurally related to the native, MW 150K binding protein (BP) in serum. This has now been examined using a polyclonal antiserum (R8) raised against highly purified inhibitor. Western blotting analysis of inhibitor using R8 gave 4 immunoreactive (ir-) bands (MW 34.5K, 23K, 16K and 12K), the most intense being the MW 16K band, identical to the MW of the inhibitor. Ligand blotting using /sup 125/I-IGF-I indicated specific IGF-binding activity at MW 29K, 26.5K, 16K and 12K, indicating that at least 2 of the ir-bands (16K and 12K) were IGF-BPs. Western blotting of a salt-precipitated fraction of serum gave 8 ir-bands of which 3 (MW 42K, 38K and 34K) were identical with BP bands detected previously by Hossenlopp et al. These immunological crossreactivities indicate that the inhibitor is structurally related to the higher MW IGF-BPs in serum.

  3. The associations of diet with serum insulin-like growth factor I and its main binding proteins in 292 women meat-eaters, vegetarians, and vegans.

    PubMed

    Allen, Naomi E; Appleby, Paul N; Davey, Gwyneth K; Kaaks, Rudolf; Rinaldi, Sabina; Key, Timothy J

    2002-11-01

    The lower rates of some cancers in Asian countries than in Western countries may be partly because of diet, although the mechanisms are unknown. The aim of this cross-sectional study was to determine whether a plant-based (vegan) diet is associated with a lower circulating level of insulin-like growth factor I (IGF-I) compared with a meat-eating or lacto-ovo-vegetarian diet among 292 British women, ages 20-70 years. The mean serum IGF-I concentration was 13% lower in 92 vegan women compared with 99 meat-eaters and 101 vegetarians (P = 0.0006). The mean concentrations of both serum IGF-binding protein (IGFBP)-1 and IGFBP-2 were 20-40% higher in vegan women compared with meat-eaters and vegetarians (P = 0.005 and P = 0.0008 for IGFBP-1 and IGFBP-2, respectively). There were no significant differences in IGFBP-3, C-peptide, or sex hormone-binding globulin concentrations between the diet groups. Intake of protein rich in essential amino acids was positively associated with serum IGF-I (Pearson partial correlation coefficient; r = 0.27; P < 0.0001) and explained most of the differences in IGF-I concentration between the diet groups. These data suggest that a plant-based diet is associated with lower circulating levels of total IGF-I and higher levels of IGFBP-1 and IGFBP-2. PMID:12433724

  4. Nitric oxide protects neuroblastoma cells from apoptosis induced by serum deprivation through cAMP-response element-binding protein (CREB) activation.

    PubMed

    Ciani, Elisabetta; Guidi, Sandra; Della Valle, Giuliano; Perini, Giovanni; Bartesaghi, Renata; Contestabile, Antonio

    2002-12-20

    The transcription factor cAMP-response element-binding protein (CREB) mediates survival in many cells, including neurons. Recently, death of cerebellar granule neurons due to nitric oxide (NO) deprivation was shown to be accompanied by down-regulation of CREB activity (). We now provide evidence that overproduction of endogenous NO or supplementation with exogenous NO renders SK-N-BE human neuroblastoma cells more resistant to apoptosis induced by serum deprivation. Parental cells underwent apoptosis after 24 h of serum deprivation, an outcome largely absent in clones overexpressing human neuronal nitric oxide synthase (nNOS). This protective effect was reversed by the inhibition of NOS itself or soluble guanylyl cyclase, pointing at cGMP as an intermediate effector of NO-mediated rescue. A slow-releasing NO donor protected parental cells to a significant extent, thus confirming the survival effect of NO. The impaired viability of serum-deprived parental cells was accompanied by a strong decrease of CREB phosphorylation and transcriptional activity, effects significantly attenuated in nNOS-overexpressing clones. To confirm the role of CREB in survival, the ectopic expression of CREB and/or protein kinase A largely counteracted serum deprivation-induced cell death of SK-N-BE cells, whereas transfection with a CREB negative mutant was ineffective. These experiments indicate that CREB activity is an important step for NO-mediated survival in neuronal cells. PMID:12368293

  5. The role of fibronectin in the development of experimental amyloidosis. Evidence of immunohistochemical codistribution and binding property with serum amyloid protein A.

    PubMed Central

    Kawahara, E.; Shiroo, M.; Nakanishi, I.; Migita, S.

    1989-01-01

    Azocasein-induced amyloid A (AA) amyloidosis in CBA/K1Jms mice was investigated to elucidate a preference of serum amyloid A (SAA) deposition in the spleen. By indirect immunofluorescence using anti-SAA/AA antibodies the initial deposition of SAA/AA was recognized in the marginal zone of spleen at 20 days after azocasein injection. Indirect immunofluorescence using anti-fibronectin antibodies also showed meshwork positivity in the corresponding area more intensely than that in controls. Immunoelectron microscopy using anti-SAA/AA revealed the presence of positively stained flocculent materials on cell surfaces of macrophages in the marginal area in addition to amyloid fibril. The tissue fibronectin rapidly increased in the spleen and maintained 10 times more than that of controls until the 20th day. Binding assay of SAA on frozen sections revealed the presence of SAA-binding substances in the perifollicular area. Affinity chromatographic assay showed fibronectin have a binding capacity to SAA1 and SAA2. By binding assay on the microtiter plate, SAA had more affinity to fibronectin than those of heparan sulfate, collagen type I, or serum amyloid P component. These results indicate that fibronectin plays an important role in the development of amyloidosis by working as a linking protein between SAA and the cell surface of macrophages. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 7 Figure 8 PMID:2757120

  6. Studies of the Interaction between Isoimperatorin and Human Serum Albumin by Multispectroscopic Method: Identification of Possible Binding Site of the Compound Using Esterase Activity of the Protein

    PubMed Central

    Ranjbar, Samira; Shokoohinia, Yalda; Ghobadi, Sirous; Gholamzadeh, Saeed; Moradi, Nastaran; Ashrafi-Kooshk, Mohammad Reza; Aghaei, Abbas

    2013-01-01

    Isoimperatorin is one of the main components of Prangos ferulacea as a linear furanocoumarin and used as anti-inflammatory, analgesic, antispasmodic, and anticancer drug. Human serum albumin (HSA) is a principal extracellular protein with a high concentration in blood plasma and carrier for many drugs to different molecular targets. Since the carrying of drug by HSA may affect on its structure and action, we decided to investigate the interaction between HSA and isoimperatorin using fluorescence and UV spectroscopy. Fluorescence data indicated that isoimperatorin quenches the intrinsic fluorescence of the HSA via a static mechanism and hydrophobic interaction play the major role in the drug binding. The binding average distance between isoimperatorin and Trp 214 of HSA was estimated on the basis of the theory of Förster energy transfer. Decrease of protein surface hydrophobicity (PSH) was also documented upon isoimperatorin binding. Furthermore, the synchronous fluorescence spectra show that the microenvironment of the tryptophan residues does not have obvious changes. Site marker compettive and fluorescence experiments revealed that the binding of isoimperatorin to HSA occurred at or near site I. Finally, the binding details between isoimperatorin and HSA were further confirmed by molecular docking and esterase activity inhibition studies which revealed that drug was bound at subdomain IIA. PMID:24319355

  7. Effects of vitamin D binding protein phenotypes and vitamin D supplementation on serum total 25(OH)D and directly measured free 25(OH)D

    PubMed Central

    Sollid, Stina T; Hutchinson, Moira Y S; Berg, Vivian; Fuskevåg, Ole M; Figenschau, Yngve; Thorsby, Per M; Jorde, Rolf

    2016-01-01

    Objective To determine the relationship between serum total 25-hydroxyvitamin D (25(OH)D), directly measured free 25(OH)D and calculated free 25(OH)D with regard to vitamin D-binding protein (DBP) phenotypes, sex, BMI, age and season, and their interrelationship to vitamin D supplementation. Design, patients and interventions A randomized controlled trial with 20 000 IU of vitamin D3 per week or placebo for 12 months was designed. A total of 472 subjects, 236 in each of the intervention groups, were included in the analyses. Main outcome measures Baseline serum concentrations and increases in serum total 25(OH)D, directly measured free 25(OH)D, calculated free 25(OH)D and DBP. Results Serum total 25(OH)D and DBP concentrations were significantly lower in subjects with the phenotype Gc2/Gc2 compared to phenotypes with the Gc1S allele, and lower in males compared to females. When using directly measured free 25(OH)D, the differences related to DBP phenotypes and sexes were clearly diminished. All calculated free 25(OH)D concentrations were overestimated compared to the directly measured free 25(OH)D. Serum parathyroid hormone showed an inverse correlation with all vitamin D parameters analyzed. The increases after 12 months of vitamin D supplementation were not significantly different for any of the vitamin D parameters regardless of DBP phenotype, sex or age. Supplementation with vitamin D did not affect serum DBP. Conclusion Direct measurements of free 25(OH)D reduce the differences seen in total 25(OH)D between DBP phenotype groups and sexes, probably caused by differences in DBP concentrations. With conditions affecting serum DBP concentrations, direct measurements of free 25(OH)D should be considered. PMID:26733479

  8. Photoaffinity labeling of human serum vitamin D binding protein and chemical cleavage of the labeled protein: Identification of an 11. 5-kDa peptide containing the putative 25-hydroxyvitamin D sub 3 binding site

    SciTech Connect

    Ray, R.; Holick, M.F. ); Bouillon, R.; Baelen, H.V. )

    1991-07-30

    In this paper, the authors describe photoaffinity labeling and related studies of human serum vitamin D binding protein (hDBP) with 25-hydroxyvitamin D{sub 3} 3{beta}-3{prime}-(N-(4-azido-2-nitrophenyl)amino)propyl ether (25-ANE) and its radiolabeled counterpart, i.e., 25-hydroxyvitamin D{sub 3} 3{beta}-3{prime}-(N-(4-azido-2-nitro-(3,5-{sup 3}H)phenyl)amino)propyl ether ({sup 3}H-25-ANE). They have carried out studies to demonstrate that (1) 25-ANE competes with 25-OH-D{sub 3} for the binding site of the latter in hDBP and (2) {sup 3}H-25-ANE is capable of covalently labeling the hDBP molecule when exposed ot UV light. Treatment of a sample of purified hDBP, labeled with {sup 3}H-25-ANE, with BNPS-skatole produced two Coomassie Blue stained peptide fragments, and the majority of the radioactivity was assoicated with the smaller of the two peptide fragments (16.5 kDa). On the other hand, cleavage of the labeled protein with cyanogen bromide produced a peptide (11.5 kDa) containing most of the covalently attached radioactivity. Considering the primary amino acid structure of hDBP, this peptide fragment (11.5 kDa) represents the N-terminus through residue 108 of the intact protein. Thus, the results tentatively identify this segment of the protein containing the binding pocket for 25-OH-D{sub 3}.

  9. Hepatic uptake of (TH)retinol bound to the serum retinol binding protein involves both parenchymal and perisinusoidal stellate cells

    SciTech Connect

    Blomhoff, R.; Norum, K.R.; Berg, T.

    1985-11-05

    We have studied the hepatic uptake of retinol bound to the circulating retinol binding protein-transthyretin complex. Labeled complex was obtained from the plasma of donor rats that were fed radioactive retinol. When labeled retinol-retinol binding protein-transthyretin complex was injected intravenously into control rats, about 45% of the administered dose was recovered in liver after 56 h. Parenchymal liver cells were responsible for an initial rapid uptake. Perisinusoidal stellate cells initially accumulated radioactivity more slowly than did the parenchymal cells, but after 16 h, these cells contained more radioactivity than the parenchymal cells. After 56 h, about 70% of the radioactivity recovered in liver was present in stellate cells. For the first 2 h after injection, most of the radioactivity in parenchymal cells was recovered as unesterified retinol. The radioactivity in the retinyl ester fraction increased after a lag period of about 2 h, and after 5 h more than 60% of the radioactivity was recovered as retinyl esters. In stellate cells, radioactivity was mostly present as retinyl esters at all time points examined. Uptake of retinol in both parenchymal cells and stellate cells was reduced considerably in vitamin A-deficient rats. Less than 5% of the injected dose of radioactivity was found in liver after 5-6 h (as compared to 25% in control rats), and the radioactivity recovered in liver from these animals was mostly in the unesterified retinol fraction. Studies with separated cells in vitro suggested that both parenchymal and stellate cells isolated from control rats were able to take up retinol from the retinol-retinol binding protein-transthyretin complex. This uptake was temperature dependent.

  10. Deglycosylation of serum vitamin D3-binding protein by alpha-N-acetylgalactosaminidase detected in the plasma of patients with systemic lupus erythematosus.

    PubMed

    Yamamoto, N; Naraparaju, V R; Moore, M; Brent, L H

    1997-03-01

    A serum glycoprotein, Gc protein (vitamin D3-binding protein), can be converted by beta-galactosidase of B cells and sialidase of T cells to a potent macrophage-activating factor (MAF), a protein with N-acetylgalactosamine as the remaining sugar moiety. Thus, Gc protein is the precursor for MAF. Treatment of Gc protein with immobilized beta-galactosidase and sialidase generates a remarkably high titered macrophage-activating factor (GcMAF). When peripheral blood monocytes/ macrophages (designated macrophages) of 33 systemic lupus erythematosus patients were incubated with GcMAF (100 pg/ml), the macrophages of all patients were activated as determined by superoxide generation. However, the precursor activity of patient plasma Gc protein was lost or reduced in these patients. Loss of the precursor activity was the result of deglycosylation of plasma Gc protein by alpha-N-acetylgalactosaminidase activity found in the patient plasma. Levels of plasma alpha-N-acetylgalactosaminidase activity in individual patients had an inverse correlation with the MAF precursor activity of their plasma Gc protein. Deglycosylated Gc protein cannot be converted to macro-phage-activating factor. The resulting defect in macro-phage activation may lead to an inability to clear pathogenic immune complexes. Thus, elevated plasma alpha-N-acetylgalactosaminidase activity resulting in the loss of MAF precursor activity and reduced macro-phage activity may play a role in the pathogenesis of systemic lupus erythematosus. PMID:9073553

  11. (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.

  12. 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

  13. Associations of serum carotenoid concentrations and fruit or vegetable consumption with serum insulin-like growth factor (IGF)-1 and IGF binding protein-3 concentrations in the Third National Health and Nutrition Examination Survey (NHANES III).

    PubMed

    Diener, Anja; Rohrmann, Sabine

    2016-01-01

    Dietary intervention may alter the insulin-like growth factor (IGF) system and thereby cancer risk. In a qualitative review, eleven of twenty studies showed a link between one or more carotenoids, vegetable or fruit intake and the IGF system, however, with partly contrary findings, such that no firm conclusion can be drawn. Therefore, we evaluated associations between serum carotenoid concentrations or the intake of fruits and vegetables with IGF-1, IGF binding protein (BP)-3 and their molar ratio (IGF-1:IGFBP-3) within the Third National Health and Nutrition Examination Survey (NHANES III, 1988-1994). In our analysis, we included 6061 NHANES III participants and used multivariable-adjusted linear regression models. IGF-1 concentrations were significantly positively associated with serum concentrations of lycopene, β-carotene, α-carotene, β-cryptoxanthin and lutein/zeaxanthin in men and women. Statistically significant positive associations were observed for serum concentrations of α-carotene and lutein/zeaxanthin and intake of fruits with serum IGFBP-3 concentrations in women, but not in men. The IGF-1:IGFBP-3 molar ratio was significantly positively associated with serum concentrations of lycopene, β-carotene and α-carotene in men and with β-carotene in women. In conclusion, dietary interventions with carotenoids, fruits and vegetables may affect the IGF system, although the direction of these effects is currently unclear. PMID:27313849

  14. Analysis of multi-site drug-protein interactions by high-performance affinity chromatography: Binding by glimepiride to normal or glycated human serum albumin.

    PubMed

    Matsuda, Ryan; Li, Zhao; Zheng, Xiwei; Hage, David S

    2015-08-21

    High-performance affinity chromatography (HPAC) was used in a variety of formats to examine multi-site interactions between glimepiride, a third-generation sulfonylurea drug, and normal or in vitro glycated forms of the transport protein human serum albumin (HSA). Frontal analysis revealed that glimepiride interacts with normal HSA and glycated HSA at a group of high affinity sites (association equilibrium constant, or Ka, 9.2-11.8×10(5)M(-1) at pH 7.4 and 37°C) and a group of lower affinity regions (Ka, 5.9-16×10(3)M(-1)). Zonal elution competition studies were designed and carried out in both normal- and reversed-role formats to investigate the binding by this drug at specific sites. These experiments indicated that glimepiride was interacting at both Sudlow sites I and II. Allosteric effects were also noted with R-warfarin at Sudlow site I and with tamoxifen at the tamoxifen site on HSA. The binding at Sudlow site I had a 2.1- to 2.3-fold increase in affinity in going from normal HSA to the glycated samples of HSA. There was no significant change in the affinity for glimepiride at Sudlow site II in going from normal HSA to a moderately glycated sample of HSA, but a slight decrease in affinity was seen in going to a more highly glycated HSA sample. These results demonstrated how various HPAC-based methods can be used to profile and characterize multi-site binding by a drug such as glimepiride to a protein and its modified forms. The information obtained from this study should be useful in providing a better understanding of how drug-protein binding may be affected by glycation and of how separation and analysis methods based on HPAC can be employed to study systems with complex interactions or that involve modified proteins. PMID:26189669

  15. Analysis of Multi-Site Drug-Protein Interactions by High-Performance Affinity Chromatography: Binding by Glimepiride to Normal or Glycated Human Serum Albumin

    PubMed Central

    Matsuda, Ryan; Li, Zhao; Zheng, Xiwei; Hage, David S.

    2015-01-01

    High-performance affinity chromatography (HPAC) was used in a variety of formats to examine multi-site interactions between glimepiride, a third-generation sulfonylurea drug, and normal or in vitro glycated forms of the transport protein human serum albumin (HSA). Frontal analysis revealed that glimepiride interacts with normal HSA and glycated HSA at a group of high affinity sites (association equilibrium constant, or Ka, 9.2–11.8 × 105 M−1 at pH 7.4 and 37°C) and a group of lower affinity regions (Ka, 5.9–16.2 × 103 M−1). Zonal elution competition studies were designed and carried out in both normal- and reversed-role formats to investigate the binding by this drug at specific sites. These experiments indicated that glimepiride was interacting at both Sudlow sites I and II. Allosteric effects were also noted with R-warfarin at Sudlow site I and with tamoxifen at the tamoxifen site on HSA. The binding at Sudlow site I had a 2.1- to 2.3-fold increase in affinity in going from normal HSA to the glycated samples of HSA. There was no significant change in the affinity for glimepiride at Sudlow site II in going from normal HSA to a moderately glycated sample of HSA, but a slight decrease in affinity was seen in going to a more highly glycated HSA sample. These results demonstrated how various HPAC-based methods can be used to profile and characterize multi-site binding by a drug such as glimepiride to a protein and its modified forms. The information obtained from this study should be useful in providing a better understanding of how drug-protein binding may be affected by glycation and of how separation and analysis methods based on HPAC can be employed to study systems with complex interactions or that involve modified proteins. PMID:26189669

  16. Protein Binding Pocket Dynamics.

    PubMed

    Stank, Antonia; Kokh, Daria B; Fuller, Jonathan C; Wade, Rebecca C

    2016-05-17

    The dynamics of protein binding pockets are crucial for their interaction specificity. Structural flexibility allows proteins to adapt to their individual molecular binding partners and facilitates the binding process. This implies the necessity to consider protein internal motion in determining and predicting binding properties and in designing new binders. Although accounting for protein dynamics presents a challenge for computational approaches, it expands the structural and physicochemical space for compound design and thus offers the prospect of improved binding specificity and selectivity. A cavity on the surface or in the interior of a protein that possesses suitable properties for binding a ligand is usually referred to as a binding pocket. The set of amino acid residues around a binding pocket determines its physicochemical characteristics and, together with its shape and location in a protein, defines its functionality. Residues outside the binding site can also have a long-range effect on the properties of the binding pocket. Cavities with similar functionalities are often conserved across protein families. For example, enzyme active sites are usually concave surfaces that present amino acid residues in a suitable configuration for binding low molecular weight compounds. Macromolecular binding pockets, on the other hand, are located on the protein surface and are often shallower. The mobility of proteins allows the opening, closing, and adaptation of binding pockets to regulate binding processes and specific protein functionalities. For example, channels and tunnels can exist permanently or transiently to transport compounds to and from a binding site. The influence of protein flexibility on binding pockets can vary from small changes to an already existent pocket to the formation of a completely new pocket. Here, we review recent developments in computational methods to detect and define binding pockets and to study pocket dynamics. We introduce five

  17. Retinol binding protein and vitamin D associations with serum antibody isotypes, serum influenza virus-specific neutralizing activities and airway cytokine profiles.

    PubMed

    Jones, B G; Oshansky, C M; Bajracharya, R; Tang, L; Sun, Y; Wong, S S; Webby, R; Thomas, P G; Hurwitz, J L

    2016-02-01

    Vitamin A supports the induction of immunoglobulin (Ig)A responses at mucosal surfaces in mice, but much less is known about the influence of vitamins on antibody isotype expression in humans. To address this knowledge gap, we examined 46 residual blood samples from adults and children, some of whom were experiencing influenza virus infections of the respiratory tract. Assays were performed for retinol binding protein (RBP, a surrogate for vitamin A), vitamin D (a related vitamin) and antibody isotypes. Results showed that all but two tested samples exhibited RBP and/or vitamin D insufficiencies or deficiencies. Vitamin D correlated with blood IgM and IgG3, while RBP correlated with IgG4 and IgA. RBP also correlated positively with age and with influenza virus-specific antibody neutralization titres. Individuals with low blood RBP levels exhibited the highest frequencies of over-expressed cytokines and growth factors in nasal wash samples, an indication of inflamed mucosal tissues. While cause-effect relationships were not discerned, results support a hypothesis that vitamins directly influence B cell isotype expression in humans, and by so doing may help protect mucosal surfaces from respiratory viral disease. PMID:26425827

  18. Growth hormone-binding protein directly depends on serum leptin levels in adults with different nutritional status.

    PubMed

    Llopis, M A; Granada, M L; Cuatrecasas, G; Formiguera, X; Sánchez-Planell, L; Sanmartí, A; Alastrué, A; Rull, M; Corominas, A; Foz, M

    1998-06-01

    The aim of this work was to assess the relationship between GH-binding protein (GHBP) and leptin. Both peptides are nutritionally regulated, but the recent implication of a role for leptin in the GH axis requires further study. To avoid the sexual dimorphism in leptin values, we performed leptin standardization according to gender (SD score-leptin). The relationship between SD score-leptin and GHBP was studied in 128 adults with different nutritional status [8 groups according to body mass index (BMI)], ranging from severely underweight anorexia nervosa to highly morbid obesity. Both GHBP and SD score-leptin significantly increased according to BMI within the range from 18-27 kg/m2, whereas no significant differences were found among underweight groups (BMI, < 18 kg/m2) or among obesity grades (BMI, > 27 kg/m2). We found a strong correlation between GHBP and SD score-leptin (r = 0.8; P < 0.0001). Multiple regression analysis revealed SD score-leptin to be a significant determinant of GHBP, accounting for 64% of the variation, whereas BMI did not contribute further to explaining changes in GHBP. This suggests a physiological pathway involving both GHBP (the soluble fraction of GH receptor) and leptin. Thus, we might speculate that leptin could be the signal that induces the related nutritional changes observed in GHBP/GH receptor expression. PMID:9626132

  19. Serum Level of Heart-Type Fatty Acid Binding Protein (H-FABP) Before and After Treatment of Congestive Heart Failure in Children.

    PubMed

    Zoair, Amr; Mawlana, Wegdan; Abo-Elenin, Amany; Korrat, Mostafa

    2015-12-01

    Remodeling of the heart following injury affects the morbidity and mortality in children presented with heart failure (HF). Heart-type fatty acid binding protein (H-FABP) is a novel biomarker that could be of help to predict the prognosis and risk stratification in those children. We aimed to evaluate the diagnostic and prognostic value of H-FABP in children with heart failure before and after treatment. The study was conducted as a prospective cohort study. It included 30 children with HF as a patient group and 20 healthy children matched for age and sex as a control group. Echocardiographic assessment of the heart was done using conventional Doppler echocardiography. Serum levels of (H-FABP) were measured using enzyme-linked immunosorbent assay before and after treatment of HF. All patients were observed during follow-up period of 3 months. There was a significant difference in the serum level of H-FABP in our patients before treatment (5.278 ± 3.253 ng/ml) compared with after treatment (2.089 ± 0.160 ng/ml) with significant difference compared with the control group. There was a significant increase in the serum level of H-FABP with increase in the severity of heart failure according to Ross classification. Significant increase in the H-FABP was associated with adverse outcome. Serum levels of H-FABP strongly correlated with clinical and echocardiographic assessment of LV performance of children with HF, and its levels significantly increased in children with adverse outcome suggesting its value as a useful diagnostic and prognostic predictor (with high sensitivity and specificity). PMID:26123812

  20. Serum Levels of Vascular Endothelial Growth Factor and Insulin-like Growth Factor Binding Protein-3 in Obstructive Sleep Apnea Patients: Effect of Continuous Positive Airway Pressure Treatment

    PubMed Central

    Archontogeorgis, Kostas; Nena, Evangelia; Papanas, Nikolaos; Xanthoudaki, Maria; Hatzizisi, Olga; Kyriazis, Georgios; Tsara, Venetia; Maltezos, Efstratios; Froudarakis, Marios; Steiropoulos, Paschalis

    2015-01-01

    Background and Aim: Hypoxia, a major feature of obstructive sleep apnea (OSA), modifies Vascular Endothelial Growth Factor (VEGF) and Insulin-like Growth Factor Binding Protein-3 (IGFBP-3) levels, which contribute to atherogenesis and occurrence of cardiovascular (CV) events. We assessed and compared serum levels of VEGF and IGFBP-3 in newly diagnosed OSA patients and controls, to explore associations with anthropometric and sleep parameters and to study the effect of continuous positive airway pressure (CPAP) treatment on these levels. Materials and Methods: Serum levels of VEGF and IGFBP-3 were measured in 65 OSA patients and 31 age- and body mass index- matched controls. In OSA patients, measurements were repeated after 6 months of CPAP therapy. All participants were non-smokers, without any comorbidities or systemic medication use. Results: At baseline, serum VEGF levels in OSA patients were higher compared with controls (p<0.001), while IGFBP-3 levels were lower (1.41±0.56 vs. 1.61±0.38 μg/ml, p=0.039). VEGF levels correlated with apnea-hypopnea index (r=0.336, p=0.001) and oxygen desaturation index (r=0.282, p=0.007). After 6 months on CPAP treatment, VEGF levels decreased in OSA patients (p<0.001), while IGFBP-3 levels increased (p<0.001). Conclusion: In newly diagnosed OSA patients, serum levels of VEGF are elevated, while IGFBP-3 levels are low. After 6 months of CPAP treatment these levels change. These results may reflect an increased CV risk in untreated OSA patients, which is ameliorated after CPAP therapy. PMID:27006717

  1. Evaluation of Fucosylated Haptoglobin and Mac-2 Binding Protein as Serum Biomarkers to Estimate Liver Fibrosis in Patients with Chronic Hepatitis C

    PubMed Central

    Tawara, Seiichi; Tatsumi, Tomohide; Iio, Sadaharu; Kobayashi, Ichizou; Shigekawa, Minoru; Hikita, Hayato; Sakamori, Ryotaro; Hiramatsu, Naoki; Miyoshi, Eiji; Takehara, Tetsuo

    2016-01-01

    Fucosylated haptoglobin (Fuc-Hpt) and Mac-2 binding protein (Mac-2 bp) are identified as cancer biomarkers, based on the results from a glyco-proteomic analysis. Recently, we reported that these glyco-biomarkers were associated with liver fibrosis and/or ballooning hepatocytes in patients with nonalcoholic fatty liver disease (NAFLD). We evaluated the ability of these glycoproteins to estimate liver fibrosis in 317 patients with chronic hepatitis C. We measured the serum Fuc-Hpt and Mac-2 bp levels using a lectin-antibody ELISA and ELISA, respectively. The serum levels of both Fuc-Hpt and Mac-2 bp increased with the progression of liver fibrosis. The multivariate analysis revealed that Mac-2 bp was an independent factor associated with moderate liver fibrosis (F ≥ 2). In contrast, Fuc-Hpt was an independent factor associated with advanced liver fibrosis (F ≥ 3). In terms of evaluating liver fibrosis, the serum levels of these glycomarkers were correlated with well-known liver fibrosis indexes, such as the aspartate aminotransferase to platelet ratio index (APRI) and Fibrosis-4 (FIB4) index. An assay that combined the APRI or FIB4 index and the Fuc-Hpt or Mac-2 bp levels increased the AUC value for diagnosing hepatic fibrosis. Interestingly, the cumulative incidence of hepatocellular carcinoma (HCC) was significantly higher in the patients with elevated serum levels of Fuc-Hpt and Mac-2 bp. In conclusion, both Fuc-Hpt and Mac-2 bp could be useful glyco-biomarkers of liver fibrosis and predictors of HCC in patients with chronic hepatitis C. PMID:27002630

  2. ANALYSIS OF DRUG INTERACTIONS WITH MODIFIED PROTEINS BY HIGH-PERFORMANCE AFFINITY CHROMATOGRAPHY: BINDING OF GLIBENCLAMIDE TO NORMAL AND GLYCATED HUMAN SERUM ALBUMIN

    PubMed Central

    Matsuda, Ryan; Anguizola, Jeanethe; Joseph, K.S.; Hage, David S.

    2012-01-01

    High-performance affinity chromatography (HPAC) was used to examine the changes in binding that occur for the sulfonylurea drug glibenclamide with human serum albumin (HSA) at various stages of glycation for HSA. Frontal analysis on columns containing normal HSA or glycated HSA indicated glibenclamide was interacting through both high affinity sites (association equilibrium constant, Ka, 1.4–1.9 × 106 M−1 at pH 7.4 and 37°C) and lower affinity sites (Ka, 4.4–7.2 × 104 M−1). Competition studies were used to examine the effect of glycation at specific binding sites of HSA. An increase in affinity of 1.7- to 1.9-fold was seen at Sudlow site I with moderate to high levels of glycation. An even larger increase of 4.3- to 6.0-fold in affinity was noted at Sudlow site II for all of the tested samples of glycated HSA. A slight decrease in affinity may have occurred at the digitoxin site, but this change was not significant for any individual glycated HSA sample. These results illustrate how HPAC can be used as tool for examining the interactions of relatively non-polar drugs like glibenclamide with modified proteins and should lead to a more complete understanding of how glycation can alter the binding of drugs in blood. PMID:23092871

  3. Studies of the binding of different iron donors to human serum transferrin and isolation of iron-binding fragments from the N- and C-terminal regions of the protein.

    PubMed Central

    Evans, R W; Williams, J

    1978-01-01

    1. Trypsin digestion of human serum transferrin partially saturated with iron(III)-nitrilotriacetate at pH 5.5 or pH 8.5 produces a carbohydrate-containing iron-binding fragment of mol.wt. 43000. 2. When iron(III) citrate, FeCl3, iron (III) ascorabate and (NH4)2SO4,FeSO4 are used as iron donors to saturate the protein partially, at pH8.5, proteolytic digestion yields a fragment of mol.wt. 36000 that lacks carbohydrate. 3. The two fragments differ in their antigenic structures, amino acid compositions and peptide 'maps'. 4. The fragment with mol.wt. 36000 was assigned to the N-terminal region of the protein and the other to the C-terminal region. 5. The distribution of iron in human serum transferrin partially saturated with various iron donors was examined by electrophoresis in urea/polyacrylamide gels and the two possible monoferric forms were unequivocally identified. 6. The site designated A on human serum transferrin [Harris (1977) Biochemistry 16, 560--564] was assigned to the C-terminal region of the protein and the B site to the N-terminal region. 7. The distribution of iron on transferrin in human plasma was determined. Images Fig. 1. Fig. 3. Fig. 5. Fig. 6. PMID:100104

  4. HIGH-PERFORMANCE AFFINITY CHROMATOGRAPHY AND THE ANALYSIS OF DRUG INTERACTIONS WITH MODIFIED PROTEINS: BINDING OF GLICLAZIDE WITH GLYCATED HUMAN SERUM ALBUMIN

    PubMed Central

    Matsuda, Ryan; Anguizola, Jeanethe; Joseph, K.S.; Hage, David S.

    2011-01-01

    This study used high-performance affinity chromatography (HPAC) to examine the binding of gliclazide (i.e., a sulfonylurea drug used to treat diabetes) with the protein human serum albumin (HSA) at various stages of modification due to glycation. Frontal analysis conducted with small HPAC columns was first used to estimate the number of binding sites and association equilibrium constants (Ka) for gliclazide with normal HSA and glycated HSA. Both normal and glycated HSA interacted with gliclazide according to a two-site model, with a class of high affinity sites (average Ka, 7.1-10 × 104 M−1) and a group of lower affinity sites (average Ka, 5.7-8.9 × 103 M−1) at pH 7.4 and 37°C. Competition experiments indicated that Sudlow sites I and II of HSA were both involved in these interactions, with the Ka values for gliclazide at these sites being 1.9 × 104 M−1 and 6.0 × 104 M−1, respectively, for normal HSA. Two samples of glycated HSA had similar affinities to normal HSA for gliclazide at Sudlow site I, but one sample had a 1.9-fold increase in affinity at this site. All three glycated HSA samples differed from normal HSA in their affinity for gliclazide at Sudlow site II. This work illustrated how HPAC can be used to examine both the overall binding of a drug with normal or modified proteins and the site-specific changes that can occur in these interactions as a result of protein modification. PMID:21922305

  5. A glycoprotein secreted by lung cancer cells is present in human serum as an immunoglobulin-binding protein.

    PubMed

    Nonaka, N; Kobayashi, K; Hirai, H

    1994-01-01

    The 6B3-Ag recognized by a monoclonal antibody 6B3 to human large cell lung carcinoma cell line (HLC-2) is a high-molecular-weight glycoprotein of 1,000,000. Its serum level is increased in various adenocarcinoma patients. When a patient's serum with a high concentration of 6B3.Ag (54 micrograms/ml) or concentrated 6B3.Ag from normal human serum was analyzed by immunoelectrophoresis, 6B3.Ag showed a long bimodal precipitin line extending from the per-beta to beta globulin region. However, the precipitin line of 6B3.Ag in the HLC-2 culture medium was formed only in the pre-beta globulin region. The 6B3.Ag was purified from pooled patients' serum by salting out, precipitation by acidification at pH 4.5 and Sepharose 4B and immunoaffinity chromatographies. Western blotting indicated that the 6B3.Ag from human serum contained IgG and/or IgM. The 6B3.Ag from human serum showed a dose-dependent reaction in a sandwich enzyme-linked immunosorbent assay with anti-6B3.Ag antibody as a solid-phase antibody and anti-human IgG or anti-human IgM antibody labeled with alkaline phosphatase. The 6B3.Ag was concluded to be partly present as a complex with IgG and/or IgM in human serum, and this complex showed a precipitin line in the beta globulin region on immunoelectrophoresis. PMID:7508905

  6. Protein electrophoresis - serum

    MedlinePlus

    ... digestive tract to absorb proteins ( protein-losing enteropathy ) Malnutrition Kidney disorder called nephrotic syndrome Scarring of the ... may indicate: Abnormally low level of LDL cholesterol Malnutrition Increased gamma globulin proteins may indicate: Bone marrow ...

  7. Alternative binding modes identified for growth and differentiation factor-associated serum protein (GASP) family antagonism of myostatin.

    PubMed

    Walker, Ryan G; Angerman, Elizabeth B; Kattamuri, Chandramohan; Lee, Yun-Sil; Lee, Se-Jin; Thompson, Thomas B

    2015-03-20

    Myostatin, a member of the TGF-β family of ligands, is a strong negative regulator of muscle growth. As such, it is a prime therapeutic target for muscle wasting disorders. Similar to other TGF-β family ligands, myostatin is neutralized by binding one of a number of structurally diverse antagonists. Included are the antagonists GASP-1 and GASP-2, which are unique in that they specifically antagonize myostatin. However, little is known from a structural standpoint describing the interactions of GASP antagonists with myostatin. Here, we present the First low resolution solution structure of myostatin-free and myostatin-bound states of GASP-1 and GASP-2. Our studies have revealed GASP-1, which is 100 times more potent than GASP-2, preferentially binds myostatin in an asymmetrical 1:1 complex, whereas GASP-2 binds in a symmetrical 2:1 complex. Additionally, C-terminal truncations of GASP-1 result in less potent myostatin inhibitors that form a 2:1 complex, suggesting that the C-terminal domains of GASP-1 are the primary mediators for asymmetric complex formation. Overall, this study provides a new perspective on TGF-β antagonism, where closely related antagonists can utilize different ligand-binding strategies. PMID:25657005

  8. Alternative Binding Modes Identified for Growth and Differentiation Factor-associated Serum Protein (GASP) Family Antagonism of Myostatin*

    PubMed Central

    Walker, Ryan G.; Angerman, Elizabeth B.; Kattamuri, Chandramohan; Lee, Yun-Sil; Lee, Se-Jin; Thompson, Thomas B.

    2015-01-01

    Myostatin, a member of the TGF-β family of ligands, is a strong negative regulator of muscle growth. As such, it is a prime therapeutic target for muscle wasting disorders. Similar to other TGF-β family ligands, myostatin is neutralized by binding one of a number of structurally diverse antagonists. Included are the antagonists GASP-1 and GASP-2, which are unique in that they specifically antagonize myostatin. However, little is known from a structural standpoint describing the interactions of GASP antagonists with myostatin. Here, we present the First low resolution solution structure of myostatin-free and myostatin-bound states of GASP-1 and GASP-2. Our studies have revealed GASP-1, which is 100 times more potent than GASP-2, preferentially binds myostatin in an asymmetrical 1:1 complex, whereas GASP-2 binds in a symmetrical 2:1 complex. Additionally, C-terminal truncations of GASP-1 result in less potent myostatin inhibitors that form a 2:1 complex, suggesting that the C-terminal domains of GASP-1 are the primary mediators for asymmetric complex formation. Overall, this study provides a new perspective on TGF-β antagonism, where closely related antagonists can utilize different ligand-binding strategies. PMID:25657005

  9. Serum 25-hydroxyvitamin D, vitamin D binding protein, and risk of colorectal cancer in the Prostate, Lung, Colorectal, and Ovarian Cancer Screening Trial

    PubMed Central

    Weinstein, Stephanie J.; Purdue, Mark P.; Smith-Warner, Stephanie A.; Mondul, Alison M.; Black, Amanda; Ahn, Jiyoung; Huang, Wen-Yi; Horst, Ronald L.; Kopp, William; Rager, Helen; Ziegler, Regina G.; Albanes, Demetrius

    2014-01-01

    The potential role of vitamin D in cancer prevention has generated substantial interest, and laboratory experiments indicate several anti-cancer properties for vitamin D compounds. Prospective studies of circulating 25-hydroxyvitamin D [25(OH)D], the accepted biomarker of vitamin D status, suggest an inverse association with colorectal cancer risk, but with some inconsistencies. Furthermore, the direct or indirect impact of the key transport protein, vitamin D binding protein (DBP), has not been examined. We conducted a prospective study of serum 25(OH)D and DBP concentrations and colorectal cancer risk in the Prostate, Lung, Colorectal, and Ovarian Cancer Screening Trial, based on 476 colorectal cancer cases and 476 controls, matched on age, sex, race, and date of serum collection. All subjects underwent sigmoidoscopic screening at baseline and once during follow-up. Conditional logistic regression estimated odds ratios (ORs) and 95% confidence intervals (CIs). Circulating 25(OH)D was inversely associated with colorectal cancer (OR=0.60, 95% CI 0.38-0.94 for highest versus lowest quintile, p-trend 0.01). Adjusting for recognized colorectal cancer risk factors and accounting for seasonal vitamin D variation did not alter the findings. Neither circulating DBP nor the 25(OH)D:DBP molar ratio, a proxy for free circulating 25(OH)D, was associated with risk (OR=0.82, 95% CI 0.54-1.26, and OR=0.79, 95% CI 0.52-1.21, respectively), and DBP did not modify the 25(OH)D association. The current study eliminated confounding by colorectal cancer screening behavior, and supports an association between higher vitamin D status and substantially lower colorectal cancer risk, but does not indicate a direct or modifying role for DBP. PMID:25156182

  10. In Vitro Enhancement of Carvedilol Glucuronidation by Amiodarone-Mediated Altered Protein Binding in Incubation Mixture of Human Liver Microsomes with Bovine Serum Albumin.

    PubMed

    Sekimoto, Makoto; Takamori, Toru; Nakamura, Saki; Taguchi, Masato

    2016-01-01

    Carvedilol is mainly metabolized in the liver to O-glucuronide (O-Glu). We previously found that the glucuronidation activity of racemic carvedilol in pooled human liver microsomes (HLM) was increased, R-selectively, in the presence of amiodarone. The aim of this study was to clarify the mechanisms for the enhancing effect of amiodarone on R- and S-carvedilol glucuronidation. We evaluated O-Glu formation of R- and S-carvedilol enantiomers in a reaction mixture of HLM including 0.2% bovine serum albumin (BSA). In the absence of amiodarone, glucuronidation activity of R- and S-carvedilol for 25 min was 0.026, and 0.51 pmol/min/mg protein, and that was increased by 6.15 and 1.60-fold in the presence of 50 µM amiodarone, respectively. On the other hand, in the absence of BSA, or when BSA was replaced with human serum albumin, no enhancing effect of amiodarone on glucuronidation activity was observed, suggesting that BSA played a role in the mechanisms for the enhancement of glucuronidation activity. Unbound fraction of S-carvedilol in the reaction mixture was greater than that of R-carvedilol in the absence of amiodarone. Also, the addition of amiodarone caused a greater increase of unbound fraction of R-carvedilol than that of S-carvedilol. These results suggest that the altered protein binding by amiodarone is a key mechanism for R-selective stimulation of carvedilol glucuronidation. PMID:27476943

  11. Serum 25-hydroxyvitamin D, vitamin D binding protein and risk of colorectal cancer in the Prostate, Lung, Colorectal and Ovarian Cancer Screening Trial.

    PubMed

    Weinstein, Stephanie J; Purdue, Mark P; Smith-Warner, Stephanie A; Mondul, Alison M; Black, Amanda; Ahn, Jiyoung; Huang, Wen-Yi; Horst, Ronald L; Kopp, William; Rager, Helen; Ziegler, Regina G; Albanes, Demetrius

    2015-03-15

    The potential role of vitamin D in cancer prevention has generated substantial interest, and laboratory experiments indicate several anti-cancer properties for vitamin D compounds. Prospective studies of circulating 25-hydroxyvitamin D [25(OH)D], the accepted biomarker of vitamin D status, suggest an inverse association with colorectal cancer risk, but with some inconsistencies. Furthermore, the direct or indirect impact of the key transport protein, vitamin D binding protein (DBP), has not been examined. We conducted a prospective study of serum 25(OH)D and DBP concentrations and colorectal cancer risk in the Prostate, Lung, Colorectal and Ovarian Cancer Screening Trial, based on 476 colorectal cancer cases and 476 controls, matched on age, sex, race and date of serum collection. All subjects underwent sigmoidoscopic screening at baseline and once during follow-up. Conditional logistic regression estimated odds ratios (ORs) and 95% confidence intervals (CIs). Circulating 25(OH)D was inversely associated with colorectal cancer (OR = 0.60, 95% CI 0.38-0.94 for highest versus lowest quintile, p trend 0.01). Adjusting for recognized colorectal cancer risk factors and accounting for seasonal vitamin D variation did not alter the findings. Neither circulating DBP nor the 25(OH)D:DBP molar ratio, a proxy for free circulating 25(OH)D, was associated with risk (OR = 0.82, 95% CI 0.54-1.26, and OR = 0.79, 95% CI 0.52-1.21, respectively), and DBP did not modify the 25(OH)D association. The current study eliminated confounding by colorectal cancer screening behavior, and supports an association between higher vitamin D status and substantially lower colorectal cancer risk, but does not indicate a direct or modifying role for DBP. PMID:25156182

  12. Vitamin D Binding Protein Genotype Is Associated with Serum 25-Hydroxyvitamin D and PTH Concentrations, as Well as Bone Health in Children and Adolescents in Finland

    PubMed Central

    Viljakainen, Heli T.; Kokkonen, Elina; Jakobsen, Jette; Cashman, Kevin; Mäkitie, Outi; Lamberg-Allardt, Christel

    2014-01-01

    Vitamin D binding protein (DBP)/group-specific component (Gc), correlates positively with serum vitamin D metabolites, and phenotype influences serum 25-hydroxyvitamin D (S-25(OH)D) concentration. The protein isoform has been associated with decreased bone mineral density (BMD) and increased fracture risk. We examined the role of GC genotypes in S-25(OH)D status and BMD in 231 Finnish children and adolescents aged 7−19 yr. BMD was measured with DXA from lumbar spine (LS), total hip, and whole body, and for 175 subjects, radial volumetric BMD was measured with pQCT. Background characteristic and total dietary intakes of vitamin D and calcium were collected. The concentrations of 25(OH)D, parathyroid hormone (PTH), calcium and other markers of calcium homeostasis were determined from blood and urine. Genotyping was based on single-nucleotide polymorphism (rs4588) in the GC gene. The genotype distribution was: GC 1/1 68%, GC 1/2 26% and GC 2/2 6%. A significant difference emerged in 25(OH)D and PTH concentrations between the genotypes, (p = 0.001 and 0.028 respectively, ANCOVA). There was also a linear trend in: Gc 2/2 had the lowest 25(OH)D and PTH concentrations (p = 0.025 and 0.012, respectively). Total hip bone mineral content was associated with GC genotype (BMC) (p = 0.05, ANCOVA) in boys. In regression analysis, after adjusting for relevant covariates, GC genotype was associated with LS BMC and strength and strain index (SSI) Z-score in both genders, and LS BMD in boys. In conclusion, the present study demonstrates the association between GC genotypes and S-25(OH)D and PTH concentrations. The results show the influence of DBP genetic variation on bone mass accrual in adolescence. PMID:24498064

  13. Protein electrophoresis - serum

    MedlinePlus

    Normal value ranges are: Total protein: 6.4 to 8.3 g/dL (grams per deciliter) Albumin: 3.5 to 5.0 g/dL Alpha-1 ... Decreased total protein may indicate: Abnormal loss of protein from the digestive tract or the inability of the digestive tract ...

  14. Human serum amyloid A3 (SAA3) protein, expressed as a fusion protein with SAA2, binds the oxidized low density lipoprotein receptor.

    PubMed

    Tomita, Takeshi; Ieguchi, Katsuaki; Sawamura, Tatsuya; Maru, Yoshiro

    2015-01-01

    Serum amyloid A3 (SAA3) possesses characteristics distinct from the other serum amyloid A isoforms, SAA1, SAA2, and SAA4. High density lipoprotein contains the latter three isoforms, but not SAA3. The expression of mouse SAA3 (mSAA3) is known to be up-regulated extrahepatically in inflammatory responses, and acts as an endogenous ligand for the toll-like receptor 4/MD-2 complex. We previously reported that mSAA3 plays an important role in facilitating tumor metastasis by attracting circulating tumor cells and enhancing hyperpermeability in the lungs. On the other hand, human SAA3 (hSAA3) has long been regarded as a pseudogene, which is in contrast to the abundant expression levels of the other isoforms. Although the nucleotide sequence of hSAA3 is very similar to that of the other SAAs, a single oligonucleotide insertion in exon 2 causes a frame-shift to generate a unique amino acid sequence. In the present study, we identified that hSAA3 was transcribed in the hSAA2-SAA3 fusion transcripts of several human cell lines. In the fusion transcript, hSAA2 exon 3 was connected to hSAA3 exon 1 or hSAA3 exon 2, located approximately 130kb downstream from hSAA2 exon 3 in the genome, which suggested that it is produced by alternative splicing. Furthermore, we succeeded in detecting and isolating hSAA3 protein for the first time by an immunoprecipitation-enzyme linked immune assay system using monoclonal and polyclonal antibodies that recognize the hSAA3 unique amino acid sequence. We also demonstrated that hSAA3 bound oxidized low density lipoprotein receptor (oxLDL receptor, LOX-1) and elevated the phosphorylation of ERK, the intracellular MAP-kinase signaling protein. PMID:25738827

  15. Human Serum Amyloid A3 (SAA3) Protein, Expressed as a Fusion Protein with SAA2, Binds the Oxidized Low Density Lipoprotein Receptor

    PubMed Central

    Tomita, Takeshi; Ieguchi, Katsuaki; Sawamura, Tatsuya; Maru, Yoshiro

    2015-01-01

    Serum amyloid A3 (SAA3) possesses characteristics distinct from the other serum amyloid A isoforms, SAA1, SAA2, and SAA4. High density lipoprotein contains the latter three isoforms, but not SAA3. The expression of mouse SAA3 (mSAA3) is known to be up-regulated extrahepatically in inflammatory responses, and acts as an endogenous ligand for the toll-like receptor 4/MD-2 complex. We previously reported that mSAA3 plays an important role in facilitating tumor metastasis by attracting circulating tumor cells and enhancing hyperpermeability in the lungs. On the other hand, human SAA3 (hSAA3) has long been regarded as a pseudogene, which is in contrast to the abundant expression levels of the other isoforms. Although the nucleotide sequence of hSAA3 is very similar to that of the other SAAs, a single oligonucleotide insertion in exon 2 causes a frame-shift to generate a unique amino acid sequence. In the present study, we identified that hSAA3 was transcribed in the hSAA2-SAA3 fusion transcripts of several human cell lines. In the fusion transcript, hSAA2 exon 3 was connected to hSAA3 exon 1 or hSAA3 exon 2, located approximately 130kb downstream from hSAA2 exon 3 in the genome, which suggested that it is produced by alternative splicing. Furthermore, we succeeded in detecting and isolating hSAA3 protein for the first time by an immunoprecipitation-enzyme linked immune assay system using monoclonal and polyclonal antibodies that recognize the hSAA3 unique amino acid sequence. We also demonstrated that hSAA3 bound oxidized low density lipoprotein receptor (oxLDL receptor, LOX-1) and elevated the phosphorylation of ERK, the intracellular MAP-kinase signaling protein. PMID:25738827

  16. Cellulose binding domain proteins

    DOEpatents

    Shoseyov, O.; Shpiegl, I.; Goldstein, M.; Doi, R.

    1998-11-17

    A cellulose binding domain (CBD) having a high affinity for crystalline cellulose and chitin is disclosed, along with methods for the molecular cloning and recombinant production. Fusion products comprising the CBD and a second protein are likewise described. A wide range of applications are contemplated for both the CBD and the fusion products, including drug delivery, affinity separations, and diagnostic techniques. 16 figs.

  17. Cellulose binding domain proteins

    DOEpatents

    Shoseyov, Oded; Shpiegl, Itai; Goldstein, Marc; Doi, Roy

    1998-01-01

    A cellulose binding domain (CBD) having a high affinity for crystalline cellulose and chitin is disclosed, along with methods for the molecular cloning and recombinant production thereof. Fusion products comprising the CBD and a second protein are likewise described. A wide range of applications are contemplated for both the CBD and the fusion products, including drug delivery, affinity separations, and diagnostic techniques.

  18. Spectroscopic study on binding of rutin to human serum albumin

    NASA Astrophysics Data System (ADS)

    Pastukhov, Alexander V.; Levchenko, Lidiya A.; Sadkov, Anatoli P.

    2007-10-01

    Steady-state and time-resolved fluorescence spectroscopy techniques were used to study the interaction of the flavonoid rutin with human serum albumin (HSA) as well as spectral properties of the protein-bound flavonoid. Both quenching of the intrinsic fluorescence of the protein (Trp214) and the ligand fluorescence, appearing upon complexation with HSA, were used to determine binding parameters. The binding constant determined from the quenching of the Trp214 fluorescence by rutin is equal to 6.87 ± 0.22 × 10 4 M -1 and that obtained from the fluorescence of HSA-bound rutin is 3.8 ± 0.4 × 10 4 M -1. Based on the Job plot analysis, the 1:1 binding stoichiometry for the HSA-rutin complex was determined. The efficient quenching of the Trp214 fluorescence by rutin, fluorescence resonance energy transfer from excited Trp214 to rutin, and competitive binding of warfarin indicate that the binding site for the flavonoid is situated within subdomain IIA of HSA. The presence of the sugar moiety in the flavonoid molecule reduces affinity of rutin for binding to HSA but does not affect the binding stoichiometry and location of the binding site compared with aglycone analogues.

  19. Autoantibodies against protective molecules--C1q, C-reactive protein, serum amyloid P, mannose-binding lectin, and apolipoprotein A1: prevalence in systemic lupus erythematosus.

    PubMed

    Shoenfeld, Yehuda; Szyper-Kravitz, Martine; Witte, Torsten; Doria, Andrea; Tsutsumi, Akito; Tatsuya, Abe; Dayer, Jean-Michel; Roux-Lombard, Pascale; Fontao, Lionel; Kallenberg, Cees G M; Bijl, Marc; Matthias, Torsten; Fraser, Abigail; Zandman-Goddard, Gisele; Blank, Miri; Gilburd, Boris; Meroni, Pier Luigi

    2007-06-01

    Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the production of several autoantibodies. Among the multiple factors involved in SLE development, apoptotic defects and impaired clearance of cellular debris have gained considerable interest, as they contribute to autoantigen overload. Several molecules of the innate immunity, also participate in the removal of damaged and apoptotic cells. Among them are C1q, C-reactive protein (CRP), serum amyloid P protein (SAP), mannose-binding lectin (MBL), and apolipoprotein A1 (APO A1). To evaluate the prevalence of autoantibodies against CRP, SAP, MBL, APO A1, and C1q among SLE patients, and their relationship with disease activity, a total of 150 SLE patients were screened for the presence of elevated antibody titers against C1q, CRP, SAP, MBL, and APO A1, utilizing the enzyme-linked immunosorbent assay (ELISA) method. Disease activity was assessed using the ECLAM or SLEDAI scores. The study population comprised two groups of patients: 100 patients with quiescent disease (median ECLAM score 2) comprised the first group, and 50 patients with active disease (median SLEDAI score 16) comprised group 2. Elevated titers of anti-CRP antibodies were significantly elevated only in group 1 (10% versus 4% of controls). Antibodies against SAP were evaluated only among patients in group 1, and were found at a significant high prevalence (20%). Elevated titers of anti-MBL antibodies were significantly elevated only in group 1 (15% versus 3.6%); and antibodies directed against APO A1 were significantly elevated in 21% of group 1, and 50% of group 2 patients. Elevated titers of anti-C1q were evaluated only in group 2, and were found at a significant prevalence of 66%. Significant correlation with disease activity was found only for anti-APO A1 antibodies, and only in group 1. Several patients harbored more than one of the autoantibodies tested. In patients with SLE, autoantibodies directed against protective

  20. The value of serum Wisteria floribunda agglutinin-positive human Mac-2-binding protein as a predictive marker for hepatitis C virus-related complications after systemic chemotherapy.

    PubMed

    Totani, Haruhito; Kusumoto, Shigeru; Tanaka, Yasuhito; Suzuki, Nana; Hagiwara, Shinya; Kinoshita, Shiori; Iio, Etsuko; Ito, Asahi; Ri, Masaki; Ishida, Takashi; Komatsu, Hirokazu; Iida, Shinsuke

    2016-09-01

    Wisteria floribunda agglutinin-positive human Mac-2-binding protein (WFA(+)-M2BP) was developed recently as a predictive marker of progression to liver fibrosis and hepatocellular carcinoma (HCC) in patients seropositive for hepatitis C virus (HCV). We retrospectively analyzed 16 HCV-seropositive patients who received systemic chemotherapy for hematologic malignancies to evaluate the usefulness of WFA(+)-M2BP for predicting HCV-related complications. These were defined as the onset of significant liver damage (LD) with increased HCV RNA levels, leading to interrupted or discontinued chemotherapy or the occurrence of HCC after chemotherapy. Baseline WFA(+)-M2BP levels were determined using preserved serum samples. The median level of WFA(+)-M2BP was 1.59 [cutoff index (C.O.I.) value range 0.38-6.66]. With a median follow-up of 623 days (range 120-2404), LD and HCC were observed in three and two patients, respectively. Detectable HCV RNA and WFA(+)-M2BP ≥2.0 C.O.I. at baseline were identified as risk factors for these HCV-related complications (P = 0.034 and P = 0.005, respectively). The sensitivity, specificity, and positive and negative predictive values of the WFA(+)-M2BP level (cutoff point: 2.0 C.O.I.) for the occurrence of HCV-related complications were 100.0, 81.8, 71.4, and 100.0 %, respectively. WFA(+)-M2BP may be a useful marker for the prediction of HCV-related complications in HCV-seropositive patients following systemic chemotherapy. PMID:27255233

  1. Serum insulin-like growth factor-I (IGF-I) and IGF-binding protein-3 levels in severe iodine deficiency.

    PubMed

    Alikaşifoğlu, Ayfer; Ozön, Alev; Yordam, Nurşen

    2002-01-01

    Iodine deficiency is an important public health problem worldwide. It is well known that it has severe consequences such as brain damage, developmental delay, deficits in hearing and learning and lower intellectual attainment. It also has a negative impact on growth. In this study, we aimed to address this issue and we assessed height standard deviation scores of children living in an area of severe iodine deficiency in comparison to those living in a mild iodine deficiency area. Serum levels of insulin-like growth factor-I (IGF-I), IGF-binding protein-3 (IGFBP-3), thyroxine (T4), and thyroid stimulating hormone (TSH) were also analyzed to investigate the mechanisms by which iodine depletion leads to growth failure. Pubertal children in a severe iodine deficient SID area had lower height standard deviation scores (HSDS), IGF-I and IGFBP-3 levels than those living in mild iodine deficient MID area. Similar findings could not be elucidated in the prepubertal age group. The major determinants of HSDS were age, IGF-I, IGFBP-3 and TSH. IGF-I and IGFBP-3 were negatively correlated with T4. These findings suggest that iodine deficiency has a negative impact on growth, as well as IGF-I and IGFBP-3 levels. This effect seems to be due to the derangements in thyroid hormone economy arising from iodine depletion. The degree of this impact may be related to the duration of iodine depletion or may be dependent on the developmental stage of the organism at the time of iodine depletion. PMID:12405432

  2. Serum concentrations of insulin-like growth factor-I and insulin-like growth factor binding protein-2 and -3 in eight hoofstock species.

    PubMed

    Govoni, Kristen E; Goodman, Danielle; Maclure, Rebecca M; Penfold, Linda M; Zinn, Steven A

    2011-01-01

    The somatotropic axis, which includes growth hormone, insulin-like growth factor (IGF)-I, and IGF binding proteins (IGFBP), is involved in the regulation of growth and metabolism. Measures of the somatotropic axis can be predictive of nutritional status and growth rate that can be utilized to identify nutritional status of individual animals. Before the somatotropic axis can be a predictive tool, concentrations of hormones of the somatotropic axis need to be established in healthy individuals. To begin to establish these data, we quantified IGF-I, IGFBP-2, and IGFBP-3 in males and females of eight threatened hoofstock species at various ages. Opportunistic blood samples were collected from Bos javanicus (Java banteng), Tragelaphus eurycerus isaaci (bongo), Gazella dama ruficollis (addra gazelle), Taurotragus derbianus gigas (giant eland), Kobus megaceros (Nile lechwe), Hippotragus equines cottoni (roan antelope), Ceratotherium simum simum (white rhinoceros), and Elephas maximus (Asian elephant). Serum IGF-I and IGFBPs were determined by radioimmunoassay and ligand blot, respectively. Generally, IGF-I and IGFBP-3 were greater in males, and IGFBP-2 was greater in females. In banteng (P = 0.08) and male Nile lechwe (P < 0.05), IGF-I increased with age, but decreased in rhinoceros (P = 0.07) and female Nile lechwe (P < 0.05). In banteng, IGFBP-3 was greater (P < 0.01) in males. In elephants (P < 0.05) and antelope (P = 0.08), IGFBP-2 were greater in females. Determination of concentrations of hormones in the somatotropic axis in healthy animals makes it possible to develop models that can identify the nutritional status of these threatened hoofstock species. PMID:20853408

  3. Heparin-binding properties of human serum spreading factor.

    PubMed

    Barnes, D W; Reing, J E; Amos, B

    1985-08-01

    Human serum spreading factor (SF) is a blood glycoprotein that promotes attachment and spreading and influences growth, migration, and differentiation of a variety of animal cells in culture. SF purified from human plasma or serum by chromatographic methods reported previously (Barnes, D. W., and Silnutzer, J. (1983) J. Biol. Chem. 258, 12548-12552) does not bind to heparin-Sepharose under conditions of physiological ionic strength and pH. In a further examination of the heparin-binding properties of human serum SF, we found that exposure of purified SF to 8 M urea altered several properties of the protein, including heparin affinity, and these alterations remained after removal of the urea from SF solutions. Urea-treated SF bound to heparin under physiological conditions, and salt concentrations of 0.4 M or higher were required for elution of urea-treated SF from heparin-Sepharose at pH 7.0. The alteration of heparin-binding properties of SF also was observed upon exposure of the protein to heat or acid. Treatment of SF with urea, heat, or acid resulted additionally in greatly decreased cell spreading-promoting activity of the molecule. The decreased biological activity was associated with a reduced ability of the treated SF to bind to the cell culture substratum, a prerequisite for the attachment-promoting activity of the molecule. Experiments examining the heparin-binding properties of native SF in unfractionated human plasma indicated that the major portion of SF in blood did not bind to heparin under conditions of physiological ionic strength and pH. PMID:2410408

  4. [Spectroscopic studies on the binding of phenazopyridine hydrochloride and bovine serum albumin].

    PubMed

    Zhou, Hong; Chen, Chang-Yun; Xie, An-Jian

    2007-09-01

    The binding of phenazopyridine hydrochloride and bovine serum albumin under physiological conditions was studied by spectroscopic method. The quenching mechanism of the fluorescence of bovine serum albumin by phenazopyridine hydrochloride was studied with fluorescence and absorption spectroscopy. The binding constant Kb and the number of binding sites n were determined at different temperatures according to Scatchard equation, and the main binding force was discussed by thermodynamic equations. The effect of the drug on bovine serum albumin conformation was also studied by using synchronous fluorescence spectroscopy. The quenching mechanism of phenazopyridine hydrochloride to bovine serum albumin is static quenching and non-radiation energy transfer. The binding constants Kb at 15, 25 and 37 degrees C are 2.47 x 10(7), 9.15 x 10(6) and 4.36 x 10(6) mol(-1) with one binding site, respectively. The thermodynamic parameters of the reaction are DeltaH = -71.2 kJ x mol(-1), and DeltaS = 124.8 J x mol(-1) x K(-1). Binding phenazopyridine hydrochloride to bovine serum albumin is a spontaneous inter-molecular interaction in which entropy increases and Gibbs free energy decreases. The binding distance r between phenazopyridine hydrochloride and bovine serum albumin is 1.61 nm according to Forster theory of non-radiation energy transfer. The binding force is electrostatic interaction. Phenazopyridine hydrochloride can be deposited and transported by serum protein in vivo. Phenazopyridine hydrochloride does affect the serum protein conformation. PMID:18051539

  5. Piezoelectric microcantilever serum protein detector

    NASA Astrophysics Data System (ADS)

    Capobianco, Joseph A.

    The development of a serum protein detector will provide opportunities for better screening of at-risk cancer patients, tighter surveillance of disease recurrence and better monitoring of treatment. An integrated system that can process clinical samples for a number of different types of biomarkers would be a useful tool in the early detection of cancer. Also, screening biomarkers such as antibodies in serum would provide clinicians with information regarding the patient's response to treatment. Therefore, the goal of this study is to develop a sensor which can be used for rapid, all-electrical, real-time, label-fee, in-situ, specific quantification of cancer markers, e.g., human epidermal receptor 2 (Her2) or antibodies, in serum. To achieve this end, piezoelectric microcantilever sensors (PEMS) were constructed using an 8 mum thick lead magnesium niobate-lead titanate (PMN-PT) freestanding film as the piezoelectric layer. The desired limit of detection is on the order of pg/mL. In order to achieve this goal the higher frequency lateral extension modes were used. Also, as the driving and sensing of the PEMS is electrical, the PEMS must be insulated in a manner that allows it to function in aqueous solutions. The insulation layer must also be compatible with standardized bioconjugation techniques. Finally, detection of both cancer antigens and antibodies in serum was carried out, and the results were compared to a standard commercialized protocol. PEMS have demonstrated the capability of detecting Her2 at a concentration of 5 pg/mL in diluted human serum (1:40) in less than 1 hour. The approach can be easily translated into the clinical setting because the sensitivity is more than sufficient for monitoring prognosis of breast cancer patients. In addition to Her2 detection, antibodies in serum were assayed in order to demonstrate the feasibility of monitoring the immune response for antibody-dependent cellular cytotoxicity (ADCC) in patients on antibody therapies

  6. 21 CFR 866.5765 - Retinol-binding protein immunological test system.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Retinol-binding protein immunological test system....5765 Retinol-binding protein immunological test system. (a) Identification. A retinol-binding protein... the retinol-binding protein that binds and transports vitamin A in serum and urine. Measurement...

  7. 21 CFR 866.5765 - Retinol-binding protein immunological test system.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Retinol-binding protein immunological test system....5765 Retinol-binding protein immunological test system. (a) Identification. A retinol-binding protein... the retinol-binding protein that binds and transports vitamin A in serum and urine. Measurement...

  8. 21 CFR 866.5765 - Retinol-binding protein immunological test system.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Retinol-binding protein immunological test system....5765 Retinol-binding protein immunological test system. (a) Identification. A retinol-binding protein... the retinol-binding protein that binds and transports vitamin A in serum and urine. Measurement...

  9. 21 CFR 866.5765 - Retinol-binding protein immunological test system.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Retinol-binding protein immunological test system....5765 Retinol-binding protein immunological test system. (a) Identification. A retinol-binding protein... the retinol-binding protein that binds and transports vitamin A in serum and urine. Measurement...

  10. 21 CFR 866.5765 - Retinol-binding protein immunological test system.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Retinol-binding protein immunological test system....5765 Retinol-binding protein immunological test system. (a) Identification. A retinol-binding protein... the retinol-binding protein that binds and transports vitamin A in serum and urine. Measurement...

  11. Effects of glycation on meloxicam binding to human serum albumin

    NASA Astrophysics Data System (ADS)

    Trynda-Lemiesz, Lilianna; Wiglusz, Katarzyna

    2011-05-01

    The current study reports a binding of meloxicam a pharmacologically important new generation, non-steroidal anti-inflammatory drug to glycated form of the human serum albumin (HSA). The interaction of the meloxicam with nonglycated and glycated albumin has been studied at pH 7.4 in 0.05 M sodium phosphate buffer with 0.1 M NaCl, using fluorescence quenching technique and circular dichroism spectroscopy. Results of the present study have shown that the meloxicam could bind both forms of albumin glycated and nonglycated at a site, which was close to the tryptophan residues. Similarly, how for native albumin glycated form has had one high affinity site for the drug with association constants of the order of 10 5 M -1. The glycation process of the HSA significantly has affected the impact of the meloxicam on the binding of other ligands such as warfarin and bilirubin. The affinity of the glycated albumin for bilirubin as for native albumin has been reduced by meloxicam but observed effect was weaker by half (about 20%) compared with nonglycated albumin. In contrast to the native albumin meloxicam binding to glycated form of the protein only slightly affected the binding of warfarin. It seemed possible that the effects on warfarin binding might be entirely attributable to the Lys 199 modification which was in site I.

  12. Changes in circulating immune complexes in tumour patient serum after in vitro or ex vivo affinity chromatography of blood plasma or whole blood over immunoglobulin-binding staphylococcal protein A-Sepharose.

    PubMed

    Håkansson, L; Hed, J; Baldetorp, L; Eneström, S; Jonsson, S; Liedén, G

    1984-01-01

    Circulating immune complexes (CIC) were determined in tumour patient sera using three methods. One is based on PEG-precipitation, one on C1q-reactivity, and one on protein A-reactivity. About 25-30% of the sera were positive in at least one of the tests. Incubation of serum with protein A-Sepharose in vitro removed PEG-precipitable CIC from most sera, whereas C1q-reactive CICs had a much lower affinity to protein A. The protein A-reactive complexes showed considerable variation in their binding to protein A-Sepharose, and in some sera the amount of these CICs was actually increased. Similar changes in protein A-reactive CIC were also found during ex vivo treatment of tumour patients with immune adsorption. It is proposed that the binding of immune complexes to protein A can result in remodelling of protein A itself. Results from ultracentrifugation and fractionated PEG-precipitation support this hypothesis. PMID:6365797

  13. Spectral Changes of Erythrosin B Luminescence Upon Binding to Bovine Serum Albumin

    NASA Astrophysics Data System (ADS)

    Sablin, N. V.; Gerasimova, M. A.; Nemtseva, E. V.

    2016-04-01

    Changes in absorption, fluorescence, phosphorescence, and delayed fluorescence spectra of erythrosin B are studied in the presence of bovine serum albumin at room temperature. Spectral and chronoscopic characteristics of the observed photophysical processes are defined. The binding of erythrosin B with the protein followed by spectral changes is demonstrated. Absorption and fluorescence spectra of the dye in the bound state are described, the binding mechanism is analyzed. The binding parameters of the dye-protein complex are estimated.

  14. Cold Spots in Protein Binding.

    PubMed

    Shirian, Jason; Sharabi, Oz; Shifman, Julia M

    2016-09-01

    Understanding the energetics and architecture of protein-binding interfaces is important for basic research and could potentially facilitate the design of novel binding domains for biotechnological applications. It is well accepted that a few key residues at binding interfaces (binding hot spots) are responsible for contributing most to the free energy of binding. In this opinion article, we introduce a new concept of 'binding cold spots', or interface positions occupied by suboptimal amino acids. Such positions exhibit a potential for affinity enhancement through various mutations. We give several examples of cold spots from different protein-engineering studies and argue that identification of such positions is crucial for studies of protein evolution and protein design. PMID:27477052

  15. Exploring the binding mechanism of ondansetron hydrochloride to serum albumins: Spectroscopic approach

    NASA Astrophysics Data System (ADS)

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

    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.

  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. PMID:22112579

  17. Hafnium binding to rat serum transferrin

    NASA Astrophysics Data System (ADS)

    Then, G.; Zell, I.; Appel, H.; Thies, W.-G.; Duffield, J.; Taylor, D. M.

    1983-12-01

    Using the TDPAC-technique binding parameters for Hf were determined after in vivo uptake of181Hf in rat plasma. As much as 98.5% of the metal ions proved to be bound to protein, essentially to transferrin. The main fraction of the181Hf ions experiences a well defined electric quadrupole perturbation frequency (vQ1=(1516 ± 15)MHz, δ1=(5.3 ± 0.8)%) connected with a marked relaxation damping (λ = (46 ± 8)MHz). The remaining Hf nuclei are subject to a fairly broad distribution of electric field gradients (vQ2=(1014 ± 37)MHz, δ2=(16±3)%). The results are compared with data obtained with in vitro 181Hf-labeled human transferrin.

  18. Serum prostacyclin binding defects in thrombotic thrombocytopenic purpura.

    PubMed Central

    Wu, K K; Hall, E R; Rossi, E C; Papp, A C

    1985-01-01

    To understand the pathophysiologic significance of abnormal serum prostacyclin (PGI2) binding activities in thrombotic thrombocytopenic purpura (TTP), we evaluated the PGI2 binding characteristics in three chronic TTP sera and 19 normal sera. PGI2 binding by serum was rapid and reversible. The binding activity in TTP sera (22.1 +/- SD, 4.4%) was significantly lower than that of normal sera (42.2 +/- 6.2%). Moreover, the antiaggregating activity and 6-keto-prostaglandin F1 alpha (6KPGF1 alpha) content in the gel filtrates representing the binding peak was proportionally lower in a TTP serum than normal serum. Although normal and TTP sera bound [14C]arachidonate with similar activity, and neither bound [3H]6KPGF1 alpha, there was a difference in prostaglandin E1 (PGE1) binding. Binding of [3H]PGE1 was subnormal in two TTP sera (W.J. and T.G.) and normal in the third (H.S.). Normal serum corrected the binding defects of TTP serum. Interestingly, the mixture of two TTP sera (W.J. and H.S.) mutually corrected their PGI2 binding defects. In addition, although in vivo plasma transfusions improved the PGI2 binding activity of W.J. and H.S., there existed a striking difference in the nature of their response. These observations indicate that there is at least two types of PGI2 binding defects in TTP. Our data indicate that TTP is associated with diminished serum binding of PGI2. This defect may reduce the availability of PGI2 to damaged vascular sites and decrease an important modulator of platelet thrombus formation at times of severe vascular insult. Images PMID:3880771

  19. Binding Efficiency of Protein-Protein Complexes

    PubMed Central

    Day, Eric S.; Cote, Shaun M.; Whitty, Adrian

    2012-01-01

    We examine the relationship between binding affinity and interface size for reversible protein-protein interactions (PPI), using cytokines from the tumor necrosis factor (TNF) superfamily and their receptors as a test case. Using surface plasmon resonance, we measured single-site binding affinities for the large receptor TNFR1 binding to its ligands TNFα (KD = 1.4 ± 0.4 nM) and lymphotoxin-α (KD = 50 ± 10 nM), and also for the small receptor Fn14 binding to TWEAK (KD = 70 ± 10 nM). We additionally assembled data for all other TNF/TNFR family complexes for which reliable single site binding affinities have been reported. We used these values to calculate the binding efficiency – defined as binding energy per Å2 of surface area buried at the contact interface – for the nine of these complexes for which co-crystal structures are available, and compared the results to those for a set of 144 protein-protein complexes with published affinity values. The results show that the most efficient PPI complexes generate ~20 cal.mol−1/Å2 of binding energy. A minimum contact area of ~500 Å2 is required for a stable complex, required to generate sufficient interaction energy to pay the entropic cost of co-localizing two proteins from 1 M solution. The most compact and efficient TNF/TNFR complex was BAFF/BR3, which achieved ~80% of the maximum achievable binding efficiency. Other small receptors also gave high binding efficiencies, while the larger receptors generated only 44-49% of this limit despite interacting primarily through just a single small domain. The results provide new insight into how much binding energy can be generated by a PPI interface of a given size, and establish a quantitative method to predict how large a natural or engineered contact interface must be to achieve a given level of binding affinity. PMID:23088250

  20. GC Gene Polymorphism and Unbound Serum Retinol-Binding Protein 4 Are Related to the Risk of Insulin Resistance in Patients With Chronic Hepatitis C: A Prospective Cross-Sectional Study.

    PubMed

    Mateos-Muñoz, Beatriz; García-Martín, Elena; Torrejón, María J; Devesa-Medina, María J; Esguevillas, Gara; Cárdenas, María C; Fernández, Cristina; Carballo, Miguel; Agúndez, José A; Ladero, José M

    2016-03-01

    Insulin resistance (IR) is found in chronic hepatitis C (CHC) more frequently than in other chronic liver diseases.Prospective cross-sectional study to evaluate a wide multitest panel to identify factors related with IR in CHC and their possible interactions.In 76 patients with CHC we performed a series of routine laboratory analysis as well as specifically designed serum biochemical tests [retinol, retinol-binding protein 4 (RBP4), 25-OH vitamin D, Vitamin E, lipopolysaccharide-binding protein (LBP), interleukin-6 (IL-6), and cystatin C]. The single nucleotide polymorphisms rs7041 and rs4588 GC-DBP (group-specific component-Vitamin D-binding protein), rs738409 PNPLA3 (patatin-like phospholipase domain containing 3), and rs12979860 IL28B (interleukin-28 B) genes were determined. Insulin sensitivity was established with the HOMA-IR and IR was diagnosed when HOMA-IR > 3. Fibrosis staging was assessed with liver biopsy or transient elastography.After backward logistic regression analysis, independent variables associated with IR were Gc1s/Gc1s DBP phenotype, that results from the homozygous carriage of the rs7041G/rs4588C haplotype (P = 0.033); low retinol/RBP4 ratio, reflecting a greater rate of unbound RBP4 (P = 0.005); older age (P = 0.01); high serum tryglicerides (P = 0.026); and advanced (F3-F4) fibrosis stage. The AUROC provided by the multivariate model was 0.950 (95% CI = 0.906-0.993).In addition to previously known ones, the Gc1s/Gc1s phenotype variant of DBP and the unbound fraction of plasma RBP4 may be considered as factors related with the incidence, and possibly the risk, of IR in CHC patients. PMID:26962819

  1. Prednisolone protein binding in renal transplant patients.

    PubMed Central

    Reece, P A; Disney, A P; Stafford, I; Shastry, J C

    1985-01-01

    Prednisolone pharmacokinetics and protein binding characteristics were studied in 10 renal transplant patients with various degrees of renal function (serum creatinine: 80-380 mumol/l) who received their usual oral maintenance dose of prednisolone (0.18 +/- 0.04 mg/kg). Plasma was assayed for prednisolone and hydrocortisone by h.p.l.c. and free prednisolone concentrations were determined in each sample by a rapid ultrafiltration technique. Free prednisolone area under curve (AUCu) ranged from 101 to 436 ng ml-1 h and was 6.3 to 15.0% of total prednisolone AUC. The fraction AUCu/AUC was closely related to serum albumin and creatinine concentrations determined at the time of study (multilinear regression correlation coefficient r2 = 0.830, P less than 0.0001); elevated serum creatinine and low albumin concentrations were associated with a higher % free. These results suggest that much of the variability in prednisolone protein binding could be attributed to inter-patient variability in serum albumin and creatinine concentrations. Total prednisolone concentrations would be potentially misleading in any comparisons made between patient groups with different renal function. PMID:3899153

  2. 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. PMID:21215548

  3. Serum Protein Profile Alterations in Hemodialysis Patients

    SciTech Connect

    Murphy, G A; Davies, R W; Choi, M W; Perkins, J; Turteltaub, K W; McCutchen-Maloney, S L; Langlois, R G; Curzi, M P; Trebes, J E; Fitch, J P; Dalmasso, E A; Colston, B W; Ying, Y; Chromy, B A

    2003-11-18

    Background: Serum protein profiling patterns can reflect the pathological state of a patient and therefore may be useful for clinical diagnostics. Here, we present results from a pilot study of proteomic expression patterns in hemodialysis patients designed to evaluate the range of serum proteomic alterations in this population. Methods: Surface-Enhanced Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (SELDI-TOFMS) was used to analyze serum obtained from patients on periodic hemodialysis treatment and healthy controls. Serum samples from patients and controls were first fractionated into six eluants on a strong anion exchange column, followed by application to four array chemistries representing cation exchange, anion exchange, metal affinity and hydrophobic surfaces. A total of 144 SELDI-TOF-MS spectra were obtained from each serum sample. Results: The overall profiles of the patient and control samples were consistent and reproducible. However, 30 well-defined protein differences were observed; 15 proteins were elevated and 15 were decreased in patients compared to controls. Serum from one patient exhibited novel protein peaks suggesting possible additional changes due to a secondary disease process. Conclusion: SELDI-TOF-MS demonstrated dramatic serum protein profile differences between patients and controls. Similarity in protein profiles among dialysis patients suggests that patient physiological responses to end-stage renal disease and/or dialysis therapy have a major effect on serum protein profiles.

  4. When is protein binding important?

    PubMed

    Heuberger, Jules; Schmidt, Stephan; Derendorf, Hartmut

    2013-09-01

    The present paper is an ode to a classic citation by Benet and Hoener (2002. Clin Pharm Ther 71(3):115-121). The now classic paper had a huge impact on drug development and the way the issue of protein binding is perceived and interpreted. Although the authors very clearly pointed out the limitations and underlying assumptions for their delineations, these are too often overlooked and the classic paper's message is misinterpreted by broadening to cases that were not intended. Some members of the scientific community concluded from the paper that protein binding is not important. This was clearly not intended by the authors, as they finished their paper with a paragraph entitled: "When is protein binding important?" Misinterpretation of the underlying assumptions in the classic work can result in major pitfalls in drug development. Therefore, we revisit the topic of protein binding with the intention of clarifying when clinically relevant changes should be considered during drug development. PMID:23650013

  5. Increased serum cortisol binding in chronic active hepatitis

    SciTech Connect

    Orbach, O.; Schussler, G.C.

    1989-01-01

    A high serum cortisol concentration, apparently due to increased cortisol-binding globulin (CBG), was found in a patient (index case) with chronic active hepatitis (CAH). We therefore performed further studies to determine whether increased cortisol binding is generally associated with CAH. Serum samples were obtained from 15 hospitalized patients with long-term liver function test elevations but no evidence of cirrhosis, 15 normal subjects without a history of hepatitis, four healthy pregnant women, and 10 alcoholic patients with stigmata of cirrhosis. Serum cortisol binding was measured by an adaptation of a previously described charcoal uptake method. Thyroxine-binding globulin (TBG) and sex hormone-binding globulin were determined by radioimmunoassays. Charcoal uptake of 125I cortisol from sera of normal subjects and additional patients with CAH revealed that increased serum cortisol binding by a saturable site, presumably CBG, was associated with CAH. Cortisol binding was significantly correlated with immunoassayable TBG, suggesting that in CAH, similar mechanisms may be responsible for increasing the serum concentrations of CBG and TBG.

  6. Relationship of insulin-like growth factor (IGF) binding protein-3 (IGFBP-3) gene polymorphism with the susceptibility to development of prostate cancer and influence on serum levels of IGF-I, and IGFBP-3.

    PubMed

    Safarinejad, Mohammad Reza; Shafiei, Nayyer; Safarinejad, Shiva

    2011-06-01

    The bioavailability of IGF-I is controlled by the binding protein, IGF binding protein-3 (IGFBP-3). In addition, IGFBP-3 is a strong anti-proliferative protein that provokes apoptosis and inhibits cell proliferation in prostate cancer. We conducted this study to investigate the association between IGFBP-3 gene polymorphism and serum levels of IGF-I and IGFBP-3 and the incidence of prostate cancer (PCa) and benign prostatic hyperplasia (BPH). DNA isolation was performed in peripheral blood samples obtained from all participants. Required areas were amplified with polymerase chain reaction restriction fragment length polymorphism (PCR-RLFP) technique by using proper primers belonging to this gene area. We also measured serum IGF-I and IGFBP-3 levels. The IGFBP-3 -202 A/C polymorphism genotype frequencies showed a significant difference between PCa patients and controls (χ(2)=6.27, df=2.0, P=0.026), as well as between BPH patients and controls (χ(2)=11.57, df=4.0, P=0.014). The AA genotype frequency was significantly decreased in PCa and BPH patients compared to control group and the risk of PCa and BPH occurrence of this genotype was decreased accordingly (PCa; OR=0.28, 95% CI=0.17-0.44, P=0.0001; BPH: OR=0.48, 95% CI=0.29-0.77, P=0.001). Age-adjusted mean serum IGFBP-3 concentrations were highest in the individuals with the AA genotype and diminished significantly in a stepwise manner in the presence of 1 or 2 copies of the C allele (4577 ng/ml, 3929 ng/ml and 3349 ng/ml, respectively). Patients with PCa and BPH had lower serum IGF-1 (P=0.001, and P=0.01, respectively) and IGFBP-3 levels (P=0.001, and P=0.01, respectively) compared with controls. The AA genotype at IGFBP-3 gene polymorphism is associated with reduced risks of PCa and BPH. Both IGF-I and IGFBP-3 concentrations, are associated with modified risks of PCa and BPH. PMID:21536469

  7. Binding of dapsone and its analogues to human serum albumin.

    PubMed

    Karp, W B; Subramanyam, S B; Robertson, A F

    1985-06-01

    The binding of dapsone, 4,4'-sulfonylbis(aniline)(1), and its diacetylated derivative, 4,4"'-sulfonylbis(acetanilide)(2), to human serum albumin is reported. To assess the ability of these compounds to displace 4'-[(4-aminophenyl)sulfonyl]acetanilide (3) from albumin, a dialysis rate technique was used. Competition for the bilirubin binding site on albumin was measured with the peroxidase assay. Compounds 1 and 2 strongly displaced both 3 and bilirubin from human serum albumin. The association constants for 1 and 2 with respect to bilirubin binding were 1.29 X 10(3) and 1.15 X 10(4) M-1, respectively. These results suggest that the binding site for 3 and the bilirubin binding site are similar with respect to 1 and 2 and that the binding of dapsone and its derivatives probably does not involve the amino function. PMID:4020658

  8. Specific serum binding of morphine, levorphanol and heroin

    PubMed Central

    Herndon, B. L.; Baeder, D. H.; Ringle, D. A.

    1976-01-01

    Effects of repeated subcutaneous pellet implantation of a series of narcotic drugs on the serum binding of [14C]morphine was studied in rabbits. Three of the compounds, morphine, heroin and levorphanol, elicited production of a morphine-binding globulin in the implanted rabbits. This serum response did not occur with several other compounds tested, including the potent analgesic methadone, and the narcotic antagonist naloxone. The time course of production of this globulin response, as well as the specificity of the binding for the drug that induced the response are both characteristic of an immunological reaction.

  9. Multiplexed microfluidic quantification of proteins in serum

    NASA Astrophysics Data System (ADS)

    Rajan, Nitin; Rajauria, Sukumar; Cleland, Andrew

    2015-03-01

    Rapid and low cost immunoassays targeting proteins in blood or other bodily fluids are highly sought after for point-of-care devices and early screening of patients. Immunoturbidimetric assays utilize latex particles functionalized with antibodies, with particle aggregation in the presence of the analyte detected by a change in absorbance. Using a high throughput micro-fluidic particle analyzer based solely on electrical signals (resistive pulse sensing), we are able to accurately quantify the degree of aggregation by analyzing the changes in the particle size distribution. Thus we study the aggregation of streptavidin (SAv) coated beads in the presence of biotinylated bovine serum albumin as a proof-of-principle assay and extract the binding capacity of the SAv beads from the dose-response curve. We also use our aggregation measurement platform to characterize a commercial C-reactive protein (CRP) immunoturbidimetric assay (hsCRP, Diazyme Inc.). We obtain a linear calibration curve as well as a better limit of detection of CRP than that obtained by absorbance measurements. By using different bead sizes functionalized with different antibodies, multiplexed analyte detection is also possible. We demonstrate this by combining the commercial anti-CRP functionalized beads (0.4 microns) with biotin coated beads (1.0 microns), and carry out the simultaneous detection of SAv and CRP in a single sample.

  10. (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.

  11. 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. PMID:26212957

  12. Characterizing Active Pharmaceutical Ingredient Binding to Human Serum Albumin by Spin-Labeling and EPR Spectroscopy.

    PubMed

    Hauenschild, Till; Reichenwallner, Jörg; Enkelmann, Volker; Hinderberger, Dariush

    2016-08-26

    Drug binding to human serum albumin (HSA) has been characterized by a spin-labeling and continuous-wave (CW) EPR spectroscopic approach. Specifically, the contribution of functional groups (FGs) in a compound on its albumin-binding capabilities is quantitatively described. Molecules from different drug classes are labeled with EPR-active nitroxide radicals (spin-labeled pharmaceuticals (SLPs)) and in a screening approach CW-EPR spectroscopy is used to investigate HSA binding under physiological conditions and at varying ratios of SLP to protein. Spectral simulations of the CW-EPR spectra allow extraction of association constants (KA ) and the maximum number (n) of binding sites per protein. By comparison of data from 23 SLPs, the mechanisms of drug-protein association and the impact of chemical modifications at individual positions on drug uptake can be rationalized. Furthermore, new drug modifications with predictable protein binding tendency may be envisaged. PMID:27460503

  13. Species Dependence of [64Cu]Cu-Bis(thiosemicarbazone) Radiopharmaceutical Binding to Serum Albumins

    PubMed Central

    Basken, Nathan E.; Mathias, Carla J.; Lipka, Alexander E.; Green, Mark A.

    2008-01-01

    Introduction Interactions of three copper(II) bis(thiosemicarbazone) PET radiopharmaceuticals with human serum albumin, and the serum albumins of four additional mammalian species, were evaluated. Methods 64Cu-labeled diacetyl bis(N4-methylthiosemicarbazonato)copper(II) (Cu-ATSM), pyruvaldehyde bis(N4-methylthiosemicarbazonato)copper(II) (Cu-PTSM), and ethylglyoxal bis(thiosemicarbazonato)copper(II) (Cu-ETS) were synthesized and their binding to human, canine, rat, baboon, and porcine serum albumins quantified by ultrafiltration. Protein binding was also measured for each tracer in human, porcine, rat, and mouse serum. Results The interaction of these neutral, lipophilic copper chelates with serum albumin is highly compound- and species-dependent. Cu-PTSM and Cu-ATSM exhibit particularly high affinity for human serum albumin (HSA), while the albumin binding of Cu-ETS is relatively insensitive to species. At HSA concentrations of 40 mg/mL, “% Free” (non-albumin-bound) levels of radiopharmaceutical were 4.0 ± 0.1%; 5.3 ± 0.2%; and 38.6 ± 0.8% for Cu-PTSM; Cu-ATSM; and Cu-ETS, respectively. Conclusions Species-dependent variations in radiopharmaceutical binding to serum albumin may need to be considered when using animal models to predict the distribution and kinetics of these compounds in humans. PMID:18355683

  14. Binding of endotoxin to macrophages: distinct effects of serum constituents.

    PubMed

    Tahri-Jouti, M A; Chaby, R

    1991-07-01

    The respective roles of serum lipoproteins, and of the complement component C3, in the binding of endotoxin (LPS) to macrophages were analyzed by an in vitro assay using [3H]LPS. The addition of an anti-C3 serum in the medium induced an apparent abolishment of the specific binding of LPS to mouse macrophages, but this effect appeared to be due to an actual increase of nonspecific binding. Isolated complexes of LPS with lipoproteins of high density (HDL3) and of very high density (VHDL) did not bind to macrophages. Furthermore, addition of HDL3 and VHDL in the incubation medium inhibited the specific binding of LPS to macrophages. These results suggest that C3 reduces nonspecific interactions between LPS and macrophages whereas associations between LPS and HDL3 or VHDL inhibit specific LPS-macrophage interactions. PMID:1937584

  15. 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

  16. Identification of differentially expressed serum proteins in gastric adenocarcinoma☆

    PubMed Central

    Subbannayya, Yashwanth; Mir, Sartaj Ahmad; Renuse, Santosh; Manda, Srikanth S.; Pinto, Sneha M.; Puttamallesh, Vinuth N.; Solanki, Hitendra Singh; Manju, H.C.; Syed, Nazia; Sharma, Rakesh; Christopher, Rita; Vijayakumar, M.; Kumar, K.V. Veerendra; Prasad, T.S. Keshava; Ramaswamy, Girija; Kumar, Rekha V.; Chatterjee, Aditi; Pandey, Akhilesh; Gowda, Harsha

    2015-01-01

    Gastric adenocarcinoma is an aggressive cancer with poor prognosis. Blood based biomarkers of gastric cancer have the potential to improve diagnosis and monitoring of these tumors. Proteins that show altered levels in the circulation of gastric cancer patients could prove useful as putative biomarkers. We used an iTRAQ-based quantitative proteomic approach to identify proteins that show altered levels in the sera of patients with gastric cancer. Our study resulted in identification of 643 proteins, of which 48 proteins showed increased levels and 11 proteins showed decreased levels in serum from gastric cancer patients compared to age and sex matched healthy controls. Proteins that showed increased expression in gastric cancer included inter-alpha-trypsin inhibitor heavy chain H4 (ITIH4), Mannose-binding protein C (MBL2), sex hormone-binding globulin (SHBG), insulin-like growth factor-binding protein 2 (IGFBP2), serum amyloid A protein (SAA1), Orosomucoid 1 (ORM1) and extracellular superoxide dismutase [Cu–Zn] (SOD3). We used multiple reaction monitoring assays and validated elevated levels of ITIH4 and SAA1 proteins in serum from gastric cancer patients. Biological significance Gastric cancer is a highly aggressive cancer associated with high mortality. Serum-based biomarkers are of considerable interest in diagnosis and monitoring of various diseases including cancers. Gastric cancer is often diagnosed at advanced stages resulting in poor prognosis and high mortality. Pathological diagnosis using biopsy specimens remains the gold standard for diagnosis of gastric cancer. Serum-based biomarkers are of considerable importance as they are minimally invasive. In this study, we carried out quantitative proteomic profiling of serum from gastric cancer patients to identify proteins that show altered levels in gastric cancer patients. We identified more than 50 proteins that showed altered levels in gastric cancer patient sera. Validation in a large cohort of well

  17. The prion protein binds thiamine.

    PubMed

    Perez-Pineiro, Rolando; Bjorndahl, Trent C; Berjanskii, Mark V; Hau, David; Li, Li; Huang, Alan; Lee, Rose; Gibbs, Ebrima; Ladner, Carol; Dong, Ying Wei; Abera, Ashenafi; Cashman, Neil R; Wishart, David S

    2011-11-01

    Although highly conserved throughout evolution, the exact biological function of the prion protein is still unclear. In an effort to identify the potential biological functions of the prion protein we conducted a small-molecule screening assay using the Syrian hamster prion protein [shPrP(90-232)]. The screen was performed using a library of 149 water-soluble metabolites that are known to pass through the blood-brain barrier. Using a combination of 1D NMR, fluorescence quenching and surface plasmon resonance we identified thiamine (vitamin B1) as a specific prion ligand with a binding constant of ~60 μM. Subsequent studies showed that this interaction is evolutionarily conserved, with similar binding constants being seen for mouse, hamster and human prions. Various protein construct lengths, both with and without the unstructured N-terminal region in the presence and absence of copper, were examined. This indicates that the N-terminus has no influence on the protein's ability to interact with thiamine. In addition to thiamine, the more biologically abundant forms of vitamin B1 (thiamine monophosphate and thiamine diphosphate) were also found to bind the prion protein with similar affinity. Heteronuclear NMR experiments were used to determine thiamine's interaction site, which is located between helix 1 and the preceding loop. These data, in conjunction with computer-aided docking and molecular dynamics, were used to model the thiamine-binding pharmacophore and a comparison with other thiamine binding proteins was performed to reveal the common features of interaction. PMID:21848803

  18. Plasma protein binding of nitroxynil in several species.

    PubMed

    Alvinerie, M; Floc'h, R; Galtier, P

    1991-06-01

    The binding of nitroxynil to total plasma proteins of cows, sheep and rabbits was characterized using equilibrium dialysis. The data indicate clearly that nitroxynil was highly (97-98%) bound to plasma protein of each animal. This linear binding would be due to the particular power exerted by serum albumin. The results are in good agreement with known pharmacokinetic properties of nitroxynil in domestic species. PMID:1920604

  19. Proteomic evaluation of sheep serum proteins

    PubMed Central

    2012-01-01

    Background The applications of proteomic strategies to ovine medicine remain limited. The definition of serum proteome may be a good tool to identify useful protein biomarkers for recognising sub-clinical conditions and overt disease in sheep. Findings from bovine species are often directly translated for use in ovine medicine. In order to characterize normal protein patterns and improve knowledge of molecular species-specific characteristics, we generated a two-dimensional reference map of sheep serum. The possible application of this approach was tested by analysing serum protein patterns in ewes with mild broncho-pulmonary disease, which is very common in sheep and in the peripartum period which is a stressful time, with a high incidence of infectious and parasitic diseases. Results This study generated the first reference 2-DE maps of sheep serum. Overall, 250 protein spots were analyzed, and 138 identified. Compared with healthy sheep, serum protein profiles of animals with rhino-tracheo-bronchitis showed a significant decrease in protein spots identified as transthyretin, apolipoprotein A1 and a significant increase in spots identified as haptoglobin, endopin 1b and alpha1B glycoprotein. In the peripartum period, haptoglobin, alpha-1-acid glycoprotein, apolipoprotein A1 levels rose, while transthyretin content dropped. Conclusions This study describes applications of proteomics in putative biomarker discovery for early diagnosis as well as for monitoring the physiological and metabolic situations critical for ovine welfare. PMID:22630135

  20. Cellulose binding domain fusion proteins

    DOEpatents

    Shoseyov, O.; Yosef, K.; Shpiegl, I.; Goldstein, M.A.; Doi, R.H.

    1998-02-17

    A cellulose binding domain (CBD) having a high affinity for crystalline cellulose and chitin is disclosed, along with methods for the molecular cloning and recombinant production. Fusion products comprising the CBD and a second protein are likewise described. A wide range of applications are contemplated for both the CBD and the fusion products, including drug delivery, affinity separations, and diagnostic techniques. 16 figs.

  1. Cellulose binding domain fusion proteins

    DOEpatents

    Shoseyov, Oded; Shpiegl, Itai; Goldstein, Marc A.; Doi, Roy H.

    1998-01-01

    A cellulose binding domain (CBD) having a high affinity for crystalline cellulose and chitin is disclosed, along with methods for the molecular cloning and recombinant production thereof. Fusion products comprising the CBD and a second protein are likewise described. A wide range of applications are contemplated for both the CBD and the fusion products, including drug delivery, affinity separations, and diagnostic techniques.

  2. Calorimetric investigation of diclofenac drug binding to a panel of moderately glycated serum albumins.

    PubMed

    Indurthi, Venkata S K; Leclerc, Estelle; Vetter, Stefan W

    2014-08-01

    Glycation alters the drug binding properties of serum proteins and could affect free drug concentrations in diabetic patients with elevated glycation levels. We investigated the effect of bovine serum albumin glycation by eight physiologically relevant glycation reagents (glucose, ribose, carboxymethyllysine, acetoin, methylglyoxal, glyceraldehyde, diacetyl and glycolaldehyde) on diclofenac drug binding. We used this non-steroidal anti-inflammatory drug diclofenac as a paradigm for acidic drugs with high serum binding and because of its potential cardiovascular risks in diabetic patients. Isothermal titration calorimetry showed that glycation reduced the binding affinity Ka of serum albumin and diclofenac 2 to 6-fold by reducing structural rigidity of albumin. Glycation affected the number of drug binding sites in a glycation reagent dependent manner and lead to a 25% decrease for most reagent, expect for ribose, with decreased by 60% and for the CML-modification, increased the number of binding sites by 60%. Using isothermal titration calorimetry and differential scanning calorimetry we derived the complete thermodynamic characterization of diclofenac binding to all glycated BSA samples. Our results suggest that glycation in diabetic patients could significantly alter the pharmacokinetics of the widely used over-the-counter NSDAI drug diclofenac and with possibly negative implications for patients. PMID:24751671

  3. Clinical relevance of drug binding to plasma proteins

    NASA Astrophysics Data System (ADS)

    Ascenzi, Paolo; Fanali, Gabriella; Fasano, Mauro; Pallottini, Valentina; Trezza, Viviana

    2014-12-01

    Binding to plasma proteins highly influences drug efficacy, distribution, and disposition. Serum albumin, the most abundant protein in plasma, is a monomeric multi-domain macromolecule that displays an extraordinary ligand binding capacity, providing a depot and carrier for many endogenous and exogenous compounds, such as fatty acids and most acidic drugs. α-1-Acid glycoprotein, the second main plasma protein, is a glycoprotein physiologically involved in the acute phase reaction and is the main carrier for basic and neutral drugs. High- and low-density lipoproteins play a limited role in drug binding and are natural drug delivery system only for few lipophilic drugs or lipid-based formulations. Several factors influence drug binding to plasma proteins, such as pathological conditions, concurrent administration of drugs, sex, and age. Any of these factors, in turn, influences drug efficacy and toxicity. Here, biochemical, biomedical, and biotechnological aspects of drug binding to plasma proteins are reviewed.

  4. 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.

  5. Serum corticosteroid binding globulin expression is modulated by fasting in polar bears (Ursus maritimus).

    PubMed

    Chow, Brian A; Hamilton, Jason; Cattet, Marc R L; Stenhouse, Gordon; Obbard, Martyn E; Vijayan, Mathilakath M

    2011-01-01

    Polar bears (Ursus maritimus) from several subpopulations undergo extended fasting during the ice-free season. However, the animals appear to conserve protein despite the prolonged fasting, though the mechanisms involved are poorly understood. We hypothesized that elevated concentrations of corticosteroid binding globulin (CBG), the primary cortisol binding protein in circulation, lead to cortisol resistance and provide a mechanism for protein conservation during extended fasting. The metabolic state (feeding vs. fasting) of 16 field sampled male polar bears was determined based on their serum urea to creatinine ratio (>25 for feeding vs. <5 for fasting). There were no significant differences in serum cortisol levels between all male and female polar bears sampled. Serum CBG expression was greater in lactating females relative to non-lactating females and males. CBG expression was significantly higher in fasting males when compared to non-fasting males. This leads us to suggest that CBG expression may serve as a mechanism to conserve protein during extended fasting in polar bears by reducing systemic free cortisol concentrations. This was further supported by a lower serum glucose concentration in the fasting bears. As well, a lack of an enhanced adrenocortical response to acute capture stress supports our hypothesis that chronic hunger is not a stressor in this species. Overall, our results suggest that elevated serum CBG expression may be an important adaptation to spare proteins by limiting cortisol bioavailability during extended fasting in polar bears. PMID:20883811

  6. Search for Amyloid-Binding Proteins by Affinity Chromatography

    PubMed Central

    Calero, Miguel; Rostagno, Agueda; Ghiso, Jorge

    2013-01-01

    ‘Amyloid binging proteins’ is a generic term used to designate proteins that interact with different forms of amyloidogenic peptides or proteins and that, as a result, may modulate their physiological and pathological functions by altering solubility, transport, clearance, degradation, and fibril formation. We describe a simple affinity chromatography protocol to isolate and characterize amyloid-binding proteins based on the use of sequential elution steps that may provide further information on the type of binding interaction. As an example, we depict the application of this protocol to the study of Alzheimer’s amyloid β (Aβ) peptide-binding proteins derived from human plasma. Biochemical analysis of the proteins eluted under different conditions identified serum amyloid P component (SAP) and apolipoprotein J (clusterin) as the main plasma Aβ-binding proteins while various apolipoproteins (apoA-IV, apoE, and apoA-I), as well as albumin (HSA) and fibulin were identified as minor contributors. PMID:22528093

  7. 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.

  8. Interactions of apomorphine with serum and tissue proteins

    SciTech Connect

    Smith, R.V.; Velagapudi, R.B.; McLean, A.M.; Wilcox, R.E.

    1985-05-01

    Physical and covalent interactions of apomorphine with serum and tissue proteins could influence the drug's disposition and pharmacological activities in mammals. Ultrafiltration, equilibrium dialysis, and ultraviolet spectrophotometric methods have been used to study the reversible binding of apomorphine to bovine, human, rat, and swine plasma proteins. The degree of binding was generally greater than 90%, but variations were noted in some instances on the basis of drug concentrations and pH over the range of 6.8-7.8. Incubation of (8,9-/sup 3/H2)apomorphine with bovine serum albumin led to retention of radioactivity and a stoichiometrically controlled released of tritium which arose from the reaction of an electrophilic drug oxidation product and protein, producing drug-protein conjugates. In vitro experiments with mouse striatal brain preparations indicated parallel covalent binding reactions. In vivo experiments in mice indicated accumulation of radioactivity in brain regions and other tissues following daily injections of (8,9-/sup 3/H2)apomorphine for 14 days. The physical and covalent interactions of apomorphine with mammalian tissue proteins could be the cause of longer disposition half-lives in mammals than those previously reported. The covalent interactions, in particular, may be important in elucidating the mechanism of apomorphine-induced behavioral effects in mice.

  9. Binding of temoporfin to the lipoprotein fractions of human serum.

    PubMed Central

    Michael-Titus, A T; Whelpton, R; Yaqub, Z

    1995-01-01

    The binding of a new photosensitizer, temoporfin, to human serum lipoproteins was investigated. [14C]-Temoporfin (0.1-10 micrograms ml-1) was incubated with human serum for 30 min at room temperature or for 20 h at 4 degrees C, prior to stepwise density flotation to separate the lipoprotein fractions. The distribution of the drug was independent of the initial concentration or time and temperature of the incubation. The proportion of temoporfin in each fraction was: very low density lipoprotein 6%, low density lipoprotein 22%, lipoprotein(a) 17%, high density lipoprotein 39% and lipoprotein deficient serum 16%. Autoradiography of agarose gels showed that the drug was associated with the lipoprotein in the fractions. Fractionation of plasma samples collected from a patient after an intravenous infusion of temoporfin revealed a binding profile similar to that obtained in the in vitro study. Images Figure 1 PMID:8703668

  10. The Binding Constant of Estradiol to Bovine Serum Albumin: An Upper-Level Experiment Utilizing Tritium-Labeled Estradiol and Liquid Scintillation Counting

    ERIC Educational Resources Information Center

    Peihong Liang; Adhyaru, Bhavin; Pearson, Wright L.; Williams, Kathryn R.

    2006-01-01

    The experiment used [to the third power]H-labeled estradiol to determine the binding constant of estradiol to bovine serum albumin. Estradiol must complex with serum proteins for the transport in the blood stream because of its low solubility in aqueous systems and estradiol-protein binding constant, where K[subscript B] is important to understand…

  11. Detergent activation of the binding protein in the folate radioassay

    SciTech Connect

    Hansen, S.I.; Holm, J.; Lyngbye, J.

    1982-01-01

    A minor cow's whey protein associated with ..beta..-lactoglobulin is used as binding protein in the competitive radioassay for serum and erythrocyte folate. Seeking to optimize the assay, we tested the performance of binder solutions of increasing purity. The folate binding protein was isolated from cow's whey by means of CM-Sepharose CL-6B cation-exchange chromatography, and further purified on a methotrexate-AH-Sepharose 4B affinity matrix. In contrast to ..beta..-lactoglobulin, the purified protein did not bind folate unless the detergents cetyltrimethylammonium (10 mmol/Ll) or Triton X-100 (1 g/L) were present. Such detergent activation was not needed in the presence of serum. There seems to be a striking analogy between these phenomena and the well-known reactivation of certain purified membrane-derived enzymes by surfactants (lipids/detergents).

  12. Protein binding assay for hyaluronate

    SciTech Connect

    Lacy, B.E.; Underhill, C.B.

    1986-11-01

    A relatively quick and simple assay for hyaluronate was developed using the specific binding protein, hyaluronectin. The hyaluronectin was obtained by homogenizing the brains of Sprague-Dawley rats, and then centrifuging the homogenate. The resulting supernatant was used as a source of crude hyaluronectin. In the binding assay, the hyaluronectin was mixed with (/sup 3/H)hyaluronate, followed by an equal volume of saturated (NH/sub 4/)/sub 2/SO/sub 4/, which precipitated the hyaluronectin and any (/sup 3/H)hyaluronate associated with it, but left free (/sup 3/H)hyaluronate in solution. The mixture was then centrifuged, and the amount of bound (/sup 3/H)hyaluronate in the precipitate was determined. Using this assay, the authors found that hyaluronectin specifically bound hyaluronate, since other glycosaminoglycans failed to compete for the binding protein. In addition, the interaction between hyaluronectin and hyaluronate was of relatively high affinity, and the size of the hyaluronate did not appear to substantially alter the amount of binding. To determine the amount of hyaluronate in an unknown sample, they used a competition assay in which the binding of a set amount of (/sup 3/H)hyaluronate was blocked by the addition of unlabeled hyaluronate. By comparing the degree of competition of the unknown samples with that of known amounts of hyaluronate, it was possible to determine the amount of hyaluronate in the unknowns. They have found that this method is sensitive to 1 ..mu..g or less of hyaluronate, and is unaffected by the presence of proteins.

  13. Induced Long-Range Attractive Potentials of Human Serum Albumin by Ligand Binding

    SciTech Connect

    Sato, Takaaki; Komatsu, Teruyuki; Nakagawa, Akito; Tsuchida, Eishun

    2007-05-18

    Small-angle x-ray scattering and dielectric spectroscopy investigation on the solutions of recombinant human serum albumin and its heme hybrid revealed that heme incorporation induces a specific long-range attractive potential between protein molecules. This is evidenced by the enhanced forward intensity upon heme binding, despite no hindrance to rotatory Brownian motion, unbiased colloid osmotic pressure, and discontiguous nearest-neighbor distance, confirming monodispersity of the proteins. The heme-induced potential may play a trigger role in recognition of the ligand-filled human serum albumins in the circulatory system.

  14. Determination of the binding properties of the uremic toxin phenylacetic acid to human serum albumin.

    PubMed

    Saldanha, Juliana F; Yi, Dan; Stockler-Pinto, Milena B; Soula, Hédi A; Chambert, Stéphane; Fouque, Denis; Mafra, Denise; Soulage, Christophe O

    2016-06-01

    Uremic toxins are compounds normally excreted in urine that accumulate in patients with chronic kidney disease as a result of decreased renal clearance. Phenylacetic acid (PAA) has been identified as a new protein bound uremic toxin. The purpose of this study was to investigate in vitro the interaction between PAA and human serum albumin (HSA) at physiological and pathological concentrations. We used ultrafiltration to show that there is a single high-affinity binding site for PAA on HSA, with a binding constant on the order of 3.4 × 10(4) M(-1) and a maximal stoichiometry of 1.61 mol per mole. The PAA, at the concentration reported in end-stage renal patients, was 26% bound to albumin. Fluorescent probe competition experiments demonstrated that PAA did not bind to Sudlow's site I (in subdomain IIA) and only weakly bind to Sudlow's site II (in subdomain IIIA). The PAA showed no competition with other protein-bound uremic toxins such as p-cresyl-sulfate or indoxyl sulfate for binding to serum albumin. Our results provide evidence that human serum albumin can act as carrier protein for phenylacetic acid. PMID:26945842

  15. Evidence that Chemical Chaperone 4-Phenylbutyric Acid Binds to Human Serum Albumin at Fatty Acid Binding Sites

    PubMed Central

    James, Joel; Shihabudeen, Mohamed Sham; Kulshrestha, Shweta; Goel, Varun; Thirumurugan, Kavitha

    2015-01-01

    Endoplasmic reticulum stress elicits unfolded protein response to counteract the accumulating unfolded protein load inside a cell. The chemical chaperone, 4-Phenylbutyric acid (4-PBA) is a FDA approved drug that alleviates endoplasmic reticulum stress by assisting protein folding. It is found efficacious to augment pathological conditions like type 2 diabetes, obesity and neurodegeneration. This study explores the binding nature of 4-PBA with human serum albumin (HSA) through spectroscopic and molecular dynamics approaches, and the results show that 4-PBA has high binding specificity to Sudlow Site II (Fatty acid binding site 3, subdomain IIIA). Ligand displacement studies, RMSD stabilization profiles and MM-PBSA binding free energy calculation confirm the same. The binding constant as calculated from fluorescence spectroscopic studies was found to be kPBA = 2.69 x 105 M-1. Like long chain fatty acids, 4-PBA induces conformational changes on HSA as shown by circular dichroism, and it elicits stable binding at Sudlow Site II (fatty acid binding site 3) by forming strong hydrogen bonding and a salt bridge between domain II and III of HSA. This minimizes the fluctuation of HSA backbone as shown by limited conformational space occupancy in the principal component analysis. The overall hydrophobicity of W214 pocket (located at subdomain IIA), increases upon occupancy of 4-PBA at any FA site. Descriptors of this pocket formed by residues from other subdomains largely play a role in compensating the dynamic movement of W214. PMID:26181488

  16. Synthesis of imidazole derivatives and the spectral characterization of the binding properties towards human serum albumin

    NASA Astrophysics Data System (ADS)

    Yue, Yuanyuan; Dong, Qiao; Zhang, Yajie; Li, Xiaoge; Yan, Xuyang; Sun, Yahui; Liu, Jianming

    2016-01-01

    Small molecular drugs that can combine with target proteins specifically, and then block relative signal pathway, finally obtain the purpose of treatment. For this reason, the synthesis of novel imidazole derivatives was described and this study explored the details of imidazole derivatives binding to human serum albumin (HSA). The data of steady-state and time-resolved fluorescence showed that the conjugation of imidazole derivatives with HSA yielded quenching by a static mechanism. Meanwhile, the number of binding sites, the binding constants, and the thermodynamic parameters were also measured; the raw data indicated that imidazole derivatives could spontaneously bind with HSA through hydrophobic interactions and hydrogen bonds which agreed well with the results from the molecular modeling study. Competitive binding experiments confirmed the location of binding. Furthermore, alteration of the secondary structure of HSA in the presence of the imidazole derivatives was tested.

  17. Bezafibrate at clinically relevant doses decreases serum/liver triglycerides via down-regulation of sterol regulatory element-binding protein-1c in mice: a novel peroxisome proliferator-activated receptor alpha-independent mechanism.

    PubMed

    Nakajima, Takero; Tanaka, Naoki; Kanbe, Hiroki; Hara, Atsushi; Kamijo, Yuji; Zhang, Xiaowei; Gonzalez, Frank J; Aoyama, Toshifumi

    2009-04-01

    The triglyceride-lowering effect of bezafibrate in humans has been attributed to peroxisome proliferator-activated receptor (PPAR) alpha activation based on results from rodent studies. However, the bezafibrate dosages used in conventional rodent experiments are typically higher than those in clinical use (> or =50 versus < or =10 mg/kg/day), and thus it remains unclear whether such data can be translated to humans. Furthermore, because bezafibrate is a pan-PPAR activator, the actual contribution of PPARalpha to its triglyceride-lowering properties remains undetermined. To address these issues, bezafibrate at clinically relevant doses (10 mg/kg/day; low) was administered to wild-type and Ppara-null mice, and its effects were compared with those from conventionally used doses (100 mg/kg/day; high). Pharmacokinetic analyses showed that maximum plasma concentration and area under the concentration-time curve in bezafibrate-treated mice were similar to those in humans at low doses, but not at high doses. Low-dose bezafibrate decreased serum/liver triglycerides in a PPARalpha-independent manner by attenuation of hepatic lipogenesis and triglyceride secretion. It is noteworthy that instead of PPAR activation, down-regulation of sterol regulatory element-binding protein (SREBP)-1c was observed in mice undergoing low-dose treatment. High-dose bezafibrate decreased serum/liver triglycerides by enhancement of hepatic fatty acid uptake and beta-oxidation via PPARalpha activation, as expected. In conclusion, clinically relevant doses of bezafibrate exert a triglyceride-lowering effect by suppression of the SREBP-1c-regulated pathway in mice and not by PPARalpha activation. Our results may provide novel information about the pharmacological mechanism of bezafibrate action and new insights into the treatment of disorders involving SREBP-1c. PMID:19124612

  18. Influence of iodine supplementation on serum insulin-like growth factor-I (IGF-I) and IGF-binding protein-3 (IGFBP-3) levels in severe iodine deficiency.

    PubMed

    Ozön, Alev; Alikaşifoğlu, Ayfer; Yordam, Nurşen

    2004-01-01

    Iodine deficiency is an important public health problem worldwide. In addition to severe consequences such as brain damage, developmental delay, deficits in hearing and learning, it also has a negative impact on growth. The negative impact of severe iodine deficiency (SID) on insulin-like growth factor-I (IGF-I) and insulin-like growth factor binding protein-3 (IGFBP-3) was shown previously. In this study we aimed to analyze the impact of iodine supplementation on growth and growth factors of children with SID. One hundred and four children (63 boys and 41 girls) aged 5-15 years participated in the study. Height standard deviation scores (HSDS), and serum levels of IGF-I and IGFBP-3 were assessed both before and six months after a single dose of iodized oil. Serum levels of free thyroxine (FT4) and thyroid stimulating hormone (TSH) were also analysed to investigate the mechanisms by which alterations of iodine status may influence growth. Pubertal children had lower HSDS six months after iodine supplementation, while that of prepubertal children remained unchanged. IGF-I and IGFBP-3 levels decreased significantly and FT4 levels were suppressed six months after the supplementation, while TSH was normalized. These findings suggest a negative impact of iodine supplementation on growth factors in the short-term, which may be a direct effect of iodine repletion or an indirect effect caused by alterations in thyroid function. It may also be related to the method of supplementation used. Further studies are necessary to resolve these issues, as well as to examine the impact of iodine supplementation on growth in the long-term. PMID:15641262

  19. Quantifying Aptamer-Protein Binding via Thermofluorimetric Analysis

    PubMed Central

    Hu, Juan; Kim, Joonyul; Easley, Christopher J.

    2015-01-01

    Effective aptamer-based protein assays require coupling to a quantitative reporter of aptamer-protein binding. Typically, this involves a direct optical or electrochemical readout of DNA hybridization or an amplification step coupled to the readout. However, method development is often hampered by the multiplicity of aptamer-target binding mechanisms, which can interfere with the hybridization step. As a simpler and more generalizable readout of aptamer-protein binding, we report that thermofluorimetric analysis (TFA) can be used to quantitatively assay protein levels. Sub-nanomolar detection (0.74 nM) of platelet-derived growth factor (PDGF) with its corresponding aptamer is shown as a test case. In the presence of various DNA intercalating dyes, protein-bound aptamers exhibit a change in fluorescence intensity compared to the intercalated, unbound aptamer. This allows thermal resolution of bound and unbound aptamers using fluorescence melting analysis (−dF/dT curves). Remarkably, the homogeneous optical method allows subtraction of autofluorescence in human serum, giving PDGF detection limits of 1.8 and 10.7 nM in serum diluted 1:7 and 1:3, respectively. We have thus demonstrated that bound and unbound aptamers can be thermally resolved in a homogeneous format using a simple qPCR instrument—even in human serum. The simplicity of this approach provides an important step toward a robust, generalizable readout of aptamer-protein binding. PMID:26366207

  20. Posaconazole in Human Serum: a Greater Pharmacodynamic Effect than Predicted by the Non-Protein-Bound Serum Concentration ▿

    PubMed Central

    Lignell, Anders; Löwdin, Elisabeth; Cars, Otto; Chryssanthou, Erja; Sjölin, Jan

    2011-01-01

    It is generally accepted that only the unbound fraction of a drug is pharmacologically active. Posaconazole is an antifungal agent with a protein binding of 98 to 99%. Taking into account the degree of protein binding, plasma levels in patients, and MIC levels of susceptible strains, it can be assumed that the free concentration of posaconazole sometimes will be too low to exert the expected antifungal effect. The aim was therefore to test the activity of posaconazole in serum in comparison with that of the calculated unbound concentrations in protein-free media. Significant differences (P < 0.05) from the serum control were found at serum concentrations of posaconazole of 1.0 and 0.10 mg/liter, with calculated free concentrations corresponding to 1× MIC and 0.1× MIC, respectively, against one Candida lusitaniae strain selected for proof of principle. In RPMI 1640, the corresponding calculated unbound concentration of 0.015 mg/liter resulted in a significant effect, whereas that of 0.0015 mg/liter did not. Also, against seven additional Candida strains tested, there was an effect of the low posaconazole concentration in serum, in contrast to the results in RPMI 1640. Fluconazole, a low-grade-protein-bound antifungal, was used for comparison at corresponding concentrations in serum and RPMI 1640. No effect was observed at the serum concentration, resulting in a calculated unbound concentration of 0.1× MIC. In summary, there was a substantially greater pharmacodynamic effect of posaconazole in human serum than could be predicted by the non-protein-bound serum concentration. A flux from serum protein-bound to fungal lanosterol 14α-demethylase-bound posaconazole is suggested. PMID:21502622

  1. The measurement of serum transferrin by iron-binding capacity

    PubMed Central

    Ramsay, W. N. M.

    1973-01-01

    Two criteria which ought to be satisfied by an acceptable method for the estimation of serum transferrin by iron-binding capacity are enunciated. A screening procedure is described which involves the sequential quantitative use of ion exchange and gel filtration. Its use in testing the extent to which two published methods fulfil the recommended criteria is described. Both methods give results for total iron-binding capacity which are 1-10% high because of the inclusion of non-transferrin iron. PMID:4752411

  2. Serum protein electrophoresis in spontaneous canine hyperadrenocorticalism.

    PubMed

    van den Broek, A H; Lida, J

    1989-01-01

    The serum protein concentrations of dogs with confirmed spontaneous hyperadrenocorticalism were determined by agarose gel electrophoresis before and during treatment with mitotane. In untreated animals a significant increase was detected in the mean concentration of total protein and the mean concentration and percentage of alpha-2 globulin. The mean concentration and percentage of albumin and gamma-globulin were significantly decreased. In animals on treatment the mean concentration of total proteins and the mean concentration and percentage of beta-2 globulin were significantly reduced. PMID:2466309

  3. CHARACTERIZATION OF DRUG INTERACTIONS WITH SERUM PROTEINS BY USING HIGH-PERFORMANCE AFFINITY CHROMATOGRAPHY

    PubMed Central

    Hage, David S.; Anguizola, Jeanethe; Barnaby, Omar; Jackson, Abby; Yoo, Michelle J.; Papastavros, Efthimia; Pfaunmiller, Erika; Sobansky, Matt; Tong, Zenghan

    2011-01-01

    The binding of drugs with serum proteins can affect the activity, distribution, rate of excretion, and toxicity of pharmaceutical agents in the body. One tool that can be used to quickly analyze and characterize these interactions is high-performance affinity chromatography (HPAC). This review shows how HPAC can be used to study drug-protein binding and describes the various applications of this approach when examining drug interactions with serum proteins. Methods for determining binding constants, characterizing binding sites, examining drug-drug interactions, and studying drug-protein dissociation rates will be discussed. Applications that illustrate the use of HPAC with serum binding agents such as human serum albumin, α1-acid glycoprotein, and lipoproteins will be presented. Recent developments will also be examined, such as new methods for immobilizing serum proteins in HPAC columns, the utilization of HPAC as a tool in personalized medicine, and HPAC methods for the high-throughput screening and characterization of drug-protein binding. PMID:21395530

  4. Hyaluronan-binding properties of human serum hemopexin.

    PubMed

    Hrkal, Z; Kuzelová, K; Muller-Eberhard, U; Stern, R

    1996-03-25

    Hemopexin, the heme-binding serum glycoprotein, exhibits a complex electrophoretic pattern on two-dimensional immunoelectrophoresis on agarose gels into which hyaluronic acid is incorporated in the first and monospecific anti-hemopexin in the second dimension. This heterogeneity reflects a range of interactions of hemopexin isoforms with hyaluronic acid. Electrophoretic patterns of individual human sera greatly differ in their contents of hyaluronan-interacting hemopexin species. Hemopexin itself has no hyaluronidase activity. PMID:8612795

  5. Purification of folate binding factor in normal umbilical cord serum.

    PubMed Central

    Kamen, B A; Caston, J D

    1975-01-01

    Human umbilical cord serum was found to contain both free folate and folate complexed to a high-molecular weight factor. The complexed folate was bound to a very high affinity binder and was present in concentrations equivalent to as much as 60 ng of 5-methyltetrahydrofolic acid per ml of serum. Acidification of the serum caused disassociation of the folate-binder complex. Released folates were separated from binder by Sephadex gel filtration, zonal centrifugation through sucrose gradients, or adsorption onto activated charcoal. The separated binding factor, either saturated or unsaturated with folate, had a molecular weight of about 40,000 on Sephadex G-200 chromatography. Binding of [3H]pteroylglutamic acid was rapid and, as in the original endogenous folate-binder complex, was essentially irreversible at neutral pH. The affinity and specificity of the binder were examined by competition experiments using [3H]pteroylglutamic acid and nonradioactive folate derivatives. Oxidized folates were bound in preference to reduced derivatives, but only three to four times more unlabeled 5-methyltetrahydrofolic acid than pteroylglutamic acid was required to produce an equal level of competition. The strong affinity for 5-methyltetrahydrofolic acid, the main serum folate, suggests that the binder could be part of the mechanism by which the fetus concentrates maternally supplied folate for its growth and development. PMID:676

  6. Binding of trivalent chromium to serum transferrin is sufficiently rapid to be physiologically relevant.

    PubMed

    Deng, Ge; Wu, Kristi; Cruce, Alex A; Bowman, Michael K; Vincent, John B

    2015-02-01

    Transferrin, the major iron transport protein in the blood, also transports trivalent chromium in vivo. Recent in vitro studies have, however, suggested that the binding of chromic ions to apotransferrin is too slow to be biologically relevant. Nevertheless, the in vitro studies have generally failed to adequately take physiological bicarbonate concentrations into account. In aqueous buffer (with ambient (bi)carbonate concentrations), the binding of chromium to transferrin is too slow to be physiologically relevant, taking days to reach equilibrium with the protein's associated conformational changes. However, in the presence of 25mM (bi)carbonate, the concentration in human blood, chromic ions bind rapidly and tightly to transferrin. Details of the kinetics of chromium binding to human serum transferrin and conalbumin (egg white transferrin) in the presence of bicarbonate and other major potential chromium ligands are described and are consistent with transferrin being the major chromic ion transporter from the blood to tissues. PMID:25528477

  7. Cigarette smoke induces alterations in the drug-binding properties of human serum albumin.

    PubMed

    Clerici, Marco; Colombo, Graziano; Secundo, Francesco; Gagliano, Nicoletta; Colombo, Roberto; Portinaro, Nicola; Giustarini, Daniela; Milzani, Aldo; Rossi, Ranieri; Dalle-Donne, Isabella

    2014-04-01

    Albumin is the most abundant plasma protein and serves as a transport and depot protein for numerous endogenous and exogenous compounds. Earlier we had shown that cigarette smoke induces carbonylation of human serum albumin (HSA) and alters its redox state. Here, the effect of whole-phase cigarette smoke on HSA ligand-binding properties was evaluated by equilibrium dialysis and size-exclusion HPLC or tryptophan fluorescence. The binding of salicylic acid and naproxen to cigarette smoke-oxidized HSA resulted to be impaired, unlike that of curcumin and genistein, chosen as representative ligands. Binding of the hydrophobic fluorescent probe 4,4'-bis(1-anilino-8-naphtalenesulfonic acid) (bis-ANS), intrinsic tryptophan fluorescence, and susceptibility to enzymatic proteolysis revealed slight changes in albumin conformation. These findings suggest that cigarette smoke-induced modifications of HSA may affect the binding, transport and bioavailability of specific ligands in smokers. PMID:24388826

  8. Imatinib binding to human serum albumin modulates heme association and reactivity.

    PubMed

    Di Muzio, Elena; Polticelli, Fabio; Trezza, Viviana; Fanali, Gabriella; Fasano, Mauro; Ascenzi, Paolo

    2014-10-15

    Imatinib, an inhibitor of the Bcr-Abl tyrosine kinase, is approximately 95% bound to plasma proteins, α1-acid glycoprotein (AGP) being the primary carrier. However, human serum albumin (HSA) may represent the secondary carrier of imatinib in pathological states characterized by low AGP levels, such as pancreatic cancer, hepatic cirrhosis, hepatitis, hyperthyroidism, nephrotic syndrome, malnutrition, and cachexia. Here, thermodynamics of imatinib binding to full-length HSA and its recombinant Asp1-Glu382 truncated form (containing only the FA1, FA2, FA6, and FA7 binding sites; trHSA), in the absence and presence of ferric heme (heme-Fe(III)), and the thermodynamics of heme-Fe(III) binding to HSA and trHSA, in the absence and presence of imatinib, has been investigated. Moreover, the effect of imatinib on kinetics of peroxynitrite detoxification by ferric human serum heme-albumin (HSA-heme-Fe(III)) and ferric truncated human serum heme-albumin (trHSA-heme-Fe(III)) has been explored. All data were obtained at pH 7.0, and 20.0 °C and 37.0 °C. Imatinib binding to the FA7 site of HSA and trHSA inhibits allosterically heme-Fe(III) association to the FA1 site and vice versa, according to linked functions. Moreover, imatinib binding to the secondary FA2 site of HSA-heme-Fe(III) inhibits allosterically peroxynitrite detoxification. Docking simulations and local structural comparison with other imatinib-binding proteins support functional data indicating the preferential binding of imatinib to the FA1 and FA7 sites of HSA, and to the FA2 and FA7 sites of HSA-heme-Fe(III). Present results highlight the allosteric coupling of the FA1, FA2, and FA7 sites of HSA, and may be relevant in modulating ligand binding and reactivity properties of HSA in vivo. PMID:25057771

  9. Reciprocal Allosteric Modulation of Carbon Monoxide and Warfarin Binding to Ferrous Human Serum Heme-Albumin

    PubMed Central

    Bocedi, Alessio; De Sanctis, Giampiero; Ciaccio, Chiara; Tundo, Grazia R.; Di Masi, Alessandra; Fanali, Gabriella; Nicoletti, Francesco P.; Fasano, Mauro; Smulevich, Giulietta; Ascenzi, Paolo; Coletta, Massimo

    2013-01-01

    Human serum albumin (HSA), the most abundant protein in human plasma, could be considered as a prototypic monomeric allosteric protein, since the ligand-dependent conformational adaptability of HSA spreads beyond the immediate proximity of the binding site(s). As a matter of fact, HSA is a major transport protein in the bloodstream and the regulation of the functional allosteric interrelationships between the different binding sites represents a fundamental information for the knowledge of its transport function. Here, kinetics and thermodynamics of the allosteric modulation: (i) of carbon monoxide (CO) binding to ferrous human serum heme-albumin (HSA-heme-Fe(II)) by warfarin (WF), and (ii) of WF binding to HSA-heme-Fe(II) by CO are reported. All data were obtained at pH 7.0 and 25°C. Kinetics of CO and WF binding to the FA1 and FA7 sites of HSA-heme-Fe(II), respectively, follows a multi-exponential behavior (with the same relative percentage for the two ligands). This can be accounted for by the existence of multiple conformations and/or heme-protein axial coordination forms of HSA-heme-Fe(II). The HSA-heme-Fe(II) populations have been characterized by resonance Raman spectroscopy, indicating the coexistence of different species characterized by four-, five- and six-coordination of the heme-Fe atom. As a whole, these results suggest that: (i) upon CO binding a conformational change of HSA-heme-Fe(II) takes place (likely reflecting the displacement of an endogenous ligand by CO), and (ii) CO and/or WF binding brings about a ligand-dependent variation of the HSA-heme-Fe(II) population distribution of the various coordinating species. The detailed thermodynamic and kinetic analysis here reported allows a quantitative description of the mutual allosteric effect of CO and WF binding to HSA-heme-Fe(II). PMID:23555601

  10. Relationship between binding affinities to cellular retinoic acid-binding protein and biological potency of a new series of retinoids.

    PubMed

    Sani, B P; Dawson, M I; Hobbs, P D; Chan, R L; Schiff, L J

    1984-01-01

    Binding affinities of a new and unusual series of retinoic acid analogues to cellular retinoic acid-binding protein, a possible mediator of their biological function in the control of differentiation and tumorigenesis, and to serum albumin, their plasma transport protein, were determined. Also, biological activity of these retinoids in the reversal of keratinization in hamster tracheal organ cultures was assessed and compared with their binding affinities. Analogues that possessed high biological activity showed high binding efficiency to cellular retinoic acid-binding protein. Those that were biologically less active were poor binders to the binding protein. Three retinoids, 4657-57, 3920-59, and 4445-75, which showed 90 to 100% binding efficiency of that of retinoic acid for cellular retinoic acid-binding protein expressed high biological activity detectable in the range of 10(-10) M as against 10(-11) M for retinoic acid. The correlation noticed in these two activities not only enhances the confidence in the two assay procedures but also paves the way for design and development of potential chemopreventive agents. No apparent differences were observed in the binding affinities of the retinoids to binding proteins of a normal tissue or a tumor tissue. No correlation existed between the binding affinities of these retinoids to serum albumin and their biological activity. Structure-activity relationships of the retinoids in relation to their binding affinities and biological activities have been discussed. PMID:6317169

  11. Detergent binding as a sensor of hydrophobicity and polar interactions in the binding cavities of proteins.

    PubMed

    Peyre, Véronique; Lair, Virginie; André, Virginie; le Maire, Guerric; Kragh-Hansen, Ulrich; le Maire, Marc; Møller, Jesper V

    2005-09-13

    To evaluate the role of hydrophobic and electrostatic or other polar interactions for protein-ligand binding, we studied the interaction of human serum albumin (HSA) and beta-lactoglobulin with various aliphatic (C10-C14) cationic and zwitterionic detergents. We find that cationic detergents, at levels that do not cause unfolding, interact with a single site on beta-lactoglobulin and with two primary and five to six secondary sites on HSA with an affinity that is approximately the same as that with which zwitterionic (dimethylamineoxide) detergents interact, suggesting the absence of significant electrostatic interactions in the high-affinity binding of these compounds. The binding affinity for all of the groups of compounds was dependent upon hydrocarbon chain length, suggesting the predominant role of hydrophobic forces, supported by polar interactions at the protein surface. A distinct correlation between the binding energy and the propensity for micelle formation within the group of cationic or noncharged (nonionic and zwitterionic) detergents indicated that the critical micellar concentration (CMC) for each of these detergent groups, rather than the absolute length of the hydrocarbon chain, can be used to compare their hydrophobicities during their interaction with protein. Intrinsic fluorescence data suggest that the two primary binding sites on serum albumin for the zwitterionic and cationic compounds are located in the C-terminal part of the albumin molecule, possibly in the Sudlow II binding region. Comparisons with previous binding data on anionic amphiphiles emphasize the important contribution of ion bond formation and other polar interactions in the binding of fatty acids and dodecyl sulfate (SDS) by HSA but not by beta-lactoglobulin. Electrostatic interactions by cationic detergents played a significant role in destabilizing the protein structure at high binding levels, with beta-lactoglobulin being more susceptible to unfolding than HSA. Zwitterionic

  12. Challenges of measuring monoclonal proteins in serum.

    PubMed

    Keren, David F; Schroeder, Lee

    2016-06-01

    The measurement of monoclonal protein (M-protein) is vital for stratifying risk and following individuals with a variety of monoclonal gammopathies. Direct measurement of the M-protein spike by electrophoresis and immunochemical measurements of specific isotypes or free light chains pairs has provided useful information about the quantity of M-protein. Nonetheless, both traditional electrophoresis and immunochemical methods give poor quantification with M-proteins smaller than 10 g/L (1 g/dL) when in the presence of polyclonal immunoglobulins that co-migrate with the M-protein. In addition, measurements by electrophoresis of M-proteins migrating in the β- and α-regions are contaminated by normal serum proteins in those regions. The most precise electrophoretic method to date for quantification involves exclusion of the polyclonal immunoglobulins by using the tangent skimming method on electropherograms, which provides a 10-fold improvement in precision. So far, however, tangent measurements are limited to γ migrating M-proteins. Another way to improve M-protein measurements is the use of capillary electrophoresis (CE). With CE, one can employ immunosubtraction to select a region of interest in the β region thereby excluding much of the normal proteins from the M-protein measurement. Recent development of an immunochemical method distinguishing heavy/light chain pairs (separately measuring IgGK and IgGL, IgAK and IgAL, and IgMK and IgML) provides measurements that could exclude polyclonal contaminants of the same heavy chain with the uninvolved light chain type. Yet, even heavy/light results contain an immeasurable quantity of polyclonal heavy/light chains of the involved isotype. Finally, use of liquid chromatography-tandem mass spectrometry (LC-MS/MS) looms on the horizon as a means to provide more consistent and sensitive measurements of M-proteins. PMID:26910744

  13. Mannose-Binding Lectin Serum Levels in Patients With Candiduria

    PubMed Central

    Moslem, Maryam; Zarei Mahmoudabadi, Ali; Fatahinia, Mahnaz; Kheradmand, Alireza

    2015-01-01

    Background: Candida species are normal mycoflora of human body which are capable to cause urinary tract infection (UTI). Mannose-binding lectin (MBL) is a kind of innate immune system and decreasing plasma levels of MBL may disrupt the natural immune response and increase susceptibility to infections. Objectives: The aim of the present study was to assess MBL in the serum of patients with candiduria and compare them with control. Patients and Methods: The blood and urine samples were collected from 335 patients (hospitalized in Golestan hospital, Ahvaz) using standard methods and the growing colonies on CHROMagar were identified using routine diagnostic tests. MBL activity in the serum of 45 patients with candiduria and 45 controls was measured using Eastbiopharm enzyme-linked immunosorbent assay (ELISA) kit. Results: In this study, 45 (13.4 %) urine samples were positive for Candida species (17 males and 28 females). The most common isolated yeast was Candida albicans (34%), followed by C. glabrata (32.1%), C. tropicalis (9.4%), other Candida species (22.6%), and Rhodotorula species (1.9%). The mean serum levels of MBL were 0.85 ± 0.01 ng/mL and 1.02 ± 0.03 ng/mL among candiduric patients and controls, respectively, and there was no significant difference between the two groups (P = 0.6). Conclusions: Our results showed that there was no significant relationship between MBL serum levels and candiduria. PMID:26870314

  14. 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.

  15. Oxysterol sulfation by cytosolic sulfotransferase suppresses liver X receptor/sterol regulatory element binding protein-1c signaling pathway and reduces serum and hepatic lipids in mouse models of nonalcoholic fatty liver disease.

    PubMed

    Bai, Qianming; Zhang, Xin; Xu, Leyuan; Kakiyama, Genta; Heuman, Douglas; Sanyal, Arun; Pandak, William M; Yin, Lianhua; Xie, Wen; Ren, Shunlin

    2012-06-01

    Cytosolic sulfotransferase (SULT2B1b) catalyzes oxysterol sulfation. 5-Cholesten-3β-25-diol-3-sulfate (25HC3S), one product of this reaction, decreases intracellular lipids in vitro by suppressing liver X receptor/sterol regulatory element binding protein (SREBP)-1c signaling, with regulatory properties opposite to those of its precursor 25-hydroxycholesterol. Upregulation of SULT2B1b may be an effective strategy to treat hyperlipidemia and hepatic steatosis. The objective of the study was to explore the effect and mechanism of oxysterol sulfation by SULT2B1b on lipid metabolism in vivo. C57BL/6 and LDLR(-/-) mice were fed with high-cholesterol diet or high-fat diet for 10 weeks and infected with adenovirus encoding SULT2B1b. SULT2B1b expressions in different tissues were determined by immunohistochemistry and Western blot. Sulfated oxysterols in liver were analyzed by high-pressure liquid chromatography. Serum and hepatic lipid levels were determined by kit reagents and hematoxylin and eosin staining. Gene expressions were determined by real-time reverse transcriptase polymerase chain reaction and Western Blot. Following infection, SULT2B1b was successfully overexpressed in the liver, aorta, and lung tissues, but not in the heart or kidney. SULT2B1b overexpression, combined with administration of 25-hydroxycholesterol, significantly increased the formation of 25HC3S in liver tissue and significantly decreased serum and hepatic lipid levels, including triglycerides, total cholesterol, free cholesterol, and free fatty acids, as compared with controls in both C57BL/6 and LDLR(-/-) mice. Gene expression analysis showed that increases in SULT2B1b expression were accompanied by reduction in key regulators and enzymes involved in lipid metabolism, including liver X receptor α, SREBP-1, SREBP-2, acetyl-CoA carboxylase-1, and fatty acid synthase. These findings support the hypothesis that 25HC3S is an important endogenous regulator of lipid biosynthesis. PMID:22225954

  16. Serum protein identification and quantification of the corona of 5, 15 and 80 nm gold nanoparticles

    NASA Astrophysics Data System (ADS)

    Schäffler, Martin; Semmler-Behnke, Manuela; Sarioglu, Hakan; Takenaka, Shinji; Wenk, Alexander; Schleh, Carsten; Hauck, Stefanie M.; Johnston, Blair D.; Kreyling, Wolfgang G.

    2013-07-01

    When nanoparticles (NP) enter the body they come into contact with body fluids containing proteins which can adsorb to their surface. These proteins may influence the NP interactions with the biological vicinity, eventually determining their biological fate inside the body. Adsorption of the most abundantly binding proteins was studied after an in vitro 24 hr incubation of monodisperse, negatively charged 5, 15 and 80 nm gold spheres (AuNP) in mouse serum by a two-step analysis: proteomic protein identification and quantitative protein biochemistry. The adsorbed proteins were separated from non-adsorbed proteins by centrifugation and gel electrophoresis and identified using a MALDI-TOF-MS-Proteomics-Analyzer. Quantitative analysis of proteins in gel bands by protein densitometry, required the focus on predominantly binding serum proteins. Numerous proteins adsorbed to the AuNP depending on their size, e.g. apolipoproteins or complement C3. The qualitative and quantitative amount of adsorbed proteins differed between 5, 15 and 80 nm AuNP. Band intensities of adsorbed proteins decreased with increasing AuNP sizes based not only on their mass but also on their surface area. Summarizing, the AuNP surface is covered with serum proteins containing transport and immune related proteins among others. Hence, protein binding depends on the size, surface area and curvature of the AuNP.

  17. Calcium-binding proteins and development

    NASA Technical Reports Server (NTRS)

    Beckingham, K.; Lu, A. Q.; Andruss, B. F.; McIntire, L. V. (Principal Investigator)

    1998-01-01

    The known roles for calcium-binding proteins in developmental signaling pathways are reviewed. Current information on the calcium-binding characteristics of three classes of cell-surface developmental signaling proteins (EGF-domain proteins, cadherins and integrins) is presented together with an overview of the intracellular pathways downstream of these surface receptors. The developmental roles delineated to date for the universal intracellular calcium sensor, calmodulin, and its targets, and for calcium-binding regulators of the cytoskeleton are also reviewed.

  18. Advanced Running Performance by Genetic Predisposition in Male Dummerstorf Marathon Mice (DUhTP) Reveals Higher Sterol Regulatory Element-Binding Protein (SREBP) Related mRNA Expression in the Liver and Higher Serum Levels of Progesterone

    PubMed Central

    Brenmoehl, Julia; Walz, Christina; Ponsuksili, Siriluck; Schwerin, Manfred; Fuellen, Georg; Hoeflich, Andreas

    2016-01-01

    Long-term-selected DUhTP mice represent a non-inbred model for inborn physical high-performance without previous training. Abundance of hepatic mRNA in 70-day male DUhTP and control mice was analyzed using the Affymetrix mouse array 430A 2.0. Differential expression analysis with PLIER corrected data was performed using AltAnalyze. Searching for over-representation in biochemical pathways revealed cholesterol metabolism being most prominently affected in DUhTP compared to unselected control mice. Furthermore, pathway analysis by AltAnalyze plus PathVisio indicated significant induction of glycolysis, fatty acid synthesis and cholesterol biosynthesis in the liver of DUhTP mice versus unselected control mice. In contrast, gluconeogenesis was partially inactivated as judged from the analysis of hepatic mRNA transcript abundance in DUhTP mice. Analysis of mRNA transcripts related to steroid hormone metabolism inferred elevated synthesis of progesterone and reduced levels of sex steroids. Abundance of steroid delta isomerase-5 mRNA (Hsd3b5, FC 4.97) was increased and steroid 17-alpha-monooxygenase mRNA (Cyp17a1, FC -11.6) was massively diminished in the liver of DUhTP mice. Assessment of steroid profiles by LC-MS revealed increased levels of progesterone and decreased levels of sex steroids in serum from DUhTP mice versus controls. Analysis of hepatic mRNA transcript abundance indicates that sterol regulatory element-binding protein-1 (SREBP-1) may play a major role in metabolic pathway activation in the marathon mouse model DUhTP. Thus, results from bioinformatics modeling of hepatic mRNA transcript abundance correlated with direct steroid analysis by mass spectrometry and further indicated functions of SREBP-1 and steroid hormones for endurance performance in DUhTP mice. PMID:26799318

  19. Advanced Running Performance by Genetic Predisposition in Male Dummerstorf Marathon Mice (DUhTP) Reveals Higher Sterol Regulatory Element-Binding Protein (SREBP) Related mRNA Expression in the Liver and Higher Serum Levels of Progesterone.

    PubMed

    Ohde, Daniela; Moeller, Mark; Brenmoehl, Julia; Walz, Christina; Ponsuksili, Siriluck; Schwerin, Manfred; Fuellen, Georg; Hoeflich, Andreas

    2016-01-01

    Long-term-selected DUhTP mice represent a non-inbred model for inborn physical high-performance without previous training. Abundance of hepatic mRNA in 70-day male DUhTP and control mice was analyzed using the Affymetrix mouse array 430A 2.0. Differential expression analysis with PLIER corrected data was performed using AltAnalyze. Searching for over-representation in biochemical pathways revealed cholesterol metabolism being most prominently affected in DUhTP compared to unselected control mice. Furthermore, pathway analysis by AltAnalyze plus PathVisio indicated significant induction of glycolysis, fatty acid synthesis and cholesterol biosynthesis in the liver of DUhTP mice versus unselected control mice. In contrast, gluconeogenesis was partially inactivated as judged from the analysis of hepatic mRNA transcript abundance in DUhTP mice. Analysis of mRNA transcripts related to steroid hormone metabolism inferred elevated synthesis of progesterone and reduced levels of sex steroids. Abundance of steroid delta isomerase-5 mRNA (Hsd3b5, FC 4.97) was increased and steroid 17-alpha-monooxygenase mRNA (Cyp17a1, FC -11.6) was massively diminished in the liver of DUhTP mice. Assessment of steroid profiles by LC-MS revealed increased levels of progesterone and decreased levels of sex steroids in serum from DUhTP mice versus controls. Analysis of hepatic mRNA transcript abundance indicates that sterol regulatory element-binding protein-1 (SREBP-1) may play a major role in metabolic pathway activation in the marathon mouse model DUhTP. Thus, results from bioinformatics modeling of hepatic mRNA transcript abundance correlated with direct steroid analysis by mass spectrometry and further indicated functions of SREBP-1 and steroid hormones for endurance performance in DUhTP mice. PMID:26799318

  20. Prostate cancer serum biomarker discovery through proteomic analysis of alpha-2 macroglobulin protein complexes

    PubMed Central

    Burgess, Earle F.; Ham, Amy-Joan L.; Tabb, David L.; Billheimer, Dean; Roth, Bruce J.; Chang, Sam S.; Cookson, Michael S.; Hinton, Timothy J.; Cheek, Kristin L.; Hill, Salisha; Pietenpol, Jennifer A.

    2010-01-01

    Alpha-2 macroglobulin (A2M) functions as a universal protease inhibitor in serum and is capable of binding various cytokines and growth factors. In this study, we investigated if immunoaffinity enrichment and proteomic analysis of A2M protein complexes from human serum could improve detection of biologically relevant and novel candidate protein biomarkers in prostate cancer. Serum samples from six patients with androgen-independent, metastatic prostate cancer and six control patients without malignancy were analyzed by immunoaffinity enrichment of A2M protein complexes and MS identification of associated proteins. Known A2M substrates were reproducibly identified from patient serum in both cohorts, as well as proteins previously undetected in human serum. One example is heat shock protein 90 alpha (HSP90α), which was identified only in the serum of cancer patients in this study. Using an ELISA, the presence of HSP90α in human serum was validated on expanded test cohorts and found to exist in higher median serum concentrations in prostate cancer (n = 18) relative to control (n = 13) patients (median concentrations 50.7 versus 27.6 ng/mL, respectively, p = 0.001). Our results demonstrate the technical feasibility of this approach and support the analysis of A2M protein complexes for proteomic-based serum biomarker discovery. PMID:20107526

  1. Backbone Dynamics Of Intracellular Lipid Binding Proteins

    NASA Astrophysics Data System (ADS)

    Gutiérrez-González, Luis H.

    2005-04-01

    The family of intracellular lipid binding proteins (iLBPs) comprises a group of homologous 14-15 kDa proteins that specifically bind and facilitate the transport of fatty acids, bile acids, retinoids or eicosanoids. Members of this family include several types of fatty acid binding proteins (FABPs), ileal lipid binding protein, cellular retinoic acid binding proteins and cellular retinoid binding proteins. As a contribution to understanding the structure-function relationship in this protein family, the solution structure and backbone dynamics of human epidermal-type FABP (E-FABP) determined by NMR spectroscopy are reported. Moreover, hydrogen/deuterium exchange experiments indicated a direct correlation between the stability of the hydrogen-bonding network in the β-sheet structure and the conformational exchange in the millisecond-to-microsecond time range. The features of E-FABP backbone dynamics discussed in the present study are compared with those obtained for other phylogenetically related proteins. A strong interdependence with the overall protein stability and possibly also with the ligand-binding affinity for members of the lipid-binding protein family is shown.

  2. The detection of DNA-binding proteins by protein blotting.

    PubMed Central

    Bowen, B; Steinberg, J; Laemmli, U K; Weintraub, H

    1980-01-01

    A method, called "protein blotting," for the detection of DNA-binding proteins is described. Proteins are separated on an SDA-polyacrylamide gel. The gel is sandwiched between 2 nitrocellulose filters and the proteins allowed to diffuse out of the gel and onto the filters. The proteins are tightly bound to each filter, producing a replica of the original gel pattern. The replica is used to detect DNA-binding proteins, RNA-binding proteins or histone-binding proteins by incubation of the filter with [32P]DNA, [125I]RNA, or [125I] histone. Evidence is also presented that specific protein-DNA interactions may be detected by this technique; under appropriate conditions, the lac repressor binds only to DNA containing the lac operator. Strategies for the detection of specific protein-DNA interactions are discussed. Images PMID:6243775

  3. Tamoxifen and curcumin binding to serum albumin. Spectroscopic study

    NASA Astrophysics Data System (ADS)

    Maciążek-Jurczyk, M.; Maliszewska, M.; Pożycka, J.; Równicka-Zubik, J.; Góra, A.; Sułkowska, A.

    2013-07-01

    Tamoxifen (TMX) is widely used for the breast cancer treatment and is known as chemopreventive agent. Curcumin (CUR) is natural phenolic compound with broad spectrum of biological activity e.g. anti-inflammatory, antimicrobial, antiviral, antifungal and chemopreventive. Combination of tamoxifen and curcumin could be more effective with lower toxicity than each agent alone in use for the treatment or chemoprevention of breast cancer. Binding of drugs to serum albumin is an important factor, which determines toxicity and therapeutic dosage of the drugs. When two drugs are administered together the competition between them for the binding site on albumin can result in a decrease in bound fraction and an increase in the concentration of free biologically active fraction of drug.

  4. Interactions Between Sirolimus and Anti-Inflammatory Drugs: Competitive Binding for Human Serum Albumin

    PubMed Central

    Khodaei, Arash; Bolandnazar, Soheila; Valizadeh, Hadi; Hasani, Leila; Zakeri-Milani, Parvin

    2016-01-01

    Purpose: The aim of the present study was investigating the effects of three anti-inflammatory drugs, on Sirolimus protein biding. The binding site of Sirolimus on human serum albumin (HSA) was also determined. Methods: Six different concentrations of Sirolimus were separately exposed to HSA at pH 7.4 and 37°C. Ultrafiltration method was used for separating free drug; then free drug concentrations were measured by HPLC. Finally, Sirolimus protein binding parameters was calculated using Scatchard plots. The same processes were conducted in the presence of NSAIDs at lower concentration of albumin and different pH conditions. To characterize the binding site of Sirolimus on albumin, the free concentration of warfarin sodium and Diazepam, site I and II specific probes, bound to albumin were measured upon the addition of increasing Sirolimus concentrations. Results: Based on the obtained results presence of Diclofenac, Piroxicam and Naproxen, could significantly decrease the percentage of Sirolimus protein binding. The Binding reduction was the most in the presence of Piroxicam. Sirolimus-NSAIDs interactions were increased in higher pH values and also in lower albumin concentrations. Probe displacement study showed that Sirolimus may mainly bind to site I on albumin molecule. Conclusion: More considerations in co-administration of NSAIDs and Sirolimus is recommended. PMID:27478785

  5. Diosmin binding to human serum albumin and its preventive action against degradation due to oxidative injuries.

    PubMed

    Barreca, Davide; Laganà, Giuseppina; Bruno, Giuseppe; Magazù, Salvatore; Bellocco, Ersilia

    2013-11-01

    Diosmin is a glycosylated polyphenolic compound, commonly found in fruits and vegetables, which is utilized for the pharmacological formulation of some drugs. The interactions of diosmin to human serum albumin have been investigated by fluorescence, UV-visible, FTIR spectroscopy, native electrophoresis and protein-ligand docking studies. The fluorescence studies indicate that the binding site of the additive involves modifications of environment around Trp214 at the level of subdomain IIA. Combining the curve-fitting results of infrared Amide I' band, the modifications of protein secondary structure have been estimated, indicating a decrease in α-helix structure following flavonoid binding. Data obtained by fluorescence and UV-visible spectroscopy, FTIR experiments and molecular modeling afforded a clear picture of the association mode of diosmin to HSA, suggesting that the primary binding site of diosmin is located in Sudlow's site I. Computational mapping confirms this observation suggesting that the possible binding site of diosmin is located in the hydrophobic cavity of subdomain IIA, whose microenvironment is able to help and stabilize the binding of the ligand in non-planar conformation. Moreover the binding of diosmin to HSA significantly contributes to protect the protein against degradation due to HCLO and Fenton reaction. PMID:23886889

  6. Exploring binding properties of sertraline with human serum albumin: Combination of spectroscopic and molecular modeling studies.

    PubMed

    Shahlaei, Mohsen; Rahimi, Behnoosh; Nowroozi, Amin; Ashrafi-Kooshk, Mohammad Reza; Sadrjavadi, Komail; Khodarahmi, Reza

    2015-12-01

    Human serum albumin (HSA)-drug binding is an important factor to determine half life and bioavailability of drugs. In the present research, the interaction of sertraline (SER) to HSA was investigated using combination of spectroscopic and molecular modeling techniques. Changes in the UV-Vis, CD and FT-IR spectra as well as a significant degree of tryptophan fluorescence quenching were observed upon SER-HSA interaction. Data obtained by spectroscopic methods along with the computational studies suggest that SER binds to residues located in subdomain IIA of HSA. Analysis of spectroscopic data represented the formation of 1:1 complex, significant binding affinity, negative values of entropy and enthalpy changes and the essential role of hydrophobic interactions in binding of SER to HSA. The binding models were demonstrated in the aspects of SER's conformation, active site interactions, important amino acids and hydrogen bonding. Computational mapping of the possible binding site of SER confirmed that the ligand to be bound in a large hydrophobic cavity of HSA. In accordance with experimental data, computational analyses indicated that SER binding does not alter the secondary structure of the protein. The results not only lead to a better understanding of interaction between SER and HSA but also provide useful data about the influence of SER on the protein conformation. PMID:26471709

  7. Haptenation: Chemical Reactivity and Protein Binding

    PubMed Central

    Chipinda, Itai; Hettick, Justin M.; Siegel, Paul D.

    2011-01-01

    Low molecular weight chemical (LMW) allergens are commonly referred to as haptens. Haptens must complex with proteins to be recognized by the immune system. The majority of occupationally related haptens are reactive, electrophilic chemicals, or are metabolized to reactive metabolites that form covalent bonds with nucleophilic centers on proteins. Nonelectrophilic protein binding may occur through disulfide exchange, coordinate covalent binding onto metal ions on metalloproteins or of metal allergens, themselves, to the major histocompatibility complex. Recent chemical reactivity kinetic studies suggest that the rate of protein binding is a major determinant of allergenic potency; however, electrophilic strength does not seem to predict the ability of a hapten to skew the response between Th1 and Th2. Modern proteomic mass spectrometry methods that allow detailed delineation of potential differences in protein binding sites may be valuable in predicting if a chemical will stimulate an immediate or delayed hypersensitivity. Chemical aspects related to both reactivity and protein-specific binding are discussed. PMID:21785613

  8. Sertoli cells secrete both testis-specific and serum proteins.

    PubMed Central

    Wright, W W; Musto, N A; Mather, J P; Bardin, C W

    1981-01-01

    The secretions of the Sertoli cell were examined with two polyvalent antisera--one prepared against proteins in rat serum and the other against testis-specific proteins in rete testis fluid. These antisera detected 12 serum and 9 testis-specific proteins in rete testis fluid. To determine the origin of these proteins, primary cultures enriched in Sertoli cells were incubated with [35S]methionine, and the radiolabeled proteins in the medium were immunoprecipitated. Gel electrophoresis of the two immunoprecipitates resolved eight serum and nine testis-specific proteins. These two sets of proteins were specifically bound to their respective antiserum and were immunologically distinct. Medium from Sertoli cell cultures contained 10 times more of the testis-specific proteins than did cultures enriched for testicular myoid or interstitial cells. The concentration of the serum proteins in Sertoli cell medium was 5 and 10 times greater, respectively, than in myoid or interstitial cell preparations. The proteins from Sertoli cells were next characterized on two-dimensional gels. Seven of the proteins recognized by antiserum against serum proteins had identical molecular weights and isoelectric points as serum proteins. Three of these proteins were ceruloplasmin, transferrin, and glycoprotein 2. In addition to the proteins immunoprecipitated by the two antisera, more than 60 other proteins were detected on two-dimensional gels of the total secretory proteins. We conclude that the Sertoli cell secretes many proteins, some of which are specific to the testis and others of which are similar to serum proteins. Images PMID:6950398

  9. Lipid A binding proteins in macrophages detected by ligand blotting

    SciTech Connect

    Hampton, R.Y.; Golenbock, D.T.; Raetz, C.R.H.

    1987-05-01

    Endotoxin (LPS) stimulates a variety of eukaryotic cells. These actions are involved in the pathogenesis of Gram-negative septicemia. The site of action of the LPS toxic moiety, lipid A (LA), is unclear. Their laboratory has previously identified a bioactive LA precursor lipid IV/sub A/, which can be enzymatically labeled with /sup 32/P/sub i/ (10/sup 9/ dpm/nmole) and purified (99%). They now show that this ligand binds to specific proteins immobilized on nitrocellulose (NC) from LPS-sensitive RAW 264.7 cultured macrophages. NC blots were incubated with (/sup 32/P)-IV/sub A/ in a buffer containing BSA, NaCl, polyethylene glycol, and azide. Binding was assessed using autoradiography or scintillation counting. Dot blot binding of the radioligand was inhibited by excess cold IV/sub A/, LA, or ReLPS but not by phosphatidylcholine, cardiolipin, phosphatidylinositol, or phosphatidic acid. Binding was trypsin-sensitive and dependent on protein concentration. Particulate macrophage proteins were subjected to SDS-PAGE and then electroblotted onto NC. Several discrete binding proteins were observed. Identical treatment of fetal bovine serum or molecular weight standards revealed no detectable binding. By avoiding high nonspecific binding of intact membranes, this ligand blotting assay may be useful in elucidating the molecular actions of LPS.

  10. 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.

  11. 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.

  12. Steady-State Fluorescence Anisotropy to Investigate Flavonoids Binding to Proteins

    ERIC Educational Resources Information Center

    Ingersoll, Christine M.; Strollo, Christen M.

    2007-01-01

    The steady-state fluorescence anisotropy is employed to study the binding of protein of a model protein, human serum albumin, to a commonly used flavonoid, quercetin. The experiment describes the thermodynamics, as well as the biochemical interactions of such binding effectively.

  13. Calmodulin Binding Proteins and Alzheimer's Disease.

    PubMed

    O'Day, Danton H; Eshak, Kristeen; Myre, Michael A

    2015-01-01

    The small, calcium-sensor protein, calmodulin, is ubiquitously expressed and central to cell function in all cell types. Here the literature linking calmodulin to Alzheimer's disease is reviewed. Several experimentally-verified calmodulin-binding proteins are involved in the formation of amyloid-β plaques including amyloid-β protein precursor, β-secretase, presenilin-1, and ADAM10. Many others possess potential calmodulin-binding domains that remain to be verified. Three calmodulin binding proteins are associated with the formation of neurofibrillary tangles: two kinases (CaMKII, CDK5) and one protein phosphatase (PP2B or calcineurin). Many of the genes recently identified by genome wide association studies and other studies encode proteins that contain putative calmodulin-binding domains but only a couple (e.g., APOE, BIN1) have been experimentally confirmed as calmodulin binding proteins. At least two receptors involved in calcium metabolism and linked to Alzheimer's disease (mAchR; NMDAR) have also been identified as calmodulin-binding proteins. In addition to this, many proteins that are involved in other cellular events intimately associated with Alzheimer's disease including calcium channel function, cholesterol metabolism, neuroinflammation, endocytosis, cell cycle events, and apoptosis have been tentatively or experimentally verified as calmodulin binding proteins. The use of calmodulin as a potential biomarker and as a therapeutic target is discussed. PMID:25812852

  14. Characterization of the binding of an anticancer drug, lapatinib to human serum albumin.

    PubMed

    Kabir, Md Zahirul; Mukarram, Abdul Kadir; Mohamad, Saharuddin B; Alias, Zazali; Tayyab, Saad

    2016-07-01

    Interaction of a promising anticancer drug, lapatinib (LAP) with the major transport protein in human blood circulation, human serum albumin (HSA) was investigated using fluorescence and circular dichroism (CD) spectroscopy as well as molecular docking analysis. LAP-HSA complex formation was evident from the involvement of static quenching mechanism, as revealed by the fluorescence quenching data analysis. The binding constant, Ka value in the range of 1.49-1.01×10(5)M(-1), obtained at three different temperatures was suggestive of the intermediate binding affinity between LAP and HSA. Thermodynamic analysis of the binding data (∆H=-9.75kJmol(-1) and ∆S=+65.21Jmol(-1)K(-1)) suggested involvement of both hydrophobic interactions and hydrogen bonding in LAP-HSA interaction, which were in line with the molecular docking results. LAP binding to HSA led to the secondary and the tertiary structural alterations in the protein as evident from the far-UV and the near-UV CD spectral analysis, respectively. Microenvironmental perturbation around Trp and Tyr residues in HSA upon LAP binding was confirmed from the three-dimensional fluorescence spectral results. LAP binding to HSA improved the thermal stability of the protein. LAP was found to bind preferentially to the site III in subdomain IB on HSA, as probed by the competitive drug displacement results and supported by the molecular docking results. The effect of metal ions on the binding constant between LAP and HSA was also investigated and the results showed a decrease in the binding constant in the presence of these metal ions. PMID:27128364

  15. GRASP: a novel heparin-binding serum glycoprotein that mediates oligodendrocyte-substratum adhesion.

    PubMed

    Schirmer, E C; Farooqui, J; Polak, P E; Szuchet, S

    1994-11-01

    Cell-substratum adhesion plays a crucial part in the cascade of events that control growth or turn on and consummate a differentiation program. We are investigating the molecular basis of oligodendrocyte (OLG) cytodifferentiation, employing pure cultures of OLGs isolated from postmyelination brains. We have shown that such OLGs will regenerate in vitro and reenact the ontogenic development of myelin, but to do so they need a signal. Adherence to a polylysine surface in the presence of 20% horse serum generates such a signal. Among the events that are turned on upon OLG adhesion is the phosphorylation of myelin basic protein; no such phosphorylation takes place in the non-adhered cell. We postulated that horse serum provides an adhesion molecule. Laminin, fibronectin, collagen and native vitronectin failed to replace horse serum. Hence, we set out to fractionate horse serum by screening with an adhesion assay. We report here the identification, purification and partial characterization of a novel, heparin-binding horse serum glycoprotein that we have termed Glycine-Rich Adhesion Serum Protein--GRASP--to stress the fact that this protein has a high content of glycine and functions, in vitro, as an adhesion molecule for OLGs. There is 61% similarity at the N-terminus between GRASP and histidine-rich glycoprotein precursor (HRGP), an alpha 2-glycoprotein from human plasma. However, our data suggest that GRASP is not the horse serum homolog of HRGP. First, the two Gps are functionally distinct: HRGP does not promote the adhesion of OLGs. Second, the amino acid compositions differ significantly, e.g., GRASP is not histidine- but rather glycine-rich. Third, the region of sequence similarity between GRASP and HRGP is conserved throughout the cystatin superfamily. Fourth, anti-Gp55 polyclonal Abs recognize a similar set of polypeptides--save for slight differences in M(r)-in human serum as in horse serum, indicating that HRGP and GRASP are two distinct but related proteins

  16. Protein D of Haemophilus influenzae is not a universal immunoglobulin D-binding protein.

    PubMed Central

    Sasaki, K; Munson, R S

    1993-01-01

    Haemophilus influenzae type b and nontypeable H. influenzae have been reported to bind human immunoglobulin D (IgD). IgD myeloma sera from five patients were tested for the ability of IgD to bind to H. influenzae. Serotype b strains bound human IgD in four of the five sera tested. IgD in the fifth serum bound strongly to type b strain MinnA but poorly to other type b strains. Additionally, IgD binding was not observed when nontypeable strains were tested. The gene for protein D, the putative IgD-binding protein, was cloned from the IgD-binding H. influenzae type b strain MinnA and expressed in Escherichia coli. IgD binding to E. coli expressing protein D was not demonstrable. Recombinant protein D was purified, and antisera were generated in rabbits. Using these rabbit sera, we detected protein D in nontypeable as well as serotype b strains by Western blotting (immunoblotting). In contrast, IgD myeloma protein 4490, which was previously reported to bind to protein D by Ruan and coworkers (M. Ruan, M. Akkoyunlu, A. Grubb, and A. Forsgren, J. Immunol. 145:3379-3384), bound strongly to both type b and nontypeable H. influenzae as well as to E. coli expressing protein D. Thus, IgD binding is a general property of H. influenzae type b strains but not a general property of nontypeable strains, although both type b and nontypeable strains produce protein D. With the exception of IgD myeloma protein 4490 binding, we have no evidence for a role of protein D in IgD binding to H. influenzae. Images PMID:8514409

  17. Alteration of methotrexate binding to human serum albumin induced by oxidative stress. Spectroscopic comparative study

    NASA Astrophysics Data System (ADS)

    Maciążek-Jurczyk, M.; Sułkowska, A.; Równicka-Zubik, J.

    2016-01-01

    Changes of oxidative modified albumin conformation by comparison of non-modified (HSA) and modified (oHSA) human serum albumin absorption spectra, Red Edge Excitation Shift (REES) effect and fluorescence synchronous spectra were investigated. Studies of absorption spectra indicated that changes in the value of absorbance associated with spectral changes in the region from 200 to 250 nm involve structural alterations related to variations in peptide backbone conformation. Analysis of the REES effect allowed for the observation of changes caused by oxidation in the region of the hydrophobic pocket containing the tryptophanyl residue. Synchronous fluorescence spectroscopy confirmed changes of the position of the tryptophanyl and tyrosil residues fluorescent band. Effect of oxidative stress on binding of methotrexate (MTX) was investigated by spectrofluorescence, UV-VIS and 1HNMR spectroscopy. MTX caused the fluorescence quenching of non-modified (HSA) and modified (oHSA) human serum albumin molecule. The values of binding constants, Hill's coefficients and a number of binding sites in the protein molecule in the high affinity binding site were calculated for the binary MTX-HSA and MTX-oHSA systems. For these systems, qualitative analysis in the low affinity binding sites was performed with the use of the 1HNMR technique.

  18. A Survey of Membrane Proteins in Human Serum

    PubMed Central

    Dung, Nguyen Tien; Van Chi, Phan

    2012-01-01

    Serum and membrane proteins are two of the most attractive targets for proteomic analysis. Previous membrane protein studies tend to focus on tissue sample, while membrane protein studies in serum are still limited. In this study, an analysis of membrane proteins in normal human serum was carried out. Nano-liquid chromatography-electrospray ionization mass spectrometry (NanoLC-ESI-MS/MS) and bioinformatics tools were used to identify membrane proteins. Two hundred and seventeen membrane proteins were detected in the human serum, of which 129 membrane proteins have at least one transmembrane domain (TMD). Further characterizations of identified membrane proteins including their subcellular distributions, molecular weights, post translational modifications, transmembrane domains and average of hydrophobicity, were also implemented. Our results showed the potential of membrane proteins in serum for diagnosis and treatment of diseases. PMID:25288886

  19. Partial characterization of a proacrosin binding protein.

    PubMed

    Yi, L S; Runion, C M; Willand, J L; Polakoski, K L

    1992-01-01

    All of the acid (pH 4.0) extracted proacrosin from porcine epididymal spermatozoa was found to be tightly associated with a specific protein referred to as the binding protein. A combination of gel filterations and gel electrophoresis revealed that the binding protein is composed of a major 28 kd and a minor 29 kd protein. Both of the proteins were shown to be nonproteolytic by gelatin SDS-PAGE analysis and the amino acid composition analysis of the purified 28 kd protein revealed that it is not related to the proteolytic component of the proacrosinacrosin system. PMID:1519775

  20. Binding of amifostine to human serum albumin: a biophysical study.

    PubMed

    Sun, Yifu; Wu, Han; Zhao, Guoqing; Shi, Ying

    2015-02-01

    The aim of this present work is to investigate the interaction between amifostine and human serum albumin (HSA) in simulated physiological conditions by spectroscopic methods to reveal potential toxic effects of the drug. The results reflected that amifostine caused fluorescence quenching of HSA through a static quenching process, which was further confirmed by the electrochemical experiments. The binding constants at 290, 297 and 304 K were obtained as 2.53 × 10(5) /M, 8.13 × 10(4) /M and 3.59 × 10(4) /M, respectively. There may be one binding site of amifostine on HSA. The thermodynamic parameters indicated that the interaction between amifostine and HSA was driven mainly by hydrogen bonding and electrostatic forces. Synchronous fluorescence spectra, circular dichroism and Fourier transform infrared spectroscopy results showed amifostine binding slightly changed the conformation of HSA with secondary structural content changes. Förster resonance energy transfer study revealed high possibility of energy transfer with amifostine-Trp-214 distance of 3.48 nm. The results of the present study may provide valuable information for studying the distribution, toxicological and pharmacological mechanisms of amifostine in vivo. PMID:24962599

  1. Mercury-binding proteins of Mytilus edulis

    SciTech Connect

    Roesijadi, G.; Morris, J.E.; Calabrese, A.

    1981-11-01

    Mytilus edulis possesses low molecular weight, mercury-binding proteins. The predominant protein isolated from gill tissue is enriched in cysteinyl residues (8%) and possesses an amino acid composition similar to cadmium-binding proteins of mussels and oysters. Continuous exposure of mussels to 5 ..mu..g/l mercury results in spillover of mercury from these proteins to high molecular weight proteins. Antibodies to these proteins have been isolated, and development of immunoassays is presently underway. Preliminary studies to determine whether exposure of adult mussels to mercury will result in induction of mercury-binding proteins in offspring suggest that such proteins occur in larvae although additional studies are indicated for a conclusive demonstration.

  2. Computational Prediction of RNA-Binding Proteins and Binding Sites

    PubMed Central

    Si, Jingna; Cui, Jing; Cheng, Jin; Wu, Rongling

    2015-01-01

    Proteins and RNA interaction have vital roles in many cellular processes such as protein synthesis, sequence encoding, RNA transfer, and gene regulation at the transcriptional and post-transcriptional levels. Approximately 6%–8% of all proteins are RNA-binding proteins (RBPs). Distinguishing these RBPs or their binding residues is a major aim of structural biology. Previously, a number of experimental methods were developed for the determination of protein–RNA interactions. However, these experimental methods are expensive, time-consuming, and labor-intensive. Alternatively, researchers have developed many computational approaches to predict RBPs and protein–RNA binding sites, by combining various machine learning methods and abundant sequence and/or structural features. There are three kinds of computational approaches, which are prediction from protein sequence, prediction from protein structure, and protein-RNA docking. In this paper, we review all existing studies of predictions of RNA-binding sites and RBPs and complexes, including data sets used in different approaches, sequence and structural features used in several predictors, prediction method classifications, performance comparisons, evaluation methods, and future directions. PMID:26540053

  3. Binding of Fatty Acid Amide Amphiphiles to Bovine Serum Albumin: Role of Amide Hydrogen Bonding.

    PubMed

    Ghosh, Subhajit; Dey, Joykrishna

    2015-06-25

    The study of protein-surfactant interactions is important because of the widespread use of surfactants in industry, medicine, and pharmaceutical fields. Sodium N-lauroylsarcosinate (SL-Sar) is a widely used surfactant in cosmetics, shampoos. In this paper, we studied the interactions of bovine serum albumin (BSA) with SL-Sar and sodium N-lauroylglycinate (SL-Gly) by use of a number of techniques, including fluorescence and circular dichroism spectroscopy and isothermal titration calorimetry. The binding strength of SL-Sar is stronger than that of structurally similar SL-Gly, which differs only by the absence of a methyl group in the amide nitrogen atom. Also, these two surfactants exhibit different binding patterns with the BSA protein. The role of the amide bond and hence the surfactant headgroup in the binding mechanism is discussed in this paper. It was observed that while SL-Sar destabilized, SL-Gly stabilized the protein structure, even at concentrations less than the critical micelle concentration (cmc) value. The thermodynamics of surfactant binding to BSA was studied by use of ITC. From the ITC results, it is concluded that three molecules of SL-Sar in contrast to only one molecule of SL-Gly bind to BSA in one set of binding sites at room temperature. However, on increasing temperature four molecules of SL-Gly bind to the BSA through H-bonding and van der Waals interactions, due to loosening of the BSA structure. In contrast, with SL-Sar the binding process is enthalpy driven, and very little structural change of BSA was observed at higher temperature. PMID:26023820

  4. Evaluation of the enantioselective binding of imazalil to human serum albumin by capillary electrophoresis.

    PubMed

    Asensi-Bernardi, Lucía; Martín-Biosca, Yolanda; Escuder-Gilabert, Laura; Sagrado, Salvador; Medina-Hernández, María José

    2015-11-01

    In this work, a methodology for the evaluation of enantioselective binding of imazalil (IMA) enantiomers to human serum albumin (HSA) that does not require the separation of free and bound to HSA fractions is developed. This methodology comprises the incubation of IMA-HSA designed mixtures for 30 min directly in the capillary electrophoresis system and the subsequent direct injection and chiral separation of IMA employing highly sulfated β-cyclodextrin as chiral selector and the complete filling technique. Two mathematical approaches were used to estimate apparent affinity constants (K1), protein binding and enantioselectivity (ES) for both enantiomers of IMA. Moderate enantioselective binding of IMA enantiomers to HSA (ES = 2.0) was shown by the 1:1 stoichiometry and log K1 values of 3.4 ± 0.4 and 3.1 ± 0.3 for the first and second eluted enantiomers, respectively. PMID:25857268

  5. The molecular architecture of protein-protein binding sites.

    PubMed

    Reichmann, Dana; Rahat, Ofer; Cohen, Mati; Neuvirth, Hani; Schreiber, Gideon

    2007-02-01

    The formation of specific protein interactions plays a crucial role in most, if not all, biological processes, including signal transduction, cell regulation, the immune response and others. Recent advances in our understanding of the molecular architecture of protein-protein binding sites, which facilitates such diversity in binding affinity and specificity, are enabling us to address key questions. What is the amino acid composition of binding sites? What are interface hotspots? How are binding sites organized? What are the differences between tight and weak interacting complexes? How does water contribute to binding? Can the knowledge gained be translated into protein design? And does a universal code for binding exist, or is it the architecture and chemistry of the interface that enable diverse but specific binding solutions? PMID:17239579

  6. CspA-Mediated Binding of Human Factor H Inhibits Complement Deposition and Confers Serum Resistance in Borrelia burgdorferi▿

    PubMed Central

    Kenedy, Melisha R.; Vuppala, Santosh R.; Siegel, Corinna; Kraiczy, Peter; Akins, Darrin R.

    2009-01-01

    Borrelia burgdorferi has developed efficient mechanisms for evading the innate immune response during mammalian infection and has been shown to be resistant to the complement-mediated bactericidal activity of human serum. It is well recognized that B. burgdorferi expresses multiple lipoproteins on its surface that bind the human complement inhibitors factor H and factor H-like protein 1 (FH/FHL-1). The binding of FH/FHL-1 on the surface of B. burgdorferi is thought to enhance its ability to evade serum-mediated killing during the acute phase of infection. One of the key B. burgdorferi FH/FHL-1 binding proteins identified thus far was designated CspA. While it is known that CspA binds FH/FHL-1, it is unclear how the interaction between CspA and FH/FHL-1 specifically enhances serum resistance. To better understand how CspA mediates serum resistance in B. burgdorferi, we inactivated cspA in a virulent strain of B. burgdorferi. An affinity ligand blot immunoassay and indirect immunofluorescence revealed that the CspA mutant does not efficiently bind human FH to its surface. Consistent with the lack of FH binding, the CspA mutant was also highly sensitive to killing by human serum. Additionally, the deposition of complement components C3, C6, and C5b-9 was enhanced on the surface of the CspA mutant compared to that of the wild-type strain. The combined data lead us to conclude that the CspA-mediated binding of human FH confers serum resistance by directly inhibiting complement deposition on the surface of B. burgdorferi. PMID:19451251

  7. Structure and Function of Lipopolysaccharide Binding Protein

    NASA Astrophysics Data System (ADS)

    Schumann, Ralf R.; Leong, Steven R.; Flaggs, Gail W.; Gray, Patrick W.; Wright, Samuel D.; Mathison, John C.; Tobias, Peter S.; Ulevitch, Richard J.

    1990-09-01

    The primary structure of lipopolysaccharide binding protein (LBP), a trace plasma protein that binds to the lipid A moiety of bacterial lipopolysaccharides (LPSs), was deduced by sequencing cloned complementary DNA. LBP shares sequence identity with another LPS binding protein found in granulocytes, bactericidal/permeability-increasing protein, and with cholesterol ester transport protein of the plasma. LBP may control the response to LPS under physiologic conditions by forming high-affinity complexes with LPS that bind to monocytes and macrophages, which then secrete tumor necrosis factor. The identification of this pathway for LPS-induced monocyte stimulation may aid in the development of treatments for diseases in which Gram-negative sepsis or endotoxemia are involved.

  8. Lamin-Binding Proteins in Caenorhabditis elegans.

    PubMed

    Dobrzynska, Agnieszka; Askjaer, Peter; Gruenbaum, Yosef

    2016-01-01

    The nuclear lamina, composed of lamins and numerous lamin-associated proteins, is required for mechanical stability, mechanosensing, chromatin organization, developmental gene regulation, mRNA transcription, DNA replication, nuclear assembly, and nuclear positioning. Mutations in lamins or lamin-binding proteins cause at least 18 distinct human diseases that affect specific tissues such as muscle, adipose, bone, nerve, or skin, and range from muscular dystrophies to lipodystrophy, peripheral neuropathy, or accelerated aging. Caenorhabditis elegans has unique advantages in studying lamin-binding proteins. These advantages include the low complexity of genes encoding lamin and lamin-binding proteins, advanced transgenic techniques, simple application of RNA interference, sophisticated genetic strategies, and a large collection of mutant lines. This chapter provides detailed and comprehensive protocols for the genetic and phenotypic analysis of lamin-binding proteins in C. elegans. PMID:26778571

  9. Species differences in the binding kinetics of 25-hydroxyvitamin D3 to vitamin D binding protein.

    PubMed

    Vieth, R; Kessler, M J; Pritzker, K P

    1990-10-01

    The specific binding of 25-hydroxyvitamin D3 to its binding protein was studied in serum of the human, rhesus monkey, cow, horse, and rat. The free fraction of 25-hydroxyvitamin D3 in the rat was 0.34 +/- 0.15 pmol free/nmol total (+/- SD) and this was lower than in any of the other species (p less than 0.01). In the human, the free fraction was 1.5 +/- 0.32 pmol free/nmol total, which was higher than in any of the other species (p less than 0.001). The differences in the free fraction were mainly due to differences in dissociation constant. The relative levels of free 25-hydroxyvitamin D should be taken into account when extrapolating findings about vitamin D metabolism in animals to the human. A technical outcome of this study is that of the species tested, vitamin D binding protein from rat serum is the most suitable as a reagent component for methods used to measure total 25-hydroxyvitamin D by competitive protein binding assay. PMID:2078829

  10. Understanding the physical and chemical nature of the warfarin drug binding site in human serum albumin: experimental and theoretical studies.

    PubMed

    Abou-Zied, Osama K

    2015-01-01

    Human serum albumin (HSA) is one of the major carrier proteins in the body and constitutes approximately half of the protein found in blood plasma. It plays an important role in lipid metabolism, and its ability to reversibly bind a large variety of pharmaceutical compounds makes it a crucial determinant of drug pharmacokinetics and pharmacodynamics. This review deals with one of the protein's major binding sites "Sudlow I" which includes a binding pocket for the drug warfarin (WAR). The binding nature of this important site can be characterized by measuring the spectroscopic changes when a ligand is bound. Using several drugs, including WAR, and other drug-like molecules as ligands, the results emphasize the nature of Sudlow I as a flexible binding site, capable of binding a variety of ligands by adapting its binding pockets. The high affinity of the WAR pocket for binding versatile molecular structures stems from the flexibility of the amino acids forming the pocket. The binding site is shown to have an ionization ability which is important to consider when using drugs that are known to bind in Sudlow I. Several studies point to the important role of water molecules trapped inside the binding site in molecular recognition and ligand binding. Water inside the protein's cavity is crucial in maintaining the balance between the hydrophobic and hydrophilic nature of the binding site. Upon the unfolding and refolding of HSA, more water molecules are trapped inside the binding site which cause some swelling that prevents a full recovery from the denatured state. Better understanding of the mechanism of binding in macromolecules such as HSA and other proteins can be achieved by combining experimental and theoretical studies which produce significant synergies in studying complex biochemical phenomena. PMID:25738490

  11. Cooperative binding of drugs on human serum albumin

    NASA Astrophysics Data System (ADS)

    Varela, L. M.; Pérez-Rodríguez, M.; García, M.

    In order to explain the adsorption isotherms of the amphiphilic penicillins nafcillin and cloxacillin onto human serum albumin (HSA), a cooperative multilayer adsorption model is introduced, combining the Brunauer-Emmet-Teller (BET) adsorption isotherm with an amphiphilic ionic adsorbate, whose chemical potential is derived from Guggenheim's theory. The non-cooperative model has been previously proved to qualitatively predict the measured adsorption maxima of these drugs [Varela, L. M., García, M., Pérez-Rodríguez, M., Taboada, P., Ruso, J. M., and Mosquera, V., 2001, J. chem. Phys., 114, 7682]. The surface interactions among adsorbed drug molecules are modelled in a mean-field fashion, so the chemical potential of the adsorbate is assumed to include a term proportional to the surface coverage, the constant of proportionality being the lateral interaction energy between bound molecules. The interaction energies obtained from the empirical binding isotherms are of the order of tenths of the thermal energy, therefore suggesting the principal role of van der Waals forces in the binding process.

  12. Binding of pro-migratory serum factors to electrospun PLLA nano-fibers.

    PubMed

    Eghtesad, Saman; Nurminskaya, Maria V

    2013-01-01

    Architecture of the poly(l-lactic acid) (PLLA) scaffolds is known to affect protein affinity and binding strength. Here, we demonstrate that nanofibrous electrospun PLLA scaffolds reversibly absorb the pro-migratory serum factors that stimulate migration of vascular smooth muscle via an NFkB-dependent mechanism. Further, we demonstrate that mesenchymal stem cells seeded on the PLLA scaffolds do not enhance muscle migration but may maintain the ability of induced cells to migrate in an NFkB-independent manner. These findings further support the promising application of PLLA scaffolds for therapeutic angiogenesis and vascular graft engineering. PMID:23905695

  13. Behind the scenes of vitamin D binding protein: more than vitamin D binding.

    PubMed

    Delanghe, Joris R; Speeckaert, Reinhart; Speeckaert, Marijn M

    2015-10-01

    Although being discovered in 1959, the number of published papers in recent years reveals that vitamin D binding protein (DBP), a member of the albuminoid superfamily, is a hot research topic. Besides the three major phenotypes (DBP1F, DBP1S and DBP2), more than 120 unique variants have been described of this polymorphic protein. The presence of DBP has been demonstrated in different body fluids (serum, urine, breast milk, ascitic fluid, cerebrospinal fluid, saliva and seminal fluid) and organs (brain, heart, lungs, kidneys, placenta, spleen, testes and uterus). Although the major function is binding, solubilization and transport of vitamin D and its metabolites, the name of this glycoprotein hides numerous other important biological functions. In this review, we will focus on the analytical aspects of the determination of DBP and discuss in detail the multifunctional capacity [actin scavenging, binding of fatty acids, chemotaxis, binding of endotoxins, influence on T cell response and influence of vitamin D binding protein-macrophage activating factor (DBP-MAF) on bone metabolism and cancer] of this abundant plasma protein. PMID:26522461

  14. Evolution of Protein Binding Modes in Homooligomers

    PubMed Central

    Dayhoff, Judith E.; Shoemaker, Benjamin A.; Bryant, Stephen H.; Panchenko, Anna R.

    2009-01-01

    The evolution of protein interactions cannot be deciphered without a detailed analysis of interaction interfaces and binding modes. We performed a large-scale study of protein homooligomers in terms of their symmetry, interface sizes, and conservation of binding modes. We also focused specifically on the evolution of protein binding modes from nine families of homooligomers and mapped 60 different binding modes and oligomerization states onto the phylogenetic trees of these families. We observed a significant tendency for the same binding modes to be clustered together and conserved within clades on phylogenetic trees; this trend is especially pronounced for close homologs with 70% sequence identity or higher. Some binding modes are conserved among very distant homologs, pointing to their ancient evolutionary origin, while others are very specific for a certain phylogenetic group. Moreover, we found that the most ancient binding modes have a tendency to involve symmetrical (isologous) homodimer binding arrangements with larger interfaces, while recently evolved binding modes more often exhibit asymmetrical arrangements and smaller interfaces. PMID:19879880

  15. Drug-drug plasma protein binding interactions of ivacaftor.

    PubMed

    Schneider, Elena K; Huang, Johnny X; Carbone, Vincenzo; Baker, Mark; Azad, Mohammad A K; Cooper, Matthew A; Li, Jian; Velkov, Tony

    2015-06-01

    Ivacaftor is a novel cystic fibrosis (CF) transmembrane conductance regulator (CFTR) potentiator that improves the pulmonary function for patients with CF bearing a G551D CFTR-protein mutation. Because ivacaftor is highly bound (>97%) to plasma proteins, there is the strong possibility that co-administered CF drugs may compete for the same plasma protein binding sites and impact the free drug concentration. This, in turn, could lead to drastic changes in the in vivo efficacy of ivacaftor and therapeutic outcomes. This biochemical study compares the binding affinity of ivacaftor and co-administered CF drugs for human serum albumin (HSA) and α1 -acid glycoprotein (AGP) using surface plasmon resonance and fluorimetric binding assays that measure the displacement of site-selective probes. Because of their ability to strongly compete for the ivacaftor binding sites on HSA and AGP, drug-drug interactions between ivacaftor are to be expected with ducosate, montelukast, ibuprofen, dicloxacillin, omeprazole, and loratadine. The significance of these plasma protein drug-drug interactions is also interpreted in terms of molecular docking simulations. This in vitro study provides valuable insights into the plasma protein drug-drug interactions of ivacaftor with co-administered CF drugs. The data may prove useful in future clinical trials for a staggered treatment that aims to maximize the effective free drug concentration and clinical efficacy of ivacaftor. PMID:25707701

  16. Novel 7-(dimethylamino)fluorene-based fluorescent probes and their binding to human serum albumin.

    PubMed

    Park, Kwanghee Koh; Park, Joon Woo; Hamilton, Andrew D

    2009-10-21

    A novel solvatochromic fluorescent molecule, 9,9-dibutyl-7-(dimethylamino)-2-fluorenesulfonate 2 was synthesized from 2-nitrofluorene in moderate yield. The fluorescence spectra of 2 and 7-(dimethylamino)-2-fluorenesulfonate 1 shift to shorter wavelengths as the polarity of the medium decreases. Both 1 and 2 bind to hydrophobic sites of human serum albumin (HSA). The apparent binding constants were determined by fluorescence titration to be 0.37 x 10(6) M(-1) for 1 and 2.2 x 10(6) M(-1) for 2. The energy of the Trp-214 fluorescence of HSA is transferred to the HSA-bound fluorophores with near 100% efficiency. The covalent bonding of acrylodan (AC) to Cys-34 has little effect on the binding affinity of 2 to HSA or fluorescent behavior of HSA-bound 2. Bound 2 also has little effect on the fluorescence of AC, but 2-->AC and Trp-214-->2-->AC resonance energy transfers were observed. Competitive binding between the fluorene compounds and other ligands such as 1-anilino-8-naphthalenesulfonate, aspirin, S-(+)-ibuprofen and phenylbutazone were also studied fluorometrically. The results indicated that the primary binding site of 2 to HSA is site II in domain IIIA, whereas 1 binds to site I in domain IIA, but a different region from the phenylbutazone binding site. Because of its large molar absorptivity, strong fluorescence, sensitivity to its environment, and high binding constant to HSA, 2 can be used successfully in the study of proteins and their binding properties. PMID:19795061

  17. Protein extracts from cultured cells contain nonspecific serum albumin.

    PubMed

    Miyara, Masatsugu; Umeda, Kanae; Ishida, Keishi; Sanoh, Seigo; Kotake, Yaichiro; Ohta, Shigeru

    2016-06-01

    Serum is an important component of cell culture media. The present study demonstrates contamination of intracellular protein extract by bovine serum albumin from the culture media and illustrates how this contamination can cause the misinterpretation of western blot results. Preliminary experiments can prevent the misinterpretation of some experimental results, and optimization of the washing process may enable specific protein detection. PMID:26967711

  18. Affinity purification of proteins binding to GST fusion proteins.

    PubMed

    Swaffield, J C; Johnston, S A

    2001-05-01

    This unit describes the use of proteins fused to glutathione-S-transferase (GST fusion proteins) to affinity purify other proteins, a technique also known as GST pulldown purification. The describes a strategy in which a GST fusion protein is bound to agarose affinity beads and the complex is then used to assay the binding of a specific test protein that has been labeled with [35S]methionine by in vitro translation. However, this method can be adapted for use with other types of fusion proteins; for example, His6, biotin tags, or maltose-binding protein fusions (MBP), and these may offer particular advantages. A describes preparation of an E. coli extract that is added to the reaction mixture with purified test protein to reduce nonspecific binding. PMID:18265191

  19. Serum reserve cholesterol binding capacity (SRCBC): the relative importance of different lipoprotein classes.

    PubMed

    Børresen, A L; Berg, K

    1981-01-01

    Findings by several authors have motivated studies in our laboratory on the relationship between HDL and "serum reserve cholesterol binding capacity" (SRCBC). We found that incubation for 25 hours at 37 degrees C provided optimal conditions for uptake and saturation when 14 mg sonicated and pulverized cholesterol was added to 1 ml serum. Upon separation of serum to which 14C labelled cholesterol had been added, into lipoprotein classes, a higher cpm:protein ratio than expected (from the free cholesterol:protein ratio, assuming isotope exchange exclusively) was found in LDL and the HDL fraction. In a second series of experiments, the different lipoprotein classes were separated prior to addition of 14C-cholesterol. The highest capacity for uptake of labelled cholesterol was found in the HDL fraction. The major part of the 14C-cholesterol appeared in the LDL density area when this HDL fraction was subjected to repeated ultracentrifugation. This suggested that the uptake of added cholesterol by HDL had led to the formation of an altered particle with density characteristics similar to those of LDL particles. This was confirmed by the demonstration of a "new" particle which contained apoA-I, apoA-II, and apoE, but no apoB or Lp(a) antigen. Its electrophoretic mobility was similar to that of HDL. Electron microscopy revealed that this particle is larger than normal HDL. The findings suggest that a sub-population of HDL molecules may be responsible for serum binding of added excess cholesterol. PMID:6794547

  20. A serum amyloid P-binding hydrogel speeds healing of partial thickness wounds in pigs

    PubMed Central

    Gomer, Richard H.; Pilling, Darrell; Kauvar, Lawrence M.; Ellsworth, Stote; Ronkainen, Sanna D.; Roife, David; Davis, Stephen C.

    2010-01-01

    During wound healing, some circulating monocytes enter the wound, differentiate into fibroblast-like cells called fibrocytes, and appear to then further differentiate into myofibroblasts, cells that play a key role in collagen deposition, cytokine release, and wound contraction. The differentiation of monocytes into fibrocytes is inhibited by the serum protein serum amyloid P (SAP). Depleting SAP at a wound site thus might speed wound healing. SAP binds to some types of agarose in the presence of Ca2+. We found that human SAP binds to an agarose with a KD of 7×10−8M and a Bmax of 2.1 μg SAP/mg wet weight agarose. Mixing this agarose 1: 5 w/v with 30 μg/mL human SAP (the average SAP concentration in normal serum) in a buffer containing 2mM Ca2+ reduced the free SAP concentration to ~0.02 μg/mL, well below the concentration that inhibits fibrocyte differentiation. Compared with a hydrogel dressing and a foam dressing, dressings containing this agarose and Ca2+ significantly increased the speed of wound healing in partial thickness wounds in pigs. This suggests that agarose/Ca2+ dressings may be beneficial for wound healing in humans. PMID:19660048

  1. Localization of cellular retinol-binding protein and retinol-binding protein in cells comprising the blood-brain barrier of rat and human

    SciTech Connect

    MacDonald, P.N.; Ong, D.E. ); Bok, D. )

    1990-06-01

    Brain is not generally recognized as an organ that requires vitamin A, perhaps because no obvious histologic lesions have been observed in severely vitamin A-deficient animals. However, brain tissue does contain cellular vitamin A-binding proteins and a nuclear receptor protein for retinoic acid. In the present study, immunohistochemical techniques were used to determine the cell-specific location of cellular retinol-binding protein in human and rat brain tissue. Cellular retinol-binding protein was localized specifically within the cuboidal epithelial cells of the choroid plexus, two primary sites of the mammalian blood-brain barrier. In addition, autoradiographic procedures demonstrated binding sites for serum retinol-binding protein in the choroidal epithelium. These observations suggest that a significant movement of retinol across the blood-brain barrier may occur.

  2. Localization of Cellular Retinol-Binding Protein and Retinol-Binding Protein in Cells Comprising the Blood-Brain Barrier of Rat and Human

    NASA Astrophysics Data System (ADS)

    MacDonald, Paul N.; Bok, Dean; Ong, David E.

    1990-06-01

    Brain is not generally recognized as an organ that requiries vitamin A, perhaps because no obvious histologic lesions have been observed in severely vitamin A-deficient animals. However, brain tissue does contain cellular vitamin A-binding proteins and a nuclear receptor protein for retinoic acid. In the present study, immunohistochemical techniques were used to determine the cell-specific location of cellular retinol-binding protein in human and rat brain tissue. Cellular retinol-binding protein was localized specifically within the endothelial cells of the brain microvasculature and within the cuboidal epithelial cells of the choroid plexus, two primary sites of the mammalian blood-brain barrier. In addition, autoradiographic procedures demonstrated binding sites for serum retinol-binding protein in the choroidal epithelium. These observations suggest that a significant movement of retinol across the blood-brain barrier may occur.

  3. Copper binding in the prion protein.

    PubMed

    Millhauser, Glenn L

    2004-02-01

    A conformational change of the prion protein is responsible for a class of neurodegenerative diseases called the transmissible spongiform encephalopathies that include mad cow disease and the human afflictions kuru and Creutzfeldt-Jakob disease. Despite the attention given to these diseases, the normal function of the prion protein in healthy tissue is unknown. Research over the past few years, however, demonstrates that the prion protein is a copper binding protein with high selectivity for Cu(2+). The structural features of the Cu(2+) binding sites have now been characterized and are providing important clues about the normal function of the prion protein and perhaps how metals or loss of protein function play a role in disease. The link between prion protein and copper may provide insight into the general, and recently appreciated, role of metals in neurodegenerative disease. PMID:14967054

  4. Radioimmunoassay of beta lipoprotein—protein of rat serum

    PubMed Central

    Eaton, R. Philip; Kipnis, David M.

    1969-01-01

    A double antibody radioimmunoassay for rat serum beta lipoprotein-protein (beta Lp-protein) is described. The protein was purified by ultracentrifugation, selective heparin-manganous precipitation, and gel filtration on Sephadex G-200. Antiserum was prepared in rabbits by biweekly immunization and absorbed with nonbeta lipoprotein containing rat serum. Iodination with 125I and purification by gel filtration provided a radiolabeled protein which was > 98% displaced by purified beta lipoprotein in the immunoassay. The radioimmunoassay was sensitive to beta Lp-protein concentrations from 0.1 to 1.5 μg. Specificity of the immunoassay for beta Lp-protein was established by comparison of the displacement curves obtained with serum very low density lipoprotein (VLDL), low density lipoprotein (LDL), high density lipoprotein (HDL), and density (d) > 1.21 fractions and with the beta and alpha migrating lipoproteins eluted from paper electrophoretograms. Suitability of the assay for measuring beta Lp-protein in serum was established by demonstrating 100% recovery of beta lipoprotein added to whole serum and by the absence of immunoreactive beta Lp-protein in serum of orotic acid-treated rats. Examination of sera from six other vertebrates species revealed partial cross-reactivity. Normal rat serum was found to contain 0.25±0.01 mg/ml of beta Lp-protein and hepatic production by an isolated perfused rat liver system was determined as 0.145 mg/hr. Images PMID:4978731

  5. 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

  6. Assessment of Binding Affinity between Drugs and Human Serum Albumin Using Nanoporous Anodic Alumina Photonic Crystals.

    PubMed

    Nemati, Mahdieh; Santos, Abel; Law, Cheryl Suwen; Losic, Dusan

    2016-06-01

    In this study, we report an innovative approach aiming to assess the binding affinity between drug molecules and human serum albumin by combining nanoporous anodic alumina rugate filters (NAA-RFs) modified with human serum albumin (HSA) and reflectometric interference spectroscopy (RIfS). NAA-RFs are photonic crystal structures produced by sinusoidal pulse anodization of aluminum that present two characteristic optical parameters, the characteristic reflection peak (λPeak), and the effective optical thickness of the film (OTeff), which can be readily used as sensing parameters. A design of experiments strategy and an ANOVA analysis are used to establish the effect of the anodization parameters (i.e., anodization period and anodization offset) on the sensitivity of HSA-modified NAA-RFs toward indomethacin, a model drug. To this end, two sensing parameters are used, that is, shifts in the characteristic reflection peak (ΔλPeak) and changes in the effective optical thickness of the film (ΔOTeff). Subsequently, optimized NAA-RFs are used as sensing platforms to determine the binding affinity between a set of drugs (i.e., indomethacin, coumarin, sulfadymethoxine, warfarin, and salicylic acid) and HSA molecules. Our results verify that the combination of HSA-modified NAA-RFs with RIfS can be used as a portable, low-cost, and simple system for establishing the binding affinity between drugs and plasma proteins, which is a critical factor to develop efficient medicines for treating a broad range of diseases and medical conditions. PMID:27128744

  7. Folding funnels, binding funnels, and protein function.

    PubMed Central

    Tsai, C. J.; Kumar, S.; Ma, B.; Nussinov, R.

    1999-01-01

    Folding funnels have been the focus of considerable attention during the last few years. These have mostly been discussed in the general context of the theory of protein folding. Here we extend the utility of the concept of folding funnels, relating them to biological mechanisms and function. In particular, here we describe the shape of the funnels in light of protein synthesis and folding; flexibility, conformational diversity, and binding mechanisms; and the associated binding funnels, illustrating the multiple routes and the range of complexed conformers. Specifically, the walls of the folding funnels, their crevices, and bumps are related to the complexity of protein folding, and hence to sequential vs. nonsequential folding. Whereas the former is more frequently observed in eukaryotic proteins, where the rate of protein synthesis is slower, the latter is more frequent in prokaryotes, with faster translation rates. The bottoms of the funnels reflect the extent of the flexibility of the proteins. Rugged floors imply a range of conformational isomers, which may be close on the energy landscape. Rather than undergoing an induced fit binding mechanism, the conformational ensembles around the rugged bottoms argue that the conformers, which are most complementary to the ligand, will bind to it with the equilibrium shifting in their favor. Furthermore, depending on the extent of the ruggedness, or of the smoothness with only a few minima, we may infer nonspecific, broad range vs. specific binding. In particular, folding and binding are similar processes, with similar underlying principles. Hence, the shape of the folding funnel of the monomer enables making reasonable guesses regarding the shape of the corresponding binding funnel. Proteins having a broad range of binding, such as proteolytic enzymes or relatively nonspecific endonucleases, may be expected to have not only rugged floors in their folding funnels, but their binding funnels will also behave similarly

  8. Probing thyroglobulin in undiluted human serum based on pattern recognition and competitive adsorption of proteins

    NASA Astrophysics Data System (ADS)

    Wang, Ran; Huang, Shuai; Li, Jing; Chae, Junseok

    2014-10-01

    Thyroglobulin (Tg) is a sensitive indicator of persistent or recurrent differentiated thyroid cancer of follicular cell origin. Detection of Tg in human serum is challenging as bio-receptors, such as anti-Tg, used in immunoassay have relatively weak binding affinity. We engineer sensing surfaces using the competitive adsorption of proteins, termed the Vroman Effect. Coupled with Surface Plasmon Resonance, the "cross-responsive" interactions of Tg on the engineered surfaces produce uniquely distinguishable multiple signature patterns, which are discriminated using Linear Discriminant Analysis. Tg-spiked samples, down to 2 ng/ml Tg in undiluted human serum, are sensitively and selectively discriminated from the control (undiluted human serum).

  9. Binding of ascorbic acid and α-tocopherol to bovine serum albumin: a comparative study.

    PubMed

    Li, Xiangrong; Wang, Gongke; Chen, Dejun; Lu, Yan

    2014-02-01

    Binding of ascorbic acid (water-soluble antioxidant) and α-tocopherol (lipid-soluble antioxidant) to bovine serum albumin (BSA) has been studied using isothermal titration calorimetry (ITC), in combination with fluorescence spectroscopy, UV-vis absorption spectroscopy and Fourier transform infrared (FT-IR) spectroscopy. Thermodynamic investigations reveal that ascorbic acid/α-tocopherol binding to BSA is driven by favorable enthalpy and unfavorable entropy, and the major driving forces are hydrogen bonding and van der Waals forces. For ascorbic acid, the interaction is characterized by a high number of binding sites, which suggests that binding occurs by a surface adsorption mechanism that leads to coating of the protein surface. For α-tocopherol, one molecule of α-tocopherol combines with one molecule of BSA and no more α-tocopherol binding to BSA occurs at concentration ranges used in this study. Fluorescence experiments suggest that ascorbic acid has predominantly a "sphere of action" quenching mechanism, whereas, for α-tocopherol, the quenching mechanism is "static quenching" and due to the formation of a ground state complex. Additionally, as shown by the UV-vis absorption, synchronous fluorescence spectroscopy, and FT-IR, ascorbic acid and α-tocopherol may induce conformational and microenvironmental changes of BSA. PMID:24310979

  10. Maltose-Binding Protein (MBP), a Secretion-Enhancing Tag for Mammalian Protein Expression Systems

    PubMed Central

    Reuten, Raphael; Nikodemus, Denise; Oliveira, Maria B.; Patel, Trushar R.; Brachvogel, Bent; Breloy, Isabelle; Stetefeld, Jörg; Koch, Manuel

    2016-01-01

    Recombinant proteins are commonly expressed in eukaryotic expression systems to ensure the formation of disulfide bridges and proper glycosylation. Although many proteins can be expressed easily, some proteins, sub-domains, and mutant protein versions can cause problems. Here, we investigated expression levels of recombinant extracellular, intracellular as well as transmembrane proteins tethered to different polypeptides in mammalian cell lines. Strikingly, fusion of proteins to the prokaryotic maltose-binding protein (MBP) generally enhanced protein production. MBP fusion proteins consistently exhibited the most robust increase in protein production in comparison to commonly used tags, e.g., the Fc, Glutathione S-transferase (GST), SlyD, and serum albumin (ser alb) tag. Moreover, proteins tethered to MBP revealed reduced numbers of dying cells upon transient transfection. In contrast to the Fc tag, MBP is a stable monomer and does not promote protein aggregation. Therefore, the MBP tag does not induce artificial dimerization of tethered proteins and provides a beneficial fusion tag for binding as well as cell adhesion studies. Using MBP we were able to secret a disease causing laminin β2 mutant protein (congenital nephrotic syndrome), which is normally retained in the endoplasmic reticulum. In summary, this study establishes MBP as a versatile expression tag for protein production in eukaryotic expression systems. PMID:27029048

  11. Maltose-Binding Protein (MBP), a Secretion-Enhancing Tag for Mammalian Protein Expression Systems.

    PubMed

    Reuten, Raphael; Nikodemus, Denise; Oliveira, Maria B; Patel, Trushar R; Brachvogel, Bent; Breloy, Isabelle; Stetefeld, Jörg; Koch, Manuel

    2016-01-01

    Recombinant proteins are commonly expressed in eukaryotic expression systems to ensure the formation of disulfide bridges and proper glycosylation. Although many proteins can be expressed easily, some proteins, sub-domains, and mutant protein versions can cause problems. Here, we investigated expression levels of recombinant extracellular, intracellular as well as transmembrane proteins tethered to different polypeptides in mammalian cell lines. Strikingly, fusion of proteins to the prokaryotic maltose-binding protein (MBP) generally enhanced protein production. MBP fusion proteins consistently exhibited the most robust increase in protein production in comparison to commonly used tags, e.g., the Fc, Glutathione S-transferase (GST), SlyD, and serum albumin (ser alb) tag. Moreover, proteins tethered to MBP revealed reduced numbers of dying cells upon transient transfection. In contrast to the Fc tag, MBP is a stable monomer and does not promote protein aggregation. Therefore, the MBP tag does not induce artificial dimerization of tethered proteins and provides a beneficial fusion tag for binding as well as cell adhesion studies. Using MBP we were able to secret a disease causing laminin β2 mutant protein (congenital nephrotic syndrome), which is normally retained in the endoplasmic reticulum. In summary, this study establishes MBP as a versatile expression tag for protein production in eukaryotic expression systems. PMID:27029048

  12. Determination of human serum alpha1-acid glycoprotein and albumin binding of various marketed and preclinical kinase inhibitors.

    PubMed

    Zsila, Ferenc; Fitos, Ilona; Bencze, Gyula; Kéri, György; Orfi, László

    2009-01-01

    There are about 380 protein kinase inhibitors in drug development as of today and 15 drugs have been marketed already for the treatment of cancer. This time 139 validated kinase targets are in the focus of drug research of pharmaceutical companies and big efforts are made for the development of new, druglike kinase inhibitors. Plasma protein binding is an important factor of the ADME profiling of a drug compound. Human serum albumin (HSA) and alpha(1)-acid glycoprotein (AAG) are the most relevant drug carriers in blood plasma. Since previous literature data indicated that AAG is the principal plasma binding component of some kinase inhibitors the present work focuses on the comprehensive evaluation of AAG binding of a series of marketed and experimental kinase inhibitors by using circular dichroism (CD) spectroscopy approach. HSA binding was also evaluated by affinity chromatography. Protein binding interactions of twenty-six kinase inhibitors are characterized. The contribution of AAG and HSA binding data to the pharmacokinetic profiles of the investigated therapeutic agents is discussed. Structural, biological and drug binding properties of AAG as well as the applicability of the CD method in studying drug-protein binding interactions are also briefly reviewed. PMID:19519376

  13. Interpretation of Serum Calcium in Patients with Abnormal Serum Proteins

    PubMed Central

    Payne, R. B.; Little, A. J.; Williams, R. B.; Milner, J. R.

    1973-01-01

    Two hundred consecutive specimens received in this laboratory for “liver function tests” showed a wide range of abnormal protein concentrations. Calcium concentration correlated closely with albumin (r = 0·867) but less closely with total protein (r = 0·682). A simple formula for adjusting calcium concentration was derived from the regression equation of calcium on albumin. Adjusted calcium = calcium - albumin + 4·0, where calcium is in mg/100 ml and albumin in g/100 ml. Low calcium concentrations were found in 49 (24·5%) and raised concentrations in six (3%) of the 200 blood specimens taken for liver function tests. After adjustment, the 95% limits of the observed range were identical with the 95% limits of the normal range determined in this laboratory. Unlike adjustments based on total protein or specific gravity, the adjustment on albumin in 39 specimens which showed hypergammaglobulinaemia on electrophoresis gave normal calcium concentrations. PMID:4758544

  14. Synthetic cationic antimicrobial peptides bind with their hydrophobic parts to drug site II of human serum albumin

    PubMed Central

    2014-01-01

    Background Many biologically active compounds bind to plasma transport proteins, and this binding can be either advantageous or disadvantageous from a drug design perspective. Human serum albumin (HSA) is one of the most important transport proteins in the cardiovascular system due to its great binding capacity and high physiological concentration. HSA has a preference for accommodating neutral lipophilic and acidic drug-like ligands, but is also surprisingly able to bind positively charged peptides. Understanding of how short cationic antimicrobial peptides interact with human serum albumin is of importance for developing such compounds into the clinics. Results The binding of a selection of short synthetic cationic antimicrobial peptides (CAPs) to human albumin with binding affinities in the μM range is described. Competitive isothermal titration calorimetry (ITC) and NMR WaterLOGSY experiments mapped the binding site of the CAPs to the well-known drug site II within subdomain IIIA of HSA. Thermodynamic and structural analysis revealed that the binding is exclusively driven by interactions with the hydrophobic moieties of the peptides, and is independent of the cationic residues that are vital for antimicrobial activity. Both of the hydrophobic moieties comprising the peptides were detected to interact with drug site II by NMR saturation transfer difference (STD) group epitope mapping (GEM) and INPHARMA experiments. Molecular models of the complexes between the peptides and albumin were constructed using docking experiments, and support the binding hypothesis and confirm the overall binding affinities of the CAPs. Conclusions The biophysical and structural characterizations of albumin-peptide complexes reported here provide detailed insight into how albumin can bind short cationic peptides. The hydrophobic elements of the peptides studied here are responsible for the main interaction with HSA. We suggest that albumin binding should be taken into careful

  15. Membrane folate-binding proteins are responsible for folate-protein conjugate endocytosis into cultured cells.

    PubMed Central

    Leamon, C P; Low, P S

    1993-01-01

    Folate-protein conjugates have been shown to bind to and enter HeLa and KB cells by receptor-mediated endocytosis [Leamon and Low (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 5572-5576]. Although these cells contain a membrane folate-binding protein (FBP) involved in the uptake of free folate, no studies have been conducted to evaluate whether the folate-protein conjugates enter cells via the same protein. To address this issue, HeLa cell monolayers were treated with folate-labelled 125I-RNAase under various conditions characteristic of FBP-mediated folate uptake. Folate-labelled 125I-RNAase was found to bind to cells with high affinity (Kd = 24 nM), and like the free vitamin, its binding could be competitively blocked by excess free folate. Furthermore, binding could be reversed by either washing the cells with acid/saline, pH 3.0, or by treating the cells with phosphatidyl-inositol-specific phospholipase C, an enzyme known to release FBP from cell surfaces. Because cells pretreated with anti-FBP serum were unable to bind folate conjugates, and since the same antiserum identified a single 65 kDa band reminiscent of FBPs found in many other tissues, we conclude that a classical FBP is responsible for the uptake of folate-protein conjugates by receptor-bearing cells. Images Figure 5 PMID:8387781

  16. Serum lipids and proteins during treatment with a new oral contraceptive combination containing desogestrel.

    PubMed

    Penttilä, I M; Bergink, E W; Holma, P; Hulkko, S; Makkonen, M; Pyörälä, T; Castrén, O

    1983-12-01

    The present study was carried out to measure lipid and protein levels in serum of healthy women during treatment with a new oral contraceptive combination containing 0.075 mg desogestrel (Org 2969, 17 alpha-ethinyl-18-methyl-11-methylene-4-estren-17-ol) plus 0.050 mg ethinyloestradiol per tablet. All 30 volunteers took 1 tablet daily for 21 consecutive days, followed by a tablet-free period of 7 days. Treatment lasted 3 months. At the end of treatment serum total cholesterol had increased by 0.26 mmol/l (5.0%), high-density lipoprotein-cholesterol by 0.22 mmol/l (15.2%) and triglycerides by 0.43 mmol/l (50%); the calculated low-density lipoprotein cholesterol had decreased by 0.16 mmol/l (4.9%). All lipid concentrations had returned to initial levels 2 months after treatment stopped. After 3 months treatment serum ceruloplasmin, cortisol-binding globulin capacity, sex-hormone-binding globulin capacity and thyroxine-binding globulin had significantly increased by 85.2, 133, 206 and 101%, respectively. All protein levels returned to normal 2 months after treatment stopped. The relationship between serum lipids and hormone-binding proteins has been discussed, as well as the significance of the high-density lipoprotein level with regard to contraceptive treatment. PMID:6232161

  17. Binding of an anticancer drug, axitinib to human serum albumin: Fluorescence quenching and molecular docking study.

    PubMed

    Tayyab, Saad; Izzudin, Mohamad Mirza; Kabir, Md Zahirul; Feroz, Shevin R; Tee, Wei-Ven; Mohamad, Saharuddin B; Alias, Zazali

    2016-09-01

    Binding characteristics of a promising anticancer drug, axitinib (AXT) to human serum albumin (HSA), the major transport protein in human blood circulation, were studied using fluorescence, UV-vis absorption and circular dichroism (CD) spectroscopy as well as molecular docking analysis. A gradual decrease in the Stern-Volmer quenching constant with increasing temperature revealed the static mode of the protein fluorescence quenching upon AXT addition, thus confirmed AXT-HSA complex formation. This was also confirmed from alteration in the UV-vis spectrum of HSA upon AXT addition. Fluorescence quenching titration results demonstrated moderately strong binding affinity between AXT and HSA based on the binding constant value (1.08±0.06×10(5)M(-1)), obtained in 10mM sodium phosphate buffer, pH7.4 at 25°C. The sign and magnitude of the enthalpy change (∆H=-8.38kJmol(-1)) as well as the entropy change (∆S=+68.21Jmol(-1)K(-1)) clearly suggested involvement of both hydrophobic interactions and hydrogen bonding in AXT-HSA complex formation. These results were well supported by molecular docking results. Three-dimensional fluorescence spectral results indicated significant microenvironmental changes around Trp and Tyr residues of HSA upon complexation with AXT. AXT binding to the protein produced significant alterations in both secondary and tertiary structures of HSA, as revealed from the far-UV and the near-UV CD spectral results. Competitive drug displacement results obtained with phenylbutazone (site I marker), ketoprofen (site II marker) and hemin (site III marker) along with molecular docking results suggested Sudlow's site I, located in subdomain IIA of HSA, as the preferred binding site of AXT. PMID:27424099

  18. Binding of hydroxyquinoline probes to human serum albumin: combining molecular modeling and Förster's resonance energy transfer spectroscopy to understand flexible ligand binding.

    PubMed

    Abou-Zied, Osama K; Al-Lawatia, Najla; Elstner, Marcus; Steinbrecher, Thomas B

    2013-01-31

    Human serum albumin (HSA) is the most abundant protein in blood plasma. It has high relevance for the lipid metabolism, and its ability to bind a large variety of natural and pharmaceutical compounds makes it a crucial determinant of drug pharmaco-kinetics and -dynamics. The drug binding properties of HSA can be characterized by spectroscopic analysis of bound probe molecules. We have recently characterized the subdomain IIA binding site of HSA using three hydroxyquinoline derivatives. In this work, we extend our study by combining data from energy transfer experiments, ligand docking, and long molecular dynamics (MD) simulations. Multiple possible binding locations are found within the subdomain IIA site, and their solvent accessibility and interactions with ligands are analyzed in detail. Binding pockets appear well hydrated during simulations, with ligands in direct contact to water molecules at all times. Binding free energies in good agreement to experiment are calculated. The HSA apoprotein is found to exhibit significant conformational flexibility over 250 ns of simulation time, but individual domains remain structurally stable. Two rotamers of Trp214 were observed on a time scale longer than 50 ns in the MD simulations, supporting the experimental observation of two fluorescence lifetime components. The flexible protein structure and heterogeneous nature of its binding sites explain the ability of HSA to act as a versatile molecular transporter. The combination of experimental and computational molecular distance information allows the conclusion that hydroxyquinoline probes bind in a binding mode similar to the anticoagulant drug warfarin. PMID:23297700

  19. Astaxanthin binding protein in Atlantic salmon.

    PubMed

    Matthews, Sarah J; Ross, Neil W; Lall, Santosh P; Gill, Tom A

    2006-06-01

    The rubicund pigmentation in salmon and trout flesh is unique and is due to the deposition of dietary carotenoids, astaxanthin and canthaxanthin in the muscle. The present study was undertaken to determine which protein was responsible for pigment binding. Salmon muscle proteins were solubilized by sequential extractions with non-denaturing, low ionic strength aqueous solutions and segregated as such into six different fractions. Approximately 91% of the salmon myofibrillar proteins were solubilized under non-denaturing conditions using a protocol modified from a method described by Krishnamurthy et al. [Krishnamurthy, G., Chang, H.S., Hultin, H.O., Feng, Y., Srinivasan, S., Kelleher. S.D., 1996. Solubility of chicken breast muscle proteins in solutions of low ionic strength. J. Agric. Food Chem. 44: 408-415.] for the dissolution of avian muscle. To our knowledge, this is the first time this solubilization approach has been applied to the study of molecular interactions in myofibrillar proteins. Astaxanthin binding in each fraction was determined using an in vitro binding assay. In addition, SDS-PAGE and quantitative densitometry were used to separate and determine the relative amounts of each of the proteins in the six fractions. The results showed that alpha-actinin was the only myofibrillar protein correlating significantly (P<0.05) with astaxanthin binding. Alpha-actinin was positively identified using electrophoretic techniques and confirmed by tandem mass spectroscopy. Purified salmon alpha-actinin bound synthetic astaxanthin in a molar ratio of 1.11:1.00. The study was repeated using halibut alpha-actinin, which was found to have a molar binding ratio of astaxanthin to alpha-actinin of 0.893:1. These results suggest that the difference in pigmentation between white fish and Atlantic salmon is not due to binding capacity in the muscle, but rather differences in the metabolism or transport of pigment. PMID:16644255

  20. Five recombinant fragments of human serum albumin-tools for the characterization of the warfarin binding site.

    PubMed Central

    Dockal, M.; Chang, M.; Carter, D. C.; Rüker, F.

    2000-01-01

    Human serum albumin (HSA) interacts with a vast array of chemically diverse ligands at specific binding sites. To pinpoint the essential structural elements for the formation of the warfarin binding site on human serum albumin, a defined set of five recombinant proteins comprising combinations of domains and/or subdomains of the N-terminal part were prepared and characterized by biochemical standard procedures, tryptophanyl fluorescence, and circular dichroic measurements, indicating well-preserved secondary and tertiary structures. Affinity constants for binding to warfarin were estimated by fluorescence titration experiments and found to be highest for HSA-DOM I-II and HSA, followed by HSA-DOM IB-II, HSA-DOM II, and HSA-DOM I-IIA. In addition, ultraviolet difference spectroscopy and induced circular dichroism experiments were carried out to get an in depth understanding of the binding mechanism of warfarin to the fragments as stand-alone proteins. This systematic study indicates that the primary warfarin binding site is centered in subdomain IIA with indispensable structural contributions of subdomain IIB and domain I, while domain III is not involved in this binding site, underlining the great potential that lies in the use of combinations of recombinant fragments for the study and accurate localization of ligand binding sites on HSA. PMID:10975567

  1. Ancestral Protein Reconstruction Yields Insights into Adaptive Evolution of Binding Specificity in Solute-Binding Proteins.

    PubMed

    Clifton, Ben E; Jackson, Colin J

    2016-02-18

    The promiscuous functions of proteins are an important reservoir of functional novelty in protein evolution, but the molecular basis for binding promiscuity remains elusive. We used ancestral protein reconstruction to experimentally characterize evolutionary intermediates in the functional expansion of the polar amino acid-binding protein family, which has evolved to bind a variety of amino acids with high affinity and specificity. High-resolution crystal structures of an ancestral arginine-binding protein in complex with l-arginine and l-glutamine show that the promiscuous binding of l-glutamine is enabled by multi-scale conformational plasticity, water-mediated interactions, and selection of an alternative conformational substate productive for l-glutamine binding. Evolution of specialized glutamine-binding proteins from this ancestral protein was achieved by displacement of water molecules from the protein-ligand interface, reducing the entropic penalty associated with the promiscuous interaction. These results provide a structural and thermodynamic basis for the co-option of a promiscuous interaction in the evolution of binding specificity. PMID:26853627

  2. Clinical impact of serum proteins on drug delivery.

    PubMed

    Kratz, Felix; Elsadek, Bakheet

    2012-07-20

    Among serum proteins albumin and transferrin have attracted the most interest as drug carriers in the past two decades. Prior to that, their potential use was overshadowed by the advent of monoclonal antibodies that was initiated by Milstein and Koehler in 1975. Meanwhile intensive pursuit of exploiting transferrin, but above all albumin as an exogenous or endogenous carrier protein for treating various diseases, primarily cancer, rheumatoid arthritis, diabetes and hepatitis has resulted in several marketed products and numerous clinical trials. While the use of transferrin has clinically been primarily restricted to immunotoxins, albumin-based drug delivery systems ranging from albumin drug nanoparticles, albumin fusion protein, prodrugs and peptide derivatives that bind covalently to albumin as well as physically binding antibody fragments and therapeutically active peptides are in advanced clinical trials or approved products. For treating diabetes, Levemir and Victoza that are myristic acid derivatives of human insulin or glucagon-like peptide 1 (GLP-1) act as long-acting peptides by binding to the fatty acid binding sites on circulating albumin to control glucose levels. Levemir from Novo Nordisk has already developed into a blockbuster since its market approval in 2004. Abraxane, an albumin paclitaxel nanoparticle as a water-soluble galenic formulation avoiding the use of cremophor/ethanol, transports paclitaxel through passive targeting as an albumin paclitaxel complex to the tumor site and is superior to conventional Taxol against metastatic breast cancer. INNO-206, an albumin-binding doxorubicin prodrug that also accumulates in solid tumors due to the enhanced permeability and retention (EPR) effect but releases the parent drug through acid cleavage, either intra- or extracellularly, is entering phase II studies against sarcoma. An expanding field is the use of albumin-binding antibody moieties which do not contain the fragment crystallizable (Fc) portion

  3. Aspects of Protein, Chemistry, Part II: Oxygen-Binding Proteins

    ERIC Educational Resources Information Center

    Nixon, J. E.

    1977-01-01

    Compares differences in function and behavior of two oxygen-binding proteins, myoglobin found in muscle and hemoglobin found in blood. Describes the mechanism of oxygen-binding and allosteric effect in hemoglobin; also describes the effect of pH on the affinity of hemoglobin for oxygen. (CS)

  4. Characterization of insulin-like growth factor-binding proteins from sheep thyroid cells.

    PubMed

    Bachrach, L K; Liu, F R; Burrow, G N; Eggo, M C

    1989-12-01

    The insulin-like growth factors (IGFs) are bound by specific, high affinity binding proteins. Distinct classes of IGF-binding proteins have been described in human serum, amniotic fluid, cerebrospinal fluid, and conditioned medium from cultured cells. Sheep thyroid cells produce IGF-binding proteins under hormonal regulation. Cells grown without or with standard medium supplements (transferrin, glycyl-histidyl-lysine, hydrocortisone, somatostatin, insulin, and TSH) released binding proteins with apparent mol wt of 23, 29, and 32 kDa on Western ligand blot (nonreduced). Binding proteins from these cells appeared as 21, 26, 34, 36, and 41 kDa bands when cross-linked to [125I]IGF-I under reducing conditions. The addition of epidermal growth factor (EGF) or phorbol esters, thyroid cell mitogens stimulated the production of larger binding proteins with mol wt of 40-44 and 48-52 by ligand blot and cross-linking methods, respectively. Deglycosylation of conditioned medium cross-linked to [125I]IGF-I with endoglycosidase-F did not alter the size of the smaller binding proteins, but reduced EGF-stimulated binding proteins to 36-40 kDa. Similarly, tunicamycin treatment, which inhibits glycosylation, reduced only the size of this larger binding protein species. Polyclonal antisera directed against the human amniotic fluid binding protein (BP-28) immunoprecipitated the 32 kDa sheep thyroid binding protein seen on ligand blot and the cross-linked binding protein at 36-38 kDa. Antibody against the major human serum binding protein (BP-53) recognized only the larger EGF-stimulated binding proteins. In contrast to sheep thyroid cells, rat FRTL5 thyroid cells produced no detectable IGF-binding proteins. We conclude that the predominant binding proteins produced by sheep thyroid cells under standard culture conditions are non-glycosylated and immunoreact with antiserum directed against BP-28. EGF and phorbol esters stimulate production of larger glycosylated binding proteins

  5. Binding proteins from fish sera and intrinsic factor compared in vitamin B12 radioassay.

    PubMed

    Ithakissios, D S; Kubiatowicz, D O; Windorski, D C; Wicks, J H

    1977-11-01

    We compare serum proteins from rainbow trout, chinook salmon, coho salmon, and oyster toadfish with intrinsic factor as binding proteins in a simplified radioassay for B12. Regression analysis of B12 values, determined in 21 serum samples, shows good correlation (r greater than .975) between results for the fish sera and intrinsic factor. The accuracy of the five assays, as evaluated by analytical recovery of B12 added to pooled human serum, ranges from 90 to 110%. Intra-assay precision ranges from 2.6% for coho salmon serum to 5.5% for intrinsic factor, Ionic strength and variations in pH influence binding of [57Co]vit B12 to the fish sera. Maximum binding occurs from pH 6 to 10 at an ionic strength of 0.1 for all sera. The sera are stable for longer than two years when stored at -20 degrees C. Important advantages of fish sera are their high binding capacity (typical assay dilutions range from 1500-fold for trout serum to more than 50 000-fold for chinook salmon); high affinity for B12 (K greater than 10(12) liter/mol); their relative constant binding characteristics as compared to commercial intrinsic factor preparations; and the finding that the accuracy of radioassays with use of fish sera is not significantly affected by the amount of B12 or human serum proteins present. PMID:912869

  6. Predicting Ca(2+)-binding sites in proteins.

    PubMed Central

    Nayal, M; Di Cera, E

    1994-01-01

    The coordination shell of Ca2+ ions in proteins contains almost exclusively oxygen atoms supported by an outer shell of carbon atoms. The bond-strength contribution of each ligating oxygen in the inner shell can be evaluated by using an empirical expression successfully applied in the analysis of crystals of metal oxides. The sum of such contributions closely approximates the valence of the bound cation. When a protein is embedded in a very fine grid of points and an algorithm is used to calculate the valence of each point representing a potential Ca(2+)-binding site, a typical distribution of valence values peaked around 0.4 is obtained. In 32 documented Ca(2+)-binding proteins, containing a total of 62 Ca(2+)-binding sites, a very small fraction of points in the distribution has a valence close to that of Ca2+. Only 0.06% of the points have a valence > or = 1.4. These points share the remarkable tendency to cluster around documented Ca2+ ions. A high enough value of the valence is both necessary (58 out of 62 Ca(2+)-binding sites have a valence > or = 1.4) and sufficient (87% of the grid points with a valence > or = 1.4 are within 1.0 A from a documented Ca2+ ion) to predict the location of bound Ca2+ ions. The algorithm can also be used for the analysis of other cations and predicts the location of Mg(2+)- and Na(+)-binding sites in a number of proteins. The valence is, therefore, a tool of pinpoint accuracy for locating cation-binding sites, which can also be exploited in engineering high-affinity binding sites and characterizing the linkage between structural components and functional energetics for molecular recognition of metal ions by proteins. Images Fig. 4 PMID:8290605

  7. Carotenoid binding to proteins: Modeling pigment transport to lipid membranes.

    PubMed

    Reszczynska, Emilia; Welc, Renata; Grudzinski, Wojciech; Trebacz, Kazimierz; Gruszecki, Wieslaw I

    2015-10-15

    Carotenoid pigments play numerous important physiological functions in human organism. Very special is a role of lutein and zeaxanthin in the retina of an eye and in particular in its central part, the macula lutea. In the retina, carotenoids can be directly present in the lipid phase of the membranes or remain bound to the protein-pigment complexes. In this work we address a problem of binding of carotenoids to proteins and possible role of such structures in pigment transport to lipid membranes. Interaction of three carotenoids, beta-carotene, lutein and zeaxanthin with two proteins: bovine serum albumin and glutathione S-transferase (GST) was investigated with application of molecular spectroscopy techniques: UV-Vis absorption, circular dichroism and Fourier transform infrared spectroscopy (FTIR). Interaction of pigment-protein complexes with model lipid bilayers formed with egg yolk phosphatidylcholine was investigated with application of FTIR, Raman imaging of liposomes and electrophysiological technique, in the planar lipid bilayer models. The results show that in all the cases of protein and pigment studied, carotenoids bind to protein and that the complexes formed can interact with membranes. This means that protein-carotenoid complexes are capable of playing physiological role in pigment transport to biomembranes. PMID:26361975

  8. Bovine Serum Albumin binding to CoCrMo nanoparticles and the influence on dissolution

    NASA Astrophysics Data System (ADS)

    Simoes, T. A.; Brown, A. P.; Milne, S. J.; Brydson, R. M. D.

    2015-10-01

    CoCrMo alloys exhibit good mechanical properties, excellent biocompatibility and are widely utilised in orthopaedic joint replacements. Metal-on-metal hip implant degradation leads to the release of metal ions and nanoparticles, which persist through the implant's life and could be a possible cause of health complications. This study correlates preferential binding between proteins and metal alloy nanoparticles to the alloy's corrosion behaviour and the release of metal ions. TEM images show the formation of a protein corona in all particles immersed in albumin containing solutions. Only molybdenum release was significant in these tests, suggesting high dissolution of this element when CoCrMo alloy nanoparticles are produced as wear debris in the presence of serum albumin. The same trend was observed during extended exposure of molybdenum reference nanoparticles to albumin.

  9. The role of serum proteins in Staphylococcus aureus adhesion to ethylene glycol coated surfaces.

    PubMed

    Schuster, Swen; Yu, Wenqi; Nega, Mulugeta; Chu, Ya-Yun; Zorn, Stefan; Zhang, Fajun; Götz, Friedrich; Schreiber, Frank

    2014-11-01

    Bacterial adhesion on implants is a first step in the development of chronic foreign body associated infections. Finding strategies to minimize bacterial adhesion may contribute to minimize such infections. It is known that surfaces with oligo-ethylene-glycol (EG3OMe) or poly-ethylene-glycol (PEG2k) terminations decrease unspecific protein adsorption and bacterial adhesion. However, little is known about the influence of serum and its components on bacterial adhesion. We therefore prepared two coatings on gold surface with HS-(CH2)11EG3OMe (EG3OMe) and PEG2k-thiol and studied the role of bovine serum albumin (BSA), γ-globulins, and serum on Staphylococcus aureus adhesion. While BSA and lysozyme showed no adherence even when applied at very high concentrations (100 mg/ml), γ-globulins adsorbed already from 10 mg/ml on. The adsorption of γ-globulins was, however, significantly decreased when it was mixed with BSA in a ratio of 3:1, as it is in the serum. Pretreatment of EG3OMe and PEG2k coatings with γ-globulins or serum strongly promoted adherence of S. aureus when resuspended in buffer, suggesting that γ-globulins play a pivotal role in promoting S. aureus adhesion by its IgG binding proteins; the finding that a spa-deletion mutant, lacking the IgG binding protein A, showed decreased adherence corroborated this. Similarly, when S. aureus was pretreated with serum or γ-globulins its adherence was also significantly decreased. Our findings show that particularly γ-globulins bind to the coated surfaces thus mediating adherence of S. aureus via its protein A. As pretreatment of S. aureus with serum or γ-globulins significantly decreased adherence, treatment of patients with γ-globulins before implant surgery might lower the risk of implant-associated infections. PMID:24980510

  10. Ice-Binding Proteins and Their Function.

    PubMed

    Bar Dolev, Maya; Braslavsky, Ido; Davies, Peter L

    2016-06-01

    Ice-binding proteins (IBPs) are a diverse class of proteins that assist organism survival in the presence of ice in cold climates. They have different origins in many organisms, including bacteria, fungi, algae, diatoms, plants, insects, and fish. This review covers the gamut of IBP structures and functions and the common features they use to bind ice. We discuss mechanisms by which IBPs adsorb to ice and interfere with its growth, evidence for their irreversible association with ice, and methods for enhancing the activity of IBPs. The applications of IBPs in the food industry, in cryopreservation, and in other technologies are vast, and we chart out some possibilities. PMID:27145844

  11. Equilibrium studies on the binding of cadmium(II) to human serum transferrin

    SciTech Connect

    Harris, W.R.; Madsen, L.J.

    1988-01-12

    The binding of cadmium(II) to human serum transferrin in 0.01 M N-(2-hydroxyethyl)-piperazine-N'-2-ethanesulfonic acid with 5 mM bicarbonate at 25/sup 0/C has been evaluated by difference ultraviolet spectroscopy. Equilibrium constants were determined by competition versus three different low molecular weight chelating agents: nitrilotriacetic acid, ethylenediamine-N,N'-diacetic acid, and triethylenetetramine. Conditional equilibrium constants for the sequential binding of two cadmium ions to transferrin under the stated experimental conditions are log K/sub 1/ = 5.95 +/- 0.10 and log K/sub 2/ = 4.86 +/- 0.13. A linear free energy relationship for the complexation of cadmium and zinc has been prepared by using equilibrium data on 243 complexes of these metal ions with low molecular weight ligands. The transferrin binding constants for cadmium and zinc are in good agreement with this linear free energy relationship. This indicates that the larger size of the cadmium(II) ion does not significantly hinder its binding to the protein.

  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. PMID:26616927

  13. Spectroscopy and Molecular Modeling Study on Binding of Nickel Phthalocyanine to Human Serum Albumin.

    PubMed

    Dezhampanah, Hamid; Firouzi, Roghaye; Hasani, Leila

    2016-01-01

    The interaction of nickel tetra sulfunated phthalocyanine( NiTSPc) with human serum albumin (HSA), in 20 mM phosphate buffer pH 7.4 was investigated using advanced techniques including fluorescence, synchronous fluorescence, Fourier transform infrared (FT-IR), circular dichroism (CD) spectroscopy and molecular docking. The fluorescence quenching measurements showed a single binding site on HSA for NiTSPc with the binding constant (Kb) value equals to 1.26×106 at 25°C. The results showed that quenching mechanism of HSA by NiTSPc was of dynamic type. The results from FTIR and CD spectroscopies demonstrated that NiTSPc binds to amino acid residues of the main polypeptide chain in protein destroying the hydrogen bonding network. The corresponding thermodynamic parameters were then calculated by analysis of fluorescence data using van't Hoff plot. These data indicated that driving force for interaction was mainly hydrophobic in nature and the process was entropy driven. The information obtained from CD, FT-IR and synchronous fluorescence spectroscopies revealed that both microenvironment and conformation of HSA was changed. Molecular docking study confirmed the binding mode obtained by experimental data. PMID:27449940

  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. Spectroscopic characterization of the binding mechanism of fluorescein and carboxyfluorescein in human serum albumin

    NASA Astrophysics Data System (ADS)

    Sulaiman, Saba A. J.; Kulathunga, H. Udani; Abou-Zied, Osama K.

    2015-03-01

    Fluorescein (FL) and some of its precursors have proven to be effective fluorescent tracers in pharmaceutical and medical applications owing to their high quantum yield of fluorescence in physiological conditions and their high membrane permeability. In order to protect FL from metabolic effects during the process of its delivery, human serum albumin (HSA) has been used as a carrier because of its compatibility with the human body. In the present work, we used spectroscopic methods to characterize the binding mechanisms of FL and one of its derivatives, 5(6)- carboxyfluorescein (CFL), in the HSA protein. The absorbance change of the two ligands (FL and CFL) was quantified as a function of the HSA concentration and the results indicate a moderate binding strength for the two ligands inside HSA (1.00 +/- 0.12 x 104 M-1). The quenching effect of FL(CFL) on the fluorescence intensity of W214 (the sole tryptophan in HSA) indicates that FL and CFL occupy Site I in the protein which is known to bind several hydrophobic drugs. By performing site-competitive experiments, the location of the ligands is determined to be similar to that of the anticoagulant drug warfarin. At higher ratios of [ligand]/[HSA], we observed an upward curvature in the Stern-Volmer plots which indicates that the ligands occupy more pockets in Site I, close to W214. Our results indicate that both ligands bind in HSA with a moderate strength that should not affect their release when used as fluorescent reporters. The chemical and physical identities of the two ligands are also preserved inside the HSA binding sites.

  16. Glycosylation status of vitamin D binding protein in cancer patients.

    PubMed

    Rehder, Douglas S; Nelson, Randall W; Borges, Chad R

    2009-10-01

    On the basis of the results of activity studies, previous reports have suggested that vitamin D binding protein (DBP) is significantly or even completely deglycosylated in cancer patients, eliminating the molecular precursor of the immunologically important Gc macrophage activating factor (GcMAF), a glycosidase-derived product of DBP. The purpose of this investigation was to directly determine the relative degree of O-linked trisaccharide glycosylation of serum-derived DBP in human breast, colorectal, pancreatic, and prostate cancer patients. Results obtained by electrospray ionization-based mass spectrometric immunoassay showed that there was no significant depletion of DBP trisaccharide glycosylation in the 56 cancer patients examined relative to healthy controls. These results suggest that alternative hypotheses regarding the molecular and/or structural origins of GcMAF must be considered to explain the relative inability of cancer patient serum to activate macrophages. PMID:19642159

  17. Glycosylation status of vitamin D binding protein in cancer patients

    PubMed Central

    Rehder, Douglas S; Nelson, Randall W; Borges, Chad R

    2009-01-01

    On the basis of the results of activity studies, previous reports have suggested that vitamin D binding protein (DBP) is significantly or even completely deglycosylated in cancer patients, eliminating the molecular precursor of the immunologically important Gc macrophage activating factor (GcMAF), a glycosidase-derived product of DBP. The purpose of this investigation was to directly determine the relative degree of O-linked trisaccharide glycosylation of serum-derived DBP in human breast, colorectal, pancreatic, and prostate cancer patients. Results obtained by electrospray ionization-based mass spectrometric immunoassay showed that there was no significant depletion of DBP trisaccharide glycosylation in the 56 cancer patients examined relative to healthy controls. These results suggest that alternative hypotheses regarding the molecular and/or structural origins of GcMAF must be considered to explain the relative inability of cancer patient serum to activate macrophages. PMID:19642159

  18. Kaempferol-human serum albumin interaction: Characterization of the induced chirality upon binding by experimental circular dichroism and TDDFT calculations

    NASA Astrophysics Data System (ADS)

    Matei, Iulia; Ionescu, Sorana; Hillebrand, Mihaela

    2012-10-01

    The experimental induced circular dichroism (ICD) and absorption spectra of the achiral flavonoid kaempferol upon binding to human serum albumin (HSA) were correlated to electronic CD and UV-vis spectra theoretically predicted by time-dependent density functional theory (TDDFT). The neutral and four anionic species of kaempferol in various conformations were considered in the calculations. The appearance of the experimental ICD signal was rationalized in terms of kaempferol binding to HSA in a distorted, chiral, rigid conformation. The comparison between the experimental and simulated spectra allowed for the identification of the kaempferol species that binds to HSA, namely the anion generated by deprotonation of the hydroxyl group in position 7. This approach constitutes a convenient method for evidencing the binding species and for determining its conformation in the binding pocket of the protein. Its main advantage over the UV-vis absorption method lays in the fact that only the bound ligand species gives an ICD signal.

  19. Evaluation of the binding interaction between bovine serum albumin and dimethyl fumarate, an anti-inflammatory drug by multispectroscopic methods.

    PubMed

    Jattinagoudar, Laxmi; Meti, Manjunath; Nandibewoor, Sharanappa; Chimatadar, Shivamurti

    2016-03-01

    The information of the quenching reaction of bovine serum albumin with dimethyl fumarate is obtained by multi-spectroscopic methods. The number of binding sites, n and binding constants, KA were determined at different temperatures. The effect of increasing temperature on Stern-Volmer quenching constants (KD) indicates that a dynamic quenching mechanism is involved in the interaction. The analysis of thermodynamic quantities namely, ∆H° and ∆S° suggested hydrophobic forces playing a major role in the interaction between dimethyl fumarate and bovine serum albumin. The binding site of dimethyl fumarate on bovine serum albumin was determined by displacement studies, using the site probes viz., warfarin, ibuprofen and digitoxin. The determination of magnitude of the distance of approach for molecular interactions between dimethyl fumarate and bovine serum albumin is calculated according to the theory of Förster energy transfer. The CD, 3D fluorescence spectra, synchronous fluorescence measurements and FT-IR spectral results were indicative of the change in secondary structure of the protein. The influence of some of the metal ions on the binding interaction was also studied. PMID:26688208

  20. Evaluation of the binding interaction between bovine serum albumin and dimethyl fumarate, an anti-inflammatory drug by multispectroscopic methods

    NASA Astrophysics Data System (ADS)

    Jattinagoudar, Laxmi; Meti, Manjunath; Nandibewoor, Sharanappa; Chimatadar, Shivamurti

    2016-03-01

    The information of the quenching reaction of bovine serum albumin with dimethyl fumarate is obtained by multi-spectroscopic methods. The number of binding sites, n and binding constants, KA were determined at different temperatures. The effect of increasing temperature on Stern-Volmer quenching constants (KD) indicates that a dynamic quenching mechanism is involved in the interaction. The analysis of thermodynamic quantities namely, ∆H° and ∆S° suggested hydrophobic forces playing a major role in the interaction between dimethyl fumarate and bovine serum albumin. The binding site of dimethyl fumarate on bovine serum albumin was determined by displacement studies, using the site probes viz., warfarin, ibuprofen and digitoxin. The determination of magnitude of the distance of approach for molecular interactions between dimethyl fumarate and bovine serum albumin is calculated according to the theory of Förster energy transfer. The CD, 3D fluorescence spectra, synchronous fluorescence measurements and FT-IR spectral results were indicative of the change in secondary structure of the protein. The influence of some of the metal ions on the binding interaction was also studied.

  1. Stretching DNA to quantify nonspecific protein binding

    NASA Astrophysics Data System (ADS)

    Goyal, Sachin; Fountain, Chandler; Dunlap, David; Family, Fereydoon; Finzi, Laura

    2012-07-01

    Nonspecific binding of regulatory proteins to DNA can be an important mechanism for target search and storage. This seems to be the case for the lambda repressor protein (CI), which maintains lysogeny after infection of E. coli. CI binds specifically at two distant regions along the viral genome and induces the formation of a repressive DNA loop. However, single-molecule imaging as well as thermodynamic and kinetic measurements of CI-mediated looping show that CI also binds to DNA nonspecifically and that this mode of binding may play an important role in maintaining lysogeny. This paper presents a robust phenomenological approach using a recently developed method based on the partition function, which allows calculation of the number of proteins bound nonspecific to DNA from measurements of the DNA extension as a function of applied force. This approach was used to analyze several cycles of extension and relaxation of λ DNA performed at several CI concentrations to measure the dissociation constant for nonspecific binding of CI (˜100 nM), and to obtain a measurement of the induced DNA compaction (˜10%) by CI.

  2. Tetranectin-like protein in vertebrate serum: a comparative immunochemical analysis.

    PubMed

    Thougaard, A V; Jaliashvili, I; Christiansen, M

    2001-04-01

    The glycoprotein tetranectin (TN) found in human serum is a 90-kDa homotrimeric C-type lectin binding Ca2+, heparin and plasminogen kringle 4. TN is suggested as being implicated in tissue remodelling. The antigenic reactivity of putative TN was examined in serum from 14 different animal species using three sandwich enzyme immunoassays for human TN. Crab-eating macaque serum showed the strongest reaction, followed by horse and cat. Serum from cow, goat, pig, mouse and chicken reacted weakly, while dog, trout, and the amphibian and the reptile species did not react. The TN-like protein from macaque, horse and cat serum bound heparin and showed the same dependence on Ca2+ for interaction with the monoclonal antibodies as human TN. Gel filtration of sera from the three animal species showed that the TN-like protein eluted as single peaks with a M(r) of 70-90 kDa. Western blotting of horse and cat TN-like protein electrophoresed under reducing conditions showed that the antibodies against human TN reacted with a single band with an approximate M(r) of 30 kDa, indicating that the TN-like protein is also a homotrimer. Horse and cat TN-like protein interacted with human kringle 4-sepharose. Most likely, the reacting protein represents crab-eating macaque, horse and cat homologues of human TN. PMID:11290444

  3. Signal transduction by guanine nucleotide binding proteins.

    PubMed

    Spiegel, A M

    1987-01-01

    High affinity binding of guanine nucleotides and the ability to hydrolyze bound GTP to GDP are characteristics of an extended family of intracellular proteins. Subsets of this family include cytosolic initiation and elongation factors involved in protein synthesis, and cytoskeletal proteins such as tubulin (Hughes, S.M. (1983) FEBS Lett. 164, 1-8). A distinct subset of guanine nucleotide binding proteins is membrane-associated; members of this subset include the ras gene products (Ellis, R.W. et al. (1981) Nature 292, 506-511) and the heterotrimeric G-proteins (also termed N-proteins) (Gilman, A.G. (1984) Cell 36, 577-579). Substantial evidence indicates that G-proteins act as signal transducers by coupling receptors (R) to effectors (E). A similar function has been suggested but not proven for the ras gene products. Known G-proteins include Gs and Gi, the G-proteins associated with stimulation and inhibition, respectively, of adenylate cyclase; transducin (TD), the G-protein coupling rhodopsin to cGMP phosphodiesterase in rod photoreceptors (Bitensky, M.W. et al. (1981) Curr. Top. Membr. Transp. 15, 237-271; Stryer, L. (1986) Annu. Rev. Neurosci. 9, 87-119), and Go, a G-protein of unknown function that is highly abundant in brain (Sternweis, P.C. and Robishaw, J.D. (1984) J. Biol. Chem. 259, 13806-13813; Neer, E.J. et al. (1984) J. Biol. Chem. 259, 14222-14229). G-proteins also participate in other signal transduction pathways, notably that involving phosphoinositide breakdown. In this review, I highlight recent progress in our understanding of the structure, function, and diversity of G-proteins. PMID:2435586

  4. Accuracy of serum IgM and IgA monoclonal protein measurements by densitometry.

    PubMed

    Tseng, C Howard; Chang, Chin-Yung; Liu, Kevin S; Liu, Frank J

    2003-01-01

    We previously reported that proper dilution of sera that contain IgG monoclonal proteins (M-proteins) is necessary for accurate quantitation of serum albumin and M-protein concentrations separated by serum protein electrophoresis (SPE) using the Beckman PARAGON agarose electrophoresis system. We now report the significance of pre-electrophoretic serum dilution for M-protein quantitation of sera from patients with IgA and IgM monoclonal gammopathy. We measured M-proteins by SPE in 82 serum samples from 29 patients with IgA and 72 samples from 23 patients with IgM monodonal gammopathy. The serum M-protein concentrations (mean +/- SD) at 1:5, 1:10, and 1:20 dilutions (v/v) for all samples of both types were 49.7 +/- 12.9, 49.1 +/- 13.1, and 47.8 +/- 13.0 g/L, respectively. Thirty-two (20.8%) of 154 sera showed varying degrees of increase in M-protein concentrations with serum dilutions higher than 1:5; only 8 (5.2%) showed an increase 3 SDs. By SPE, the M-protein concentration (mean +/- SD) of these 8 sera at 1:5, 1:10, and 1:20 dilutions were 52.6 +/- 7.8, 57.1 +/- 7.2, and 57.6 +/- 7.1 g/L, respectively; the albumin concentrations (mean +/- SD) were 41.4 +/- 4.4, 37.9 +/- 3.8, and 37.1 +/- 2.9 g/L, respectively. The corresponding albumin concentration (mean +/- SD) was 36.8 +/- 3.7 g/L, assayed by the bromcresol green dye-binding method. These 8 samples were obtained from 3 patients, 2 with IgM kappa and 1 with IgA lambda monoclonal gammopathy. On the electrophoresis membranes, the M-protein bands of these 8 samples were narrow, thin, and dense; upon scanning, they appeared taller and thinner than the corresponding albumin bands. The samples of this subset contained relatively high concentrations of M-protein and total serum protein. We conclude that a pre-electrophoretic dilution of 1:5 (v/v) is adequate for most sera with IgA or IgM M-proteins. However, 1:10 or 1:20 dilution is occasionally required for a subset of sera with IgA or IgM M-proteins that show an

  5. [Carbohydrate-binding proteins of marine invertebrates].

    PubMed

    Luk'ianov, P A; Chernikov, O V; Kobelev, S S; Chikalovets, I V; Molchanova, V I; Li, W

    2007-01-01

    The information on the carbohydrate specificity and molecular organization of some carbohydrate-binding proteins (lectins) of marine invertebrates is reported. Antiviral activity of some of the lectins against human immunodeficiency virus has been studied. Lectins of marine invertebrates are promising tools for studying natural glycoconjugates and cell effectors in vitro. PMID:17375673

  6. The influence of protein binding on the antibacterial activity of faropenem against Haemophilus influenzae.

    PubMed

    Gustafsson, I; Cars, O

    2004-10-01

    The effects of albumin and human serum on the pharmacodynamics of faropenem were studied. The protein binding of faropenem was 91-95%, corresponding to the increase in MICs for Haemophilus influenzae in broth supplemented with albumin. Time-kill experiments in albumin-containing medium and in inactivated human serum 50% v/v showed that much higher drug concentrations were needed to achieve a bactericidal effect than were needed in broth. Active human serum alone exerted a strain-dependent bactericidal effect. It was concluded that it is the free fraction of faropenem in serum that has antibacterial activity against H. influenzae. PMID:15373892

  7. Revision of the biodistribution of uranyl in serum: is fetuin-A the major protein target?

    PubMed

    Basset, Christian; Averseng, Olivier; Ferron, Pierre-Jean; Richaud, Nicolas; Hagège, Agnès; Pible, Olivier; Vidaud, Claude

    2013-05-20

    Uranium is a natural actinide present as uranyl U(VI) species in aqueous environments. Its toxicity is considered to be chemical rather than radiotoxicological. Whatever the route of entry, uranyl reaches the blood, is partly eliminated via the kidneys, and accumulated in the bones. In serum, its speciation mainly involves carbonate and proteins. Direct identification of labile uranyl-protein complexes is extremely difficult because of the complexity of this matrix. Thus, until now the biodistribution of the metal in serum has not been described, and therefore, little is known about the metal transport mechanisms leading to bone accumulation. A rapid screening method based on a surface plasmon resonance (SPR) technique was used to determine the apparent affinities for U(VI) of the major serum proteins. A first biodistribution of uranyl was obtained by ranking the proteins according to the criteria of both their serum concentrations and affinities for this metal. Despite its moderate concentration in serum, fetuin-A (FETUA) was shown to exhibit an apparent affinity within the 30 nM range and to carry more than 80% of the metal. This protein involved in bone mineralization aroused interest in characterizing the U(VI) and FETUA interaction. Using complementary chromatographic and spectroscopic approaches, we demonstrated that the protein can bind 3 U(VI) at different binding sites exhibiting Kd from ∼30 nM to 10 μM. Some structural modifications and functional properties of FETUA upon uranyl complexation were also controlled. To our knowledge, this article presents the first identification of a uranyl carrier involved in bone metabolism along with the characterization of its metal binding sites. PMID:23527557

  8. Chloramphenicol binding to human serum albumin: Determination of binding constants and binding sites by steady-state fluorescence

    NASA Astrophysics Data System (ADS)

    Ding, Fei; Zhao, Guangyu; Chen, Shoucong; Liu, Feng; Sun, Ying; Zhang, Li

    2009-07-01

    The interaction between chloramphenicol and human serum albumin (HSA) was studied by fluorescence, UV/vis, circular dichroism (CD) and three-dimensional fluorescence spectroscopy. Fluorescence data revealed that the fluorescence quenching of HSA by chloramphenicol was the result of the formation of drug-HSA complex, and the effective quenching constants ( Ka) were 2.852 × 10 4, 2.765 × 10 4, 2.638 × 10 4 and 2.542 × 10 4 M -1 at 287, 295, 303 and 311 K, respectively. The thermodynamic parameters, enthalpy change (Δ H) and entropy change (Δ S) for the reaction were calculated to be -3.634 kJ mol -1 and 72.66 J mol -1 K -1 according to van't Hoff equation. The results indicated that the hydrophobic and electrostatic interactions played a major role in the binding of drug to HSA. The distance r between donor and acceptor was obtained to be 3.63 nm according to Förster's theory. Site marker competitive experiments indicated that the binding of drug to HSA primarily took place in subdomain IIA. The alterations of HSA secondary structure in the presence of chloramphenicol were confirmed by the evidences from synchronous fluorescence, CD and three-dimensional fluorescence spectra. In addition, the effect of common ions on the binding constants of drug-HSA complex was also discussed.

  9. Protein binding of chlorpromazine in vivo and in vitro: effect of chlorpromazine metabolite on chlorpromazine protein binding in rat.

    PubMed

    Sato, S; Koshiro, A

    1995-04-01

    The serum protein binding curve of chlorpromazine (CPZ) on the Scatchard plot in vitro was a two-phase downward curve. However, after i.v. administration of CPZ the curve was altered to an upward curve. To clarify the reasons for these in vivo changes, the influence of chlorpromazine S-oxide (CPZSO), chlorpromazine N-oxide (CPZNO), desmethylchlorpromazine (nor1-CPZ), chlorpromazine sulfone (sul-CPZ) and 7-hydroxychlorpromazine (7-OH-CPZ) on CPZ protein binding were studied in vitro. The results indicated that the characteristics of the CPZ protein binding are altered by the combination of CPZSO or CPZNO or by either of them. Since it was very difficult to explain the relationship between serum total and free concentrations of CPZ in vivo using mass-balance equations like Hill's equation or a competitive inhibition equation on the multiple binding sites for drug, the correlation between the ratio ot total concentration of CPZ metabolites and CPZ (CPZSO/CPZ or CPZNO/CPZ) and the free fraction of CPZ was examined using the in vitro and in vivo data. The correlation between the ratio of CPZSO/CPZ and the free fraction of CPZ was good in both the in vivo and the in vitro studies. There was no statistically significant difference between the population regression coefficient of the two studies. The values of the slope and the intercept became almost the same as those obtained using the in vivo studies when combined with CPZNO.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7655433

  10. SDS-binding assay based on tyrosine fluorescence as a tool to determine binding properties of human serum albumin in blood plasma

    NASA Astrophysics Data System (ADS)

    Zhdanova, Nadezda; Shirshin, Evgeny; Fadeev, Victor; Priezzhev, Alexander

    2016-04-01

    Among all plasma proteins human serum albumin (HSA) is the most studied one as it is the main transport protein and can bind a wide variety of ligands especially fatty acids (FAs). The concentration of FAs bound to HSA in human blood plasma differs by three times under abnormal conditions (fasting, physical exercises or in case of social important diseases). In the present study a surfactant sodium dodecyl sulfate (SDS) was used to simulate FAs binding to HSA. It was shown that the increase of Tyr fluorescence of human blood plasma due to SDS addition can be completely explained by HSA-SDS complex formation. Binding parameters of SDS-HSA complex (average number of sites and apparent constant of complex formation) were determined from titration curves based on tyrosine (Tyr) fluorescence.

  11. Fluorescence analysis of competition of phenylbutazone and methotrexate in binding to serum albumin in combination treatment in rheumatology

    NASA Astrophysics Data System (ADS)

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

    2009-04-01

    Combination of several drugs is often necessary especially during long-them therapy. The competition between drugs can cause a decrease of the amount of a drug bound to albumin. This results in an increase of the free, biological active fraction of the drug. The aim of the presented study was to describe the competition between phenylbutazone (Phe) and methotrexate (MTX), two drugs recommended for the treatment of rheumatology in binding to bovine (BSA) and human (HSA) serum albumin in the high affinity binding site. Fluorescence analysis was used to estimate the effect of drugs on the protein fluorescence and to define the binding and quenching properties of drugs-serum albumin complexes. The effect of the displacement of one drug from the complex of the other with serum albumin has been described on the basis of the comparison of the quenching curves and binding constants for the binary and ternary systems. The conclusion that both Phe and MTX form a binding site in the same subdomain (IIA) points to the necessity of using a monitoring therapy owning to the possible increase of the uncontrolled toxic effects.

  12. Analysis of Lidocaine Interactions with Serum Proteins Using High-Performance Affinity Chromatography

    PubMed Central

    Soman, Sony; Yoo, Michelle J.; Jang, Yoon Jeong; Hage, David S.

    2010-01-01

    High-performance affinity chromatography was used to study binding by the drug lidocaine to human serum albumin (HSA) and α1–acid glycoprotein (AGP). AGP had strong binding to lidocaine, with an association equilibrium constant (Ka) of 1.1-1.7 × 105 M-1 at 37 °C and pH 7.4. Lidocaine had weak-to-moderate binding to HSA, with a Ka in the range of 103 to 104 M-1. Competitive experiments with site selective probes showed that lidocaine was interacting with Sudlow site II of HSA and the propranolol site of AGP. These results agree with previous observations in the literature and provide a better quantitative understanding of how lidocaine binds to these serum proteins and is transported in the circulation. This study also demonstrates how HPAC can be used to examine the binding of a drug with multiple serum proteins and provide detailed information on the interaction sites and equilibrium constants that are involved in such processes. PMID:20138813

  13. Serum screening for oncogene proteins in workers exposed to PCBs.

    PubMed Central

    Brandt-Rauf, P W; Niman, H L

    1988-01-01

    A cohort of 16 municipal workers engaged in cleaning oil from old transformers was examined for possible health effects from exposure to polychlorinated biphenyls (PCBs). In addition to the evaluation of routine clinical parameters (history, physical examination, liver function tests, serum triglycerides, serum PCB values), a new screening technique for the presence of oncogene proteins in serum using monoclonal antibodies was used to ascertain the potential carcinogenic risk from exposure in these workers. Except for one individual, serum PCB concentrations were found to be relatively low in this cohort, probably due to the observance of appropriate protective precautions. The results of liver function test were within normal limits and serum triglyceride concentrations showed no consistent relation to PCB concentrations. Six individuals, all of whom were smokers, showed abnormal banding patterns for fes oncogene related proteins. The individual with the highest serum PCB concentration also exhibited significantly raised levels of the H-ras oncogene related P21 protein in his serum. These oncogene protein findings may be indicative of an increased risk for the development of malignant disease in these individuals. Images PMID:3143397

  14. Evolution of Protein-binding DNA Sequences through Competitive Binding

    NASA Astrophysics Data System (ADS)

    Peng, Weiqun; Gerland, Ulrich; Hwa, Terence; Levine, Herbert

    2002-03-01

    The dynamics of in vitro DNA evolution controlled via competitive binding of DNA sequences to proteins has been explored in a recent serial transfer experiment footnote B. Dubertret, S.Liu, Q. Ouyang, A. Libchaber, Phys. Rev. Lett. 86, 6022 (2001).. Motivated by the experiment, we investigate a continuum model for this evolution process in various parameter regimes. We establish a self-consistent mean-field evolution equation, determine its dynamical properties and finite population size corrections. In addition, we discuss the experimental implications of our results.

  15. Odorant-binding proteins in insects.

    PubMed

    Zhou, Jing-Jiang

    2010-01-01

    Our understanding of the molecular and biochemical mechanisms that mediate chemoreception in insects has been greatly improved after the discovery of olfactory and taste receptor proteins. However, after 50 years of the discovery of first insect sex pheromone from the silkmoth Bombyx mori, it is still unclear how hydrophobic compounds reach the dendrites of sensory neurons in vivo across aqueous space and interact with the sensory receptors. The presence of soluble polypeptides in high concentration in the lymph of chemosensilla still poses unanswered questions. More than two decades after their discovery and despite the wealth of structural and biochemical information available, the physiological function of odorant-binding proteins (OBPs) is not well understood. Here, I review the structural properties of different subclasses of insect OBPs and their binding to pheromones and other small ligands. Finally, I discuss current ideas and models on the role of such proteins in insect chemoreception. PMID:20831949

  16. Quantifying drug-protein binding in vivo.

    SciTech Connect

    Buchholz, B; Bench, G; Keating III, G; Palmblad, M; Vogel, J; Grant, P G; Hillegonds, D

    2004-02-17

    Accelerator mass spectrometry (AMS) provides precise quantitation of isotope labeled compounds that are bound to biological macromolecules such as DNA or proteins. The sensitivity is high enough to allow for sub-pharmacological (''micro-'') dosing to determine macromolecular targets without inducing toxicities or altering the system under study, whether it is healthy or diseased. We demonstrated an application of AMS in quantifying the physiologic effects of one dosed chemical compound upon the binding level of another compound in vivo at sub-toxic doses [4].We are using tissues left from this study to develop protocols for quantifying specific binding to isolated and identified proteins. We also developed a new technique to quantify nanogram to milligram amounts of isolated protein at precisions that are comparable to those for quantifying the bound compound by AMS.

  17. Functional Analysis of the Human Antibody Response to Meningococcal Factor H Binding Protein

    PubMed Central

    Beernink, Peter T.; Giuntini, Serena; Costa, Isabella; Lucas, Alexander H.

    2015-01-01

    ABSTRACT Two licensed serogroup B meningococcal vaccines contain factor H binding protein (FHbp). The antigen specifically binds human FH, which downregulates complement. In wild-type mice whose mouse FH does not bind to FHbp vaccines, the serum anti-FHbp antibody response inhibited binding of human FH to FHbp. The inhibition was important for eliciting broad anti-FHbp serum bactericidal activity. In human FH transgenic mice and some nonhuman primates, FHbp was able to form a complex with FH and FHbp vaccination elicited anti-FHbp antibodies that did not inhibit FH binding. To investigate the human anti-FHbp repertoire, we cloned immunoglobulin heavy- and light-chain-variable-region genes of individual B cells from three adults immunized with FHbp vaccines and generated 10 sequence-distinct native anti-FHbp antibody fragments (Fabs). All 10 Fabs bound to live meningococci; only 1 slightly inhibited binding of human FH, while 4 enhanced FH binding. Affinity-purified anti-FHbp antibody from serum of a fourth immunized adult also enhanced binding of human FH to live meningococcal bacteria. Despite the bound FH, the affinity-purified serum anti-FHbp antibodies elicited human complement-mediated bactericidal activity that was amplified by the alternative pathway. The lack of FH inhibition by the human anti-FHbp Fabs and serum antibodies suggests that binding of human FH to the vaccine antigen skews the anti-FHbp antibody repertoire to epitopes outside the FH-binding site. Mutant FHbp vaccines with decreased FH binding may represent a means to redirect the human antibody repertoire to epitopes within the FH binding site, which can inhibit FH binding and, potentially, increase safety and protective activity. PMID:26106082

  18. Synthesis of 25-hydroxyvitamin D sub 3 3. beta. -3 prime -(N-(4-azido-2-nitrophenyl)amino)propyl ether, a second-generation photoaffinity analogue of 25-hydroxyvitamin D sub 3 : Photoaffinity labeling of rat serum vitamin D binding protein

    SciTech Connect

    Ray, R.; Holick, M.F. ); Bouillon, R.; Van Baelen, H. )

    1991-05-14

    Vulnerability of 25-hydroxy-(26,27-{sup 3}H)vitamin D{sub 3} 3{beta}-N-(4-azido-2-nitrophenyl)glycinate, a photoaffinity analogue of 25-hydroxyvitamin D{sub 3} (25-OH-D{sub 3}) toward standard conditions of carboxymethylationin promoted the authors to synthesize 25-hydroxyvitamin D{sub 3} 3{beta}-3{prime}-(N-(4-azido-2-nitrophenyl)amino)propyl ether (25-ANE), a hydrolytically stable photoaffinity analogue of 25-OH-D{sub 3}, and 25-hydroxyvitamin D{sub 3} 3{beta}-3{prime}-(N-(4-azido-2-nitro-(3,5-{sup 3}H)phenyl)amino)propyl ether ({sup 3}H-25-ANE), the radiolabeled counterpart of 25-ANE competes for the 25-OH-D{sub 3} binding site in rat serum vitamin D binding protein (rDBP). On the other hand, UV exposure of a sample of purified rat DBP (rDBP), preincubated in the dark with {sup 3}H-25-ANE, covalently labeled the protein. However, very little covalent labeling was observed in the absence of UV light or in the presence of a large excess of 25-OH-D{sub 3}. These results provide strong evidence for the covalent labeling of the 25-OH-D{sub 3} binding site in rDPB by {sup 3}H-25-ANE.

  19. Paracetamol and cytarabine binding competition in high affinity binding sites of transporting protein

    NASA Astrophysics Data System (ADS)

    Sułkowska, A.; Bojko, B.; Równicka, J.; Sułkowski, W. W.

    2006-07-01

    Paracetamol (acetaminophen, AA) the most popular analgesic drug is commonly used in the treatment of pain in patients suffering from cancer. In our studies, we evaluated the competition in binding with serum albumin between paracetamol (AA) and cytarabine, antyleukemic drug (araC). The presence of one drug can alter the binding affinity of albumin towards the second one. Such interaction can result in changing of the free fraction of the one of these drugs in blood. Two spectroscopic methods were used to determine high affinity binding sites and the competition of the drugs. Basing on the change of the serum albumin fluorescence in the presence of either of the drugs the quenching ( KQ) constants for the araC-BSA and AA-BSA systems were calculated. Analysis of UV difference spectra allowed us to describe the changes in drug-protein complexes (araC-albumin and AA-albumin) induced by the presence of the second drug (AA and araC, respectively). The mechanism of competition between araC and AA has been proposed.

  20. Cholesterol-lowering effect of rice bran protein containing bile acid-binding proteins.

    PubMed

    Wang, Jilite; Shimada, Masaya; Kato, Yukina; Kusada, Mio; Nagaoka, Satoshi

    2015-01-01

    Dietary plant protein is well known to reduce serum cholesterol levels. Rice bran is a by-product of rice milling and is a good source of protein. The present study examined whether feeding rats a high-cholesterol diet containing 10% rice bran protein (RBP) for 10 d affected cholesterol metabolism. Rats fed dietary RBP had lower serum total cholesterol levels and increased excretion of fecal steroids, such as cholesterol and bile acids, than those fed dietary casein. In vitro assays showed that RBP strongly bound to taurocholate, and inhibited the micellar solubility of cholesterol, compared with casein. Moreover, the bile acid-binding proteins of the RBP were eluted by a chromatographic column conjugated with cholic acid, and one of them was identified as hypothetical protein OsJ_13801 (NCBI accession No. EAZ29742) using MALDI-TOF mass spectrometry analysis. These results suggest that the hypocholesterolemic action of the RBP may be caused by the bile acid-binding proteins. PMID:25374002

  1. Interaction of Serum Proteins with Surface of Hemodialysis Fiber Membranes

    NASA Astrophysics Data System (ADS)

    Afrin, Rehana; Shirako, Yuji; Kishimoto, Kikuo; Ikai, Atsushi

    2012-08-01

    The poly(vinyl pyrrolidone)-covered hydrophilic surface of hollow-fiber membranes (fiber membrane, hereafter) for hemodialysis was mechanically probed using modified tips on an atomic force microscope (AFM) with covalent crosslinkers and several types of serum protein. The retraction part of many of the force extension (F-E) curves obtained with AFM tips coated with serum albumin had a long and smooth extension up to 200-300 nm indicating forced elongation of poly(vinyl pyrrolidone) chains. When fibrinogen-coated tips were used, long extension F-E curves up to 500 nm with multiple peaks were obtained in addition to smooth curves most likely reflecting the unfolding of fibrinogen molecules. The results indicated that individual polymer chains had a significant affinity toward serum proteins. The adhesion frequency of tips coated with serum proteins was lower on the poly(vinyl pyrrolidone) surface than on the uncoated hydrophobic polysulfone surface.

  2. In vitro induction and proteomics characterisation of a uranyl-protein interaction network in bovine serum.

    PubMed

    Szyrwiel, Łukasz; Liauchuk, Viktoryia; Chavatte, Laurent; Lobinski, Ryszard

    2015-12-01

    Uranyl ions (UO2(2+)) were shown to interact with a number of foetal serum proteins, leading to the formation of a complex that could be isolated by ultracentrifugation. The molecular weight of the complex was estimated based on size-exclusion chromatography as 650 000 Da. Online ICP AES detection indicated that UO2(2+) in the complex co-eluted with minor amounts of calcium and phosphorous, but not with magnesium. A 1D gel electrophoresis of the U-complex produced more than 10 bands of similar intensity compared with only 2-3 intense bands corresponding to the main serum proteins in the control serum, indicative of the specific interaction of UO2(2+) with minor proteins. A proteomics approach allowed for the identification of 74 proteins in the complex. Analysis of the protein-protein interaction network in the UO2(2+) complex identified 32 proteins responsible for protein-protein complex formation and 34 with demonstrated ion-binding function, suggesting that UO2(2+) stimulates the formation of protein functional networks rather than using a particular molecule as its target. PMID:26506398

  3. Surface plasmon resonance and circular dichroism characterization of cucurbitacins binding to serum albumins for early pharmacokinetic profiling.

    PubMed

    Fabini, Edoardo; Fiori, Giovana Maria Lanchoti; Tedesco, Daniele; Lopes, Norberto Peporine; Bertucci, Carlo

    2016-04-15

    Cucurbitacins are a group of tetracyclic triterpenoids, known for centuries for their anti-cancer and anti-inflammatory properties, which are being actively investigated over the past decades in order to elucidate their mechanism of action. In perspective of being used as therapeutic molecules, a pharmacokinetic characterization is crucial to assess the affinity toward blood carrier proteins and extrapolate distribution volumes. Usually, pharmacokinetic data are first collected on animal models and later translated to humans; therefore, an early characterization of the interaction with carrier proteins from different species is highly desirable. In the present study, the interactions of cucurbitacins E and I with human and rat serum albumins (HSA and RSA) were investigated by means of surface plasmon resonance (SPR)-based optical biosensing and circular dichroism (CD) spectroscopy. Active HSA and RSA sensor chip surfaces were prepared through an amine coupling reaction protocol, and the equilibrium dissociation constants (Kd) for the different cucurbitacins-serum albumins complexes were then determined by SPR analysis. Further information on the binding of cucurbitacins to serum albumins was obtained by CD competition experiments with biliverdin, a specific marker binding to subdomain IB of HSA. SPR data unveiled a previously unreported binding event between CucI and HSA; the determined binding affinities of both compounds were slightly higher for RSA with respect to HSA, even though all the compounds can be ranked as high-affinity binders for both carriers. CD analysis showed that the two cucurbitacins modify the binding of biliverdin to serum albumins through opposite allosteric modulation (positive for HSA, negative for RSA), confirming the need for caution in the translation of pharmacokinetic data across species. PMID:26856457

  4. Binding of the bioactive component Aloe dihydroisocoumarin with human serum albumin

    NASA Astrophysics Data System (ADS)

    Zhang, Xiu-Feng; Xie, Ling; Liu, Yang; Xiang, Jun-Feng; Tang, Ya-Lin

    2008-11-01

    Aloe dihydroisocoumarin, one of new components isolated from Aloe vera, can scavenge reactive oxygen species. In order to explore the mechanism of drug action at a molecular level, the binding of Aloe dihydroisocoumarin with human serum albumin (HSA) has been investigated by using fluorescence, ultraviolet (UV), circular dichroism (CD) and Fourier transform infrared (FT-IR) spectroscopy, fluorescence dynamics, and molecular dynamic docking for the first time. We observed a quenching of fluorescence of HSA in the presence of Aloe dihydroisocoumarin and also analyzed the quenching results using the Stern-Volmer equation and obtained high affinity binding to HSA. An isoemissive point at 414 nm is seen, indicating that the quenching of HSA fluorescence depends on the formation of Aloe dihydroisocoumarin-HSA complex, which is further confirmed by fluorescence dynamic result. From the CD and FT-IR results, it is apparent that the interaction of Aloe dihydroisocoumarin with HSA causes a conformational change of the protein, with the gain of α-helix, β-sheet and random coil stability and the loss of β-turn content. Data obtained by fluorescence spectroscopy, fluorescence dynamics, CD, and FTIR experiments along with the docking studies suggest that Aloe dihydroisocoumarin binds to residues located in subdomain IIA of HSA.

  5. Probing the binding of morin to human serum albumin by optical spectroscopy.

    PubMed

    Qi, Zu-de; Zhang, Yue; Liao, Feng-Lin; Ou-Yang, Yi-Wen; Liu, Yi; Yang, Xi

    2008-03-13

    Morin [2-(2,4-dihydroxyphenyl)-3,5,7-trihydroxy-4H-1-benzopyran-4-one], a member of flavonols, is an important bioactive compound by interacting with nucleic acids, enzymes and protein. Its binding to human serum albumin was investigated by fluorescence quenching, fluorescence anisotropy, and UV-vis absorbance under the simulative physiological condition. Fluorescence quenching data show that the interaction of morin with HSA forms a non-fluorescent complex with the binding constants of 1.394 x 10(5), 1.489 x 10(5), 1.609 x 10(5) and 1.717 x 10(5)M(-1) at 292, 298, 303 and 310 K, respectively. The thermodynamics parameters, enthalpy change (DeltaH) and entropy change (DeltaS) were calculated to be 8.97 kJ mol(-1) and 129.15 J mol(-1)K(-1) via van't Hoff equation. From the spectroscopic results and thermodynamics parameters, it is observed that van der Waals and hydrogen bonds are predominant intermolecular forces when forming the complex. The distance r=4.25 nm between donor (Trp214) and accepter (morin) was estimated based on the Förster theory of non-radiative energy transfer. The red shift of UV-vis absorbance shows that morin is bound to several amino acids on the hydrophobic pocket of HSA. Moreover, the competitive probes, such as warfarin and ibuprofen (site I and II probes, respectively), reveal that the binding location of morin to HSA in the site I of the hydrophobic pocket, which corresponds to the results of UV-vis absorbance, while morin also binds other lower affinity binding sites on HSA from the fluorescence anisotropy spectroscopy. PMID:18178358

  6. 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.

  7. Sulfation of Lower Chlorinated Polychlorinated Biphenyls Increases Their Affinity for the Major Drug-Binding Sites of Human Serum Albumin.

    PubMed

    Rodriguez, Eric A; Li, Xueshu; Lehmler, Hans-Joachim; Robertson, Larry W; Duffel, Michael W

    2016-05-17

    The disposition of toxicants is often affected by their binding to serum proteins, of which the most abundant in humans is serum albumin (HSA). There is increasing interest in the toxicities of environmentally persistent polychlorinated biphenyls (PCBs) with lower numbers of chlorine atoms (LC-PCBs) due to their presence in both indoor and outdoor air. PCB sulfates derived from metabolic hydroxylation and sulfation of LC-PCBs have been implicated in endocrine disruption due to high affinity-binding to the thyroxine-carrying protein, transthyretin. Interactions of these sulfated metabolites of LC-PCBs with HSA, however, have not been previously explored. We have now determined the relative HSA-binding affinities for a group of LC-PCBs and their hydroxylated and sulfated derivatives by selective displacement of the fluorescent probes 5-dimethylamino-1-naphthalenesulfonamide and dansyl-l-proline from the two major drug-binding sites on HSA (previously designated as Site I and Site II). Values for half-maximal displacement of the probes indicated that the relative binding affinities were generally PCB sulfate ≥ OH-PCB > PCB, although this affinity was site- and congener-selective. Moreover, specificity for Site II increased as the numbers of chlorine atoms increased. Thus, hydroxylation and sulfation of LC-PCBs result in selective interactions with HSA which may affect their overall retention and toxicity. PMID:27116425

  8. Cadmium-binding protein (metallothionein) in carp.

    PubMed Central

    Kito, H; Ose, Y; Sato, T

    1986-01-01

    When carp (Cyprinus carpio) were exposed to 5 and 30 ppm Cd in the water, the contents of Cd-binding protein, which has low molecular weight, increased in the hepatopancreas, kidney, gills and gastrointestinal tract with the duration of exposure. This Cd-binding protein was purified from hepatopancreas, kidney, gills, and spleen of carp administered 2 mg/kg Cd (as CdCl2), intraperitoneally for 6 days. Two Cd-binding proteins were separated by DEAE-Sephadex A-25 column chromatography. These proteins had Cd-mercaptide bond, high cysteine contents (ca. 29-34%), but no aromatic amino acids or histidine. From these characteristics the Cd-binding proteins were identified as metallothionein. By using antiserum obtained from a rabbit to which carp hepatopancreas MT-II had been administered, immunological characteristics between hepatopancreas MT-I, II and kidney MT-II were studied, and a slight difference in antigenic determinant was observed among them. By immunological staining techniques with horseradish peroxidase, the localization of metallothionein was investigated. In the nontreated group, metallothionein was present in the acinar cells of hepatopancreas and renal convoluted tubules. In the Cd-treated group (2 mg/kg IP daily for 3 days), metallothionein was present in the nuclei, sinusoids, and extracellular space of hepatopancreas, in addition to the acinar cells. Carp were bred in 1 ppm Cd, 5 ppm Zn solution, and tap water for 14 days, following transfer to 15 ppm Cd solution, respectively. The survival ratio was the highest in the Zn group followed by Cd-treated and control groups. The metallothionein contents increased in hepatopancreas and kidney in the order: Zn greater than Cd greater than control group. Images FIGURE 5. FIGURE 6. PMID:3519201

  9. 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

  10. Quantitating Metabolites in Protein Precipitated Serum Using NMR Spectroscopy

    PubMed Central

    2015-01-01

    Quantitative NMR-based metabolite profiling is challenged by the deleterious effects of abundant proteins in the intact blood plasma/serum, which underscores the need for alternative approaches. Protein removal by ultrafiltration using low molecular weight cutoff filters thus represents an important step. However, protein precipitation, an alternative and simple approach for protein removal, lacks detailed quantitative assessment for use in NMR based metabolomics. In this study, we have comprehensively evaluated the performance of protein precipitation using methanol, acetonitrile, perchloric acid, and trichloroacetic acid and ultrafiltration approaches using 1D and 2D NMR, based on the identification and absolute quantitation of 44 human blood metabolites, including a few identified for the first time in the NMR spectra of human serum. We also investigated the use of a “smart isotope tag,” 15N-cholamine for further resolution enhancement, which resulted in the detection of a number of additional metabolites. 1H NMR of both protein precipitated and ultrafiltered serum detected all 44 metabolites with comparable reproducibility (average CV, 3.7% for precipitation; 3.6% for filtration). However, nearly half of the quantified metabolites in ultrafiltered serum exhibited 10–74% lower concentrations; specifically, tryptophan, benzoate, and 2-oxoisocaproate showed much lower concentrations compared to protein precipitated serum. These results indicate that protein precipitation using methanol offers a reliable approach for routine NMR-based metabolomics of human blood serum/plasma and should be considered as an alternative to ultrafiltration. Importantly, protein precipitation, which is commonly used by mass spectrometry (MS), promises avenues for direct comparison and correlation of metabolite data obtained from the two analytical platforms to exploit their combined strength in the metabolomics of blood. PMID:24796490

  11. Probing the Sudlow binding site with warfarin: how does gold nanocluster growth alter human serum albumin?

    PubMed

    Russell, B A; Mulheran, P A; Birch, D J S; Chen, Y

    2016-08-17

    The search for new fluorescent molecules is vital to the advancement of molecular imaging and sensing for the benefit of medical and biological studies. One such class of new fluorescent molecule is fluorescent gold nanoclusters encapsulated in Human Serum Albumin (HSA-AuNC). In order to use this new fluorescent molecule as a sensor or fluorescent marker in biological imaging both in vitro and in vivo it is important to understand whether/how the proteins function is changed by the synthesis and presence of the gold nanoclusters inside the protein. Natural HSA acts as the main drug carrier in the blood stream, carrying a multitude of molecules in two major binding sites (Sudlow I and II). To test the effects of gold on the ability of HSA to act as a drug carrier we employed warfarin, an anticoagulant drug, as a fluorescent probe to detect changes between natural HSA and HSA-AuNCs. AuNCs are found to inhibit the take up of warfarin by HSA. Evidence for this is found from fluorescence spectral and lifetime measurements. Interestingly, the presence of warfarin bound to HSA also inhibits the formation of gold nanoclusters within protein. This research provides valuable insight into how protein function can change upon synthesis of AuNCs and how that will affect their use as a fluorescent probe. PMID:27480626

  12. Nucleolin is a calcium-binding protein.

    PubMed

    Gilchrist, James S C; Abrenica, Bernard; DiMario, Patrick J; Czubryt, Michael P; Pierce, Grant N

    2002-01-01

    We have purified a prominent 110-kDa protein (p110) from 1.6 M NaCl extracts of rat liver nuclei that appears to bind Ca2+. p110 was originally identified by prominent blue staining with 'Stains-All' in sodium dodecyl sulfate-polyacrylamide gels and was observed to specifically bind ruthenium red and 45Ca2+ in nitrocellulose blot overlays. In spin-dialysis studies, purified p110 saturably bound approximately 75 nmol Ca2+/mg protein at a concentration of 1 mM total Ca2+ with half-maximal binding observed at 105 microM Ca2+. With purification, p110 became increasingly susceptible to proteolytic (likely autolytic) fragmentation, although most intermediary peptides between 40 and 90 kDa retained "Stains-All", ruthenium red, and 45Ca2+ binding. N-terminal sequencing of intact p110 and a 70-kDa autolytic peptide fragment revealed a strong homology to nucleolin. Two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)/IEF revealed autolysis produced increasingly acidic peptide fragments ranging in apparent pI's from 5.5 for intact p110 to 3.5 for a 40 kDa peptide fragment. Intact p110 and several peptide fragments were immunostained with a highly specific anti-nucleolin antibody, R2D2, thus confirming the identity of this protein with nucleolin. These annexin-like Ca2+-binding characteristics of nucleolin are likely contributed by its highly acidic argyrophilic N-terminus with autolysis apparently resulting in largely selective removal of its basic C-terminal domain. Although the Ca2+-dependent functions of nucleolin are unknown, we discuss the possibility that like the structurally analogous HMG-1, its Ca2+-dependent actions may regulate chromatin structure, possibly during apoptosis. PMID:11948683

  13. The Promiscuous Protein Binding Ability of Erythrosine B Studied by Metachromasy (Metachromasia)

    PubMed Central

    Ganesan, Lakshmi; Buchwald, Peter

    2013-01-01

    The present study aims to elucidate aspects of the protein binding ability of erythrosine B (ErB), a poly-iodinated xanthene dye and an FDA-approved food colorant (FD&C Red No. 3), which we have identified recently as a promiscuous inhibitor of protein–protein interactions (PPI) with a remarkably consistent median inhibitory concentration (IC50) in the 5–30 µM range. Because ErB exhibits metachromasy, i.e., color change upon binding to several proteins, we exploited this property to quantify its binding to proteins such as bovine serum albumin (BSA) and CD40L (CD154) and to determine the corresponding binding constants (Kd) and stoichiometry (nb) using spectrophotometric methods. Binding was reversible and the estimated affinities for both protein targets obtained here (Kd values of 14 and 20 µM for BSA and CD40L, respectively) were in good agreement with that expected from the protein–protein interaction (PPI) inhibitory activity of ErB. A stoichiometry greater than one was observed both for CD40L and BSA binding (nb of 5–6 and 8–9 for BSA and CD40L, respectively) indicating the possibility of nonspecific binding of the flat an rigid ErB molecule at multiple sites, which could explain the promiscuous PPI inhibitory activity if some of these overlap with the binding site of the protein partner and interfere with the binding. PMID:23456742

  14. Sterol Carrier Protein-2: Binding Protein for Endocannabinoids

    PubMed Central

    Liedhegner, Elizabeth Sabens; Vogt, Caleb D.; Sem, Daniel S.; Cunningham, Christopher W.

    2015-01-01

    The endocannabinoid (eCB) system, consisting of eCB ligands and the type 1 cannabinoid receptor (CB1R), subserves retrograde, activity-dependent synaptic plasticity in the brain. eCB signaling occurs “on-demand,” thus the processes regulating synthesis, mobilization and degradation of eCBs are also primary mechanisms for the regulation of CB1R activity. The eCBs, N-arachidonylethanolamine (AEA) and 2-arachidonoylglycerol (2-AG), are poorly soluble in water. We hypothesize that their aqueous solubility, and, therefore, their intracellular and transcellular distribution, are facilitated by protein binding. Using in silico docking studies, we have identified the nonspecific lipid binding protein, sterol carrier protein 2 (SCP-2), as a potential AEA binding protein. The docking studies predict that AEA and AM404 associate with SCP-2 at a putative cholesterol binding pocket with ΔG values of −3.6 and −4.6 kcal/mol, respectively. These values are considerably higher than cholesterol (−6.62 kcal/mol) but consistent with a favorable binding interaction. In support of the docking studies, SCP-2-mediated transfer of cholesterol in vitro is inhibited by micromolar concentrations of AEA; and heterologous expression of SCP-2 in HEK 293 cells increases time-related accumulation of AEA in a temperature-dependent fashion. These results suggest that SCP-2 facilitates cellular uptake of AEA. However, there is no effect of SCP-2 transfection on the cellular accumulation of AEA determined at equilibrium or the IC50 values for AEA, AM404 or 2-AG to inhibit steady state accumulation of radiolabelled AEA. We conclude that SCP-2 is a low affinity binding protein for AEA that can facilitate its cellular uptake but does not contribute significantly to intracellular sequestration of AEA. PMID:24510313

  15. Gene encoding herbicide safener binding protein

    SciTech Connect

    Walton, J.D.; Scott-Craig, J.S.

    1999-10-26

    The cDNA encoding safener binding protein (SafBP), also referred to as SBP1, is presented. The deduced amino acid sequence is provided. Methods of making and using SBP1 and SafBP to alter a plant's sensitivity to certain herbicides or a plant's responsiveness to certain safeners are also provided, as well as expression vectors, transgenic plants or other organisms transfected with vectors and seeds from the plants.

  16. Polynucleotides encoding TRF1 binding proteins

    DOEpatents

    Campisi, Judith; Kim, Sahn-Ho

    2002-01-01

    The present invention provides a novel telomere associated protein (Trf1-interacting nuclear protein 2 "Tin2") that hinders the binding of Trf1 to its specific telomere repeat sequence and mediates the formation of a Tin2-Trf1-telomeric DNA complex that limits telomerase access to the telomere. Also included are the corresponding nucleic acids that encode the Tin2 of the present invention, as well as mutants of Tin2. Methods of making, purifying and using Tin2 of the present invention are described. In addition, drug screening assays to identify drugs that mimic and/or complement the effect of Tin2 are presented.

  17. Evidence for covalent binding of acyl glucuronides to serum albumin via an imine mechanism as revealed by tandem mass spectrometry.

    PubMed Central

    Ding, A; Ojingwa, J C; McDonagh, A F; Burlingame, A L; Benet, L Z

    1993-01-01

    Acyl glucuronide metabolites of bilirubin and many drugs can react with serum albumin in vivo to form covalent adducts. Such adducts may be responsible for some toxic effects of carboxylic nonsteroidal antiinflammatory agents. The mechanism of formation of the adducts and their chemical structures are unknown. In this paper we describe the use of tandem mass spectrometry to locate binding sites and elucidate the binding mechanism involved in the formation of covalent adducts from tolmetin glucuronide and albumin in vitro. Human serum albumin and excess tolmetin glucuronide were coincubated in the presence of sodium cyanoborohydride to trap imine intermediates. The total protein product was reduced, carboxymethylated, and digested with trypsin. Six tolmetin-containing peptides (indicated by absorbance at 313 nm) were isolated by high-pressure liquid chromatography and analyzed by liquid secondary-ion mass spectrometry and collision-induced dissociation, using a four-sector tandem mass spectrometer. All six peptides contained tolmetin linked covalently via a glucuronic acid to protein lysine groups. Major attachment sites on the protein were Lys-195, -199, and -525; minor sites were identified as Lys-137, -351, and -541. Our results show unambiguously that the glucuronic acid moiety of acyl glucuronides can be retained within the structure when these reactive metabolites bind covalently to proteins, and they suggest that acyl migration followed by Schiff base (imine) formation is a credible mechanism for the generation of covalent adducts in vivo. PMID:8483897

  18. Calibration-free concentration analysis of protein biomarkers in human serum using surface plasmon resonance.

    PubMed

    Grover Shah, Veenita; Ray, Sandipan; Karlsson, Robert; Srivastava, Sanjeeva

    2015-11-01

    In complex biological samples such as serum, determination of specific and active concentration of target proteins, independent of a calibration curve, will be valuable in many applications. Calibration-free concentration analysis (CFCA) is a surface plasmon resonance (SPR)-based label-free approach, which calculates active concentration of proteins using their known diffusion coefficient and observed changes in binding rates at different flow rates under diffusion-limited conditions. Here, for the first time we demonstrate the application of CFCA for determining protein biomarker abundance, specifically serum amyloid A (SAA), directly in the serum samples of patients suffering from different infectious and non-infectious diseases. The assay involves preparation of appropriate reaction surfaces by immobilizing antibodies on CM5 chips via amine coupling followed by serum sample preparation and injection over activated and reference surfaces at flow-rates of 5 and 100 μL/min. The system was validated in healthy and diseased (infectious and non-infectious) serum samples by quantifying two different proteins: β2-microglobulin (β2M) and SAA. All concentration assays were performed for nearly 100 serum samples, which showed reliable quantification in unattended runs with high accuracy and sensitivity. The method could detect the serum β2M to as low as 13 ng/mL in 1000-fold serum dilution, indicating the possible utility of this approach to detect low abundance protein biomarkers in body fluids. Applying the CFCA approach, significant difference in serum abundance of SAA was identified in diseased subjects as compared to the healthy controls, which correlated well with our previous proteomic investigations. Estimation of SAA concentration for a subset of healthy and diseased sera was also performed using ELISA, and the trend was observed to be similar in both SPR assay and ELISA. The reproducibility of CFCA in various serum samples made the interpretation of assay

  19. Binding of transition metals to S100 proteins.

    PubMed

    Gilston, Benjamin A; Skaar, Eric P; Chazin, Walter J

    2016-08-01

    The S100 proteins are a unique class of EF-hand Ca(2+) binding proteins distributed in a cell-specific, tissue-specific, and cell cycle-specific manner in humans and other vertebrates. These proteins are distinguished by their distinctive homodimeric structure, both intracellular and extracellular functions, and the ability to bind transition metals at the dimer interface. Here we summarize current knowledge of S100 protein binding of Zn(2+), Cu(2+) and Mn(2+) ions, focusing on binding affinities, conformational changes that arise from metal binding, and the roles of transition metal binding in S100 protein function. PMID:27430886

  20. Coordinate estrogen-regulated instability of serum protein-coding messenger RNAs in Xenopus laevis.

    PubMed

    Pastori, R L; Moskaitis, J E; Buzek, S W; Schoenberg, D R

    1991-04-01

    Estrogen causes the cytoplasmic destabilization of albumin and gamma-fibrinogen mRNA in Xenopus laevis liver. The purpose of the present study was to determine whether mRNA destabilization is a generalized phenomenon in response to estrogen, or whether this process is restricted to a particular class of mRNAs. To address this, we have expanded our bank of serum protein-coding cDNA clones to include transferrin, the second protein of inter-alpha-trypsin inhibitor and clone 12B, for which there is no mammalian homolog. Together with albumin and gamma-fibrinogen, these represent more than 85% of the mRNAs encoding liver secreted proteins. Estrogen administration to male Xenopus or to liver explant cultures causes the generalized disappearance of all of these mRNAs. In contrast, estrogen has no effect on actin, ferritin, or poly(A)-binding protein mRNA, all of which encode intracellular proteins. We have previously demonstrated that albumin mRNA is degraded in both messenger ribonucleoprotein and polysome fractions. Sucrose gradient analysis demonstrates the same pattern for degradation of all other serum protein-coding mRNAs. Estrogen has no effect on the amounts or gradient distribution of actin, ferritin, or poly(A)-binding protein mRNA. We conclude that regulated destabilization of mRNAs encoding secreted proteins is a generalized phenomenon in response to estrogen stimulation of Xenopus liver. PMID:1922078

  1. Serum Immune-Related Proteins are Differentially Expressed during Hibernation in the American Black Bear

    PubMed Central

    Chow, Brian A.; Donahue, Seth W.; Vaughan, Michael R.; McConkey, Brendan; Vijayan, Mathilakath M.

    2013-01-01

    Hibernation is an adaptation to conserve energy in the face of extreme environmental conditions and low food availability that has risen in several animal phyla. This phenomenon is characterized by reduced metabolic rate (∼25% of the active basal metabolic rate in hibernating bears) and energy demand, while other physiological adjustments are far from clear. The profiling of the serum proteome of the American black bear (Ursus americanus) may reveal specific proteins that are differentially modulated by hibernation, and provide insight into the remarkable physiological adaptations that characterize ursid hibernation. In this study, we used differential gel electrophoresis (DIGE) analysis, liquid chromatography coupled to tandem mass spectrometry, and subsequent MASCOT analysis of the mass spectra to identify candidate proteins that are differentially expressed during hibernation in captive black bears. Seventy serum proteins were identified as changing by ±1.5 fold or more, out of which 34 proteins increased expression during hibernation. The majority of identified proteins are involved in immune system processes. These included α2-macroglobulin, complement components C1s and C4, immunoglobulin μ and J chains, clusterin, haptoglobin, C4b binding protein, kininogen 1, α2-HS-glycoprotein, and apoplipoproteins A-I and A-IV. Differential expression of a subset of these proteins identified by proteomic analysis was also confirmed by immunodetection. We propose that the observed serum protein changes contribute to the maintenance of the hibernation phenotype and health, including increased capacities for bone maintenance and wound healing during hibernation in bears. PMID:23825529

  2. Serum immune-related proteins are differentially expressed during hibernation in the American black bear.

    PubMed

    Chow, Brian A; Donahue, Seth W; Vaughan, Michael R; McConkey, Brendan; Vijayan, Mathilakath M

    2013-01-01

    Hibernation is an adaptation to conserve energy in the face of extreme environmental conditions and low food availability that has risen in several animal phyla. This phenomenon is characterized by reduced metabolic rate (∼25% of the active basal metabolic rate in hibernating bears) and energy demand, while other physiological adjustments are far from clear. The profiling of the serum proteome of the American black bear (Ursus americanus) may reveal specific proteins that are differentially modulated by hibernation, and provide insight into the remarkable physiological adaptations that characterize ursid hibernation. In this study, we used differential gel electrophoresis (DIGE) analysis, liquid chromatography coupled to tandem mass spectrometry, and subsequent MASCOT analysis of the mass spectra to identify candidate proteins that are differentially expressed during hibernation in captive black bears. Seventy serum proteins were identified as changing by ±1.5 fold or more, out of which 34 proteins increased expression during hibernation. The majority of identified proteins are involved in immune system processes. These included α2-macroglobulin, complement components C1s and C4, immunoglobulin μ and J chains, clusterin, haptoglobin, C4b binding protein, kininogen 1, α2-HS-glycoprotein, and apoplipoproteins A-I and A-IV. Differential expression of a subset of these proteins identified by proteomic analysis was also confirmed by immunodetection. We propose that the observed serum protein changes contribute to the maintenance of the hibernation phenotype and health, including increased capacities for bone maintenance and wound healing during hibernation in bears. PMID:23825529

  3. Crystal structure of equine serum albumin in complex with cetirizine reveals a novel drug binding site.

    PubMed

    Handing, Katarzyna B; Shabalin, Ivan G; Szlachta, Karol; Majorek, Karolina A; Minor, Wladek

    2016-03-01

    Serum albumin (SA) is the main transporter of drugs in mammalian blood plasma. Here, we report the first crystal structure of equine serum albumin (ESA) in complex with antihistamine drug cetirizine at a resolution of 2.1Å. Cetirizine is bound in two sites--a novel drug binding site (CBS1) and the fatty acid binding site 6 (CBS2). Both sites differ from those that have been proposed in multiple reports based on equilibrium dialysis and fluorescence studies for mammalian albumins as cetirizine binding sites. We show that the residues forming the binding pockets in ESA are highly conserved in human serum albumin (HSA), and suggest that binding of cetirizine to HSA will be similar. In support of that hypothesis, we show that the dissociation constants for cetirizine binding to CBS2 in ESA and HSA are identical using tryptophan fluorescence quenching. Presence of lysine and arginine residues that have been previously reported to undergo nonenzymatic glycosylation in CBS1 and CBS2 suggests that cetirizine transport in patients with diabetes could be altered. A review of all available SA structures from the PDB shows that in addition to the novel drug binding site we present here (CBS1), there are two pockets on SA capable of binding drugs that do not overlap with fatty acid binding sites and have not been discussed in published reviews. PMID:26896718

  4. 1HNMR study of methotrexate serum albumin (MTX SA) binding in rheumatoid arthritis

    NASA Astrophysics Data System (ADS)

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

    2008-11-01

    Rheumatoid arthritis (RA) is an immunologically depended disease. It is characterized by a chronic, progressive inflammatory process. Methotrexate (4-amino-10-methylfolic acid, MTX) is the modifying drug used to treat RA. The aim of the presented studies is to determine the low affinity binding site of MTX in bovine (BSA) and human (HSA) serum albumin with the use of proton nuclear magnetic resonance ( 1HNMR) spectroscopy. The analysis of 1HNMR spectra of MTX in the presence of serum albumin (SA) allows us to observe the interactions between aromatic rings of the drug and the rings of amino acids located in the hydrophobic subdomains of the protein. On the basis of the chemical shifts σ [ppm] and the relaxation times T1 [s] of drug protons the hydrophobic interaction between MTX-SA and the stoichiometric molar ratio of the complex was evaluated. This work is a part of a spectroscopic study on MTX-SA interactions [A. Sułkowska, M. Maciążek, J. Równicka, B. Bojko, D. Pentak, W.W. Sułkowski, J. Mol. Struct. 834-836 (2007) 162-169].

  5. Systematic discovery of Xist RNA binding proteins

    PubMed Central

    Chu, Ci; Zhang, Qiangfeng Cliff; da Rocha, Simão Teixeira; Flynn, Ryan A.; Bharadwaj, Maheetha; Calabrese, J. Mauro; Magnuson, Terry; Heard, Edith; Chang, Howard Y.

    2015-01-01

    Summary Noncoding RNAs (ncRNAs) function with associated proteins to effect complex structural and regulatory outcomes. To reveal the composition and dynamics of specific noncoding RNA- protein complexes (RNPs) in vivo, we developed comprehensive identification of RNA-binding proteins by mass spectrometry (ChIRP-MS). ChIRP-MS analysis of four ncRNAs captures key protein interactors, including a U1-specific link to the 3′ RNA processing machinery. Xist, an essential lncRNA for X-chromosome inactivation (XCI), interacts with 81 proteins from chromatin modification, nuclear matrix, and RNA remodeling pathways. The Xist RNA-protein particle assembles in two steps coupled with the transition from pluripotency to differentiation. Specific interactors include HnrnpK that participates in Xist-mediated gene silencing and histone modifications, but not Xist localization and Drosophila Split ends homolog Spen that interacts via the A-repeat domain of Xist and is required for gene silencing. Thus, Xist lncRNA engages with proteins in a modular and developmentally controlled manner to coordinate chromatin spreading and silencing. PMID:25843628

  6. Systematic discovery of Xist RNA binding proteins.

    PubMed

    Chu, Ci; Zhang, Qiangfeng Cliff; da Rocha, Simão Teixeira; Flynn, Ryan A; Bharadwaj, Maheetha; Calabrese, J Mauro; Magnuson, Terry; Heard, Edith; Chang, Howard Y

    2015-04-01

    Noncoding RNAs (ncRNAs) function with associated proteins to effect complex structural and regulatory outcomes. To reveal the composition and dynamics of specific noncoding RNA-protein complexes (RNPs) in vivo, we developed comprehensive identification of RNA binding proteins by mass spectrometry (ChIRP-MS). ChIRP-MS analysis of four ncRNAs captures key protein interactors, including a U1-specific link to the 3' RNA processing machinery. Xist, an essential lncRNA for X chromosome inactivation (XCI), interacts with 81 proteins from chromatin modification, nuclear matrix, and RNA remodeling pathways. The Xist RNA-protein particle assembles in two steps coupled with the transition from pluripotency to differentiation. Specific interactors include HnrnpK, which participates in Xist-mediated gene silencing and histone modifications but not Xist localization, and Drosophila Split ends homolog Spen, which interacts via the A-repeat domain of Xist and is required for gene silencing. Thus, Xist lncRNA engages with proteins in a modular and developmentally controlled manner to coordinate chromatin spreading and silencing. PMID:25843628

  7. Identification of C1q as a Binding Protein for Advanced Glycation End Products.

    PubMed

    Chikazawa, Miho; Shibata, Takahiro; Hatasa, Yukinori; Hirose, Sayumi; Otaki, Natsuki; Nakashima, Fumie; Ito, Mika; Machida, Sachiko; Maruyama, Shoichi; Uchida, Koji

    2016-01-26

    Advanced glycation end products (AGEs) make up a heterogeneous group of molecules formed from the nonenzymatic reaction of reducing sugars with the free amino groups of proteins. The abundance of AGEs in a variety of age-related diseases, including diabetic complications and atherosclerosis, and their pathophysiological effects suggest the existence of innate defense mechanisms. Here we examined the presence of serum proteins that are capable of binding glycated bovine serum albumin (AGEs-BSA), prepared upon incubation of BSA with dehydroascorbate, and identified complement component C1q subcomponent subunit A as a novel AGE-binding protein in human serum. A molecular interaction analysis showed the specific binding of C1q to the AGEs-BSA. In addition, we identified DNA-binding regions of C1q, including a collagen-like domain, as the AGE-binding site and established that the amount of positive charge on the binding site was the determining factor. C1q indeed recognized several other modified proteins, including acylated proteins, suggesting that the binding specificity of C1q might be ascribed, at least in part, to the electronegative potential of the ligand proteins. We also observed that C1q was involved in the AGEs-BSA-activated deposition of complement proteins, C3b and C4b. In addition, the AGEs-BSA mediated the proteolytic cleavage of complement protein 5 to release C5a. These findings provide the first evidence of AGEs as a new ligand recognized by C1q, stimulating the C1q-dependent classical complement pathway. PMID:26731343

  8. Suppression of severe lesions, myonecrosis and hemorrhage, caused by Protobothrops flavoviridis venom with its serum proteins.

    PubMed

    Chijiwa, Takahito; So, Shuhei; Hattori, Shosaku; Yoshida, Aichi; Oda-Ueda, Naoko; Ohno, Motonori

    2013-12-15

    Protobothrops flavoviridis serum proteins precipitated with ammonium sulfate were chromatographed on a DEAE-Toyopearl 650M column at pH 7.5 with stepwise increase or with linear gradient of NaCl concentration. Peaks 3 and 4 serum proteins, obtained by linear gradient elution and named Fr(de3) and Fr(de4), contained Habu serum factors (HSF) and phospholipase A2 (PLA2) inhibitors (PfPLI), respectively. The serum proteins eluted at 0.2 M NaCl by stepwise elution, named Fr(0.2NaCl), effectively suppressed myonecrosis and hemorrhage caused by P. flavoviridis venom in rat or mouse thigh muscles. The Fr(0.2NaCl) were fractionated by HPLC and the fractions, after SDS-PAGE, underwent far-western blot analysis with PLA2 ([Asp(49)]PLA2) and BPI ([Lys(49)]PLA2) as the probes. Four PfPLIs, namely, PfαPLI-A, PfαPLI-B, PfγPLI-A and PfγPLI-B, were identified together with their selective binding specificities to PLA2 species. In addition, a new 9 kDa protein, which is specifically bound to BPI, was found. Suppression of P. flavoviridis venom-induced severe lesions, such as myonecrosis, hemorrhage and edema, with its serum proteins was histopathologically observed in the present work for the first time. The cooperative use of P. flavoviridis antivenom and its serum proteins as medication for P. flavoviridis snake bites is discussed. PMID:24139850

  9. Fluorescent protein-based detection of unconjugated bilirubin in newborn serum

    PubMed Central

    Iwatani, Sota; Nakamura, Hajime; Kurokawa, Daisuke; Yamana, Keiji; Nishida, Kosuke; Fukushima, Sachiyo; Koda, Tsubasa; Nishimura, Noriyuki; Nishio, Hisahide; Iijima, Kazumoto; Miyawaki, Atsushi; Morioka, Ichiro

    2016-01-01

    Increased serum levels of unconjugated bilirubin are associated with the development of brain damage in newborns. In current clinical settings, there are no methods for directly determining serum levels of unconjugated bilirubin. UnaG, a fluorescent protein from Japanese eel muscle that specifically binds to unconjugated bilirubin was used in this study. Linear regression analysis was carried out to compare unconjugated bilirubin levels measured by UnaG and conventional bilirubin oxidase methods. Unconjugated bilirubin levels in the serum of newborns who were untreated or treated with phototherapy were compared. Effects of interfering factors in the serum (conjugated bilirubin, hemoglobin, and lipid) on unconjugated bilirubin concentration measured by the UnaG method were also evaluated. Unconjugated bilirubin levels measured by the UnaG method were highly correlated with those determined by the bilirubin oxidase assay. Unconjugated bilirubin levels determined by bilirubin oxidase and UnaG assays were similar in serum samples containing conjugated bilirubin. The performance of the UnaG assay was unaffected by phototherapy and the presence of serum hemoglobin and lipid emulsion. These results demonstrate the clinical applicability of the UnaG method for direct measurement of unconjugated bilirubin levels in newborn serum. PMID:27324682

  10. Fluorescent protein-based detection of unconjugated bilirubin in newborn serum.

    PubMed

    Iwatani, Sota; Nakamura, Hajime; Kurokawa, Daisuke; Yamana, Keiji; Nishida, Kosuke; Fukushima, Sachiyo; Koda, Tsubasa; Nishimura, Noriyuki; Nishio, Hisahide; Iijima, Kazumoto; Miyawaki, Atsushi; Morioka, Ichiro

    2016-01-01

    Increased serum levels of unconjugated bilirubin are associated with the development of brain damage in newborns. In current clinical settings, there are no methods for directly determining serum levels of unconjugated bilirubin. UnaG, a fluorescent protein from Japanese eel muscle that specifically binds to unconjugated bilirubin was used in this study. Linear regression analysis was carried out to compare unconjugated bilirubin levels measured by UnaG and conventional bilirubin oxidase methods. Unconjugated bilirubin levels in the serum of newborns who were untreated or treated with phototherapy were compared. Effects of interfering factors in the serum (conjugated bilirubin, hemoglobin, and lipid) on unconjugated bilirubin concentration measured by the UnaG method were also evaluated. Unconjugated bilirubin levels measured by the UnaG method were highly correlated with those determined by the bilirubin oxidase assay. Unconjugated bilirubin levels determined by bilirubin oxidase and UnaG assays were similar in serum samples containing conjugated bilirubin. The performance of the UnaG assay was unaffected by phototherapy and the presence of serum hemoglobin and lipid emulsion. These results demonstrate the clinical applicability of the UnaG method for direct measurement of unconjugated bilirubin levels in newborn serum. PMID:27324682

  11. Cation specific binding with protein surface charges.

    PubMed

    Hess, Berk; van der Vegt, Nico F A

    2009-08-11

    Biological organization depends on a sensitive balance of noncovalent interactions, in particular also those involving interactions between ions. Ion-pairing is qualitatively described by the law of "matching water affinities." This law predicts that cations and anions (with equal valence) form stable contact ion pairs if their sizes match. We show that this simple physical model fails to describe the interaction of cations with (molecular) anions of weak carboxylic acids, which are present on the surfaces of many intra- and extracellular proteins. We performed molecular simulations with quantitatively accurate models and observed that the order K(+) < Na(+) < Li(+) of increasing binding affinity with carboxylate ions is caused by a stronger preference for forming weak solvent-shared ion pairs. The relative insignificance of contact pair interactions with protein surfaces indicates that thermodynamic stability and interactions between proteins in alkali salt solutions is governed by interactions mediated through hydration water molecules. PMID:19666545

  12. Copper-binding protein in Mimulus guttatus

    SciTech Connect

    Robinson, N.J.; Thurman, D.A.

    1985-01-01

    A Cu-binding protein has been purified from the roots of Mimulus guttatus using gel permeation chromatography on Sephadex G-75 and anion exchange chromatography on DEAE Biogel A. The protein has similar properties to putative metallothioneins (MTS) purified from other angiosperms. Putative MT was estimated by measuring the relative percentage incorporation of (/sup 35/S) into fractions containing the protein after HPLC on SW 3000-gel. In the roots of both Cu-tolerant and non tolerant plants synthesis of putative MT is induced by increased Cu concentration in the nutrient solution. The relative percentage incorporation of (/sup 35/S) into putative MT is significantly higher in extracts from the roots of Cu-tolerant than non tolerant M. guttatus after growth in 1 ..mu..M Cu suggesting involvement in the mechanism of tolerance. 22 refs., 2 figs., 1 tab.

  13. DNA and RNA Quadruplex-Binding Proteins

    PubMed Central

    Brázda, Václav; Hároníková, Lucia; Liao, Jack C. C.; Fojta, Miroslav

    2014-01-01

    Four-stranded DNA structures were structurally characterized in vitro by NMR, X-ray and Circular Dichroism spectroscopy in detail. Among the different types of quadruplexes (i-Motifs, minor groove quadruplexes, G-quadruplexes, etc.), the best described are G-quadruplexes which are featured by Hoogsteen base-paring. Sequences with the potential to form quadruplexes are widely present in genome of all organisms. They are found often in repetitive sequences such as telomeric ones, and also in promoter regions and 5' non-coding sequences. Recently, many proteins with binding affinity to G-quadruplexes have been identified. One of the initially portrayed G-rich regions, the human telomeric sequence (TTAGGG)n, is recognized by many proteins which can modulate telomerase activity. Sequences with the potential to form G-quadruplexes are often located in promoter regions of various oncogenes. The NHE III1 region of the c-MYC promoter has been shown to interact with nucleolin protein as well as other G-quadruplex-binding proteins. A number of G-rich sequences are also present in promoter region of estrogen receptor alpha. In addition to DNA quadruplexes, RNA quadruplexes, which are critical in translational regulation, have also been predicted and observed. For example, the RNA quadruplex formation in telomere-repeat-containing RNA is involved in interaction with TRF2 (telomere repeat binding factor 2) and plays key role in telomere regulation. All these fundamental examples suggest the importance of quadruplex structures in cell processes and their understanding may provide better insight into aging and disease development. PMID:25268620

  14. DNA and RNA quadruplex-binding proteins.

    PubMed

    Brázda, Václav; Hároníková, Lucia; Liao, Jack C C; Fojta, Miroslav

    2014-01-01

    Four-stranded DNA structures were structurally characterized in vitro by NMR, X-ray and Circular Dichroism spectroscopy in detail. Among the different types of quadruplexes (i-Motifs, minor groove quadruplexes, G-quadruplexes, etc.), the best described are G-quadruplexes which are featured by Hoogsteen base-paring. Sequences with the potential to form quadruplexes are widely present in genome of all organisms. They are found often in repetitive sequences such as telomeric ones, and also in promoter regions and 5' non-coding sequences. Recently, many proteins with binding affinity to G-quadruplexes have been identified. One of the initially portrayed G-rich regions, the human telomeric sequence (TTAGGG)n, is recognized by many proteins which can modulate telomerase activity. Sequences with the potential to form G-quadruplexes are often located in promoter regions of various oncogenes. The NHE III1 region of the c-MYC promoter has been shown to interact with nucleolin protein as well as other G-quadruplex-binding proteins. A number of G-rich sequences are also present in promoter region of estrogen receptor alpha. In addition to DNA quadruplexes, RNA quadruplexes, which are critical in translational regulation, have also been predicted and observed. For example, the RNA quadruplex formation in telomere-repeat-containing RNA is involved in interaction with TRF2 (telomere repeat binding factor 2) and plays key role in telomere regulation. All these fundamental examples suggest the importance of quadruplex structures in cell processes and their understanding may provide better insight into aging and disease development. PMID:25268620

  15. A Crayfish Insulin-like-binding Protein

    PubMed Central

    Rosen, Ohad; Weil, Simy; Manor, Rivka; Roth, Ziv; Khalaila, Isam; Sagi, Amir

    2013-01-01

    Across the animal kingdom, the involvement of insulin-like peptide (ILP) signaling in sex-related differentiation processes is attracting increasing attention. Recently, a gender-specific ILP was identified as the androgenic sex hormone in Crustacea. However, moieties modulating the actions of this androgenic insulin-like growth factor were yet to be revealed. Through molecular screening of an androgenic gland (AG) cDNA library prepared from the crayfish Cherax quadricarinatus, we have identified a novel insulin-like growth factor-binding protein (IGFBP) termed Cq-IGFBP. Based on bioinformatics analyses, the deduced Cq-IGFBP was shown to share high sequence homology with IGFBP family members from both invertebrates and vertebrates. The protein also includes a sequence determinant proven crucial for ligand binding, which according to three-dimensional modeling is assigned to the exposed outer surface of the protein. Recombinant Cq-IGFBP (rCq-IGFBP) protein was produced and, using a “pulldown” methodology, was shown to specifically interact with the insulin-like AG hormone of the crayfish (Cq-IAG). Particularly, using both mass spectral analysis and an immunological tool, rCq-IGFBP was shown to bind the Cq-IAG prohormone. Furthermore, a peptide corresponding to residues 23–38 of the Cq-IAG A-chain was found sufficient for in vitro recognition by rCq-IGFBP. Cq-IGFBP is the first IGFBP family member shown to specifically interact with a gender-specific ILP. Unlike their ILP ligands, IGFBPs are highly conserved across evolution, from ancient arthropods, like crustaceans, to humans. Such conservation places ILP signaling at the center of sex-related phenomena in early animal development. PMID:23775079

  16. Identification of DNA-binding and protein-binding proteins using enhanced graph wavelet features.

    PubMed

    Zhu, Yuan; Zhou, Weiqiang; Dai, Dao-Qing; Yan, Hong

    2013-01-01

    Interactions between biomolecules play an essential role in various biological processes. For predicting DNA-binding or protein-binding proteins, many machine-learning-based techniques have used various types of features to represent the interface of the complexes, but they only deal with the properties of a single atom in the interface and do not take into account the information of neighborhood atoms directly. This paper proposes a new feature representation method for biomolecular interfaces based on the theory of graph wavelet. The enhanced graph wavelet features (EGWF) provides an effective way to characterize interface feature through adding physicochemical features and exploiting a graph wavelet formulation. Particularly, graph wavelet condenses the information around the center atom, and thus enhances the discrimination of features of biomolecule binding proteins in the feature space. Experiment results show that EGWF performs effectively for predicting DNA-binding and protein-binding proteins in terms of Matthew's correlation coefficient (MCC) score and the area value under the receiver operating characteristic curve (AUC). PMID:24334394

  17. Effect of protein binding on the in vitro activity and pharmacodynamics of faropenem.

    PubMed

    Boswell, F J; Ashby, J P; Andrews, J M; Wise, R

    2002-10-01

    The influence of protein binding upon different aspects of the in vitro activity of faropenem on recently isolated Staphylococcus aureus and respiratory pathogens was determined. The protein binding of faropenem was investigated in inactivated human serum and albumin by ultrafiltration. The effect of the presence of inactivated human serum and albumin on the in vitro activity of faropenem and amoxicillin was established and the influence of protein binding on the pharmacodynamic properties of faropenem and amoxicillin was compared. The protein binding of faropenem was 96% and 95% in pooled inactivated human serum and 99% and 98% in 45 mg/L human albumin, at 8 and 25 mg/L, respectively. The presence of inactivated human serum (20% and 70%) increased the mean faropenem MICs by two dilution steps and albumin increased the mean faropenem MICs by three dilution steps. The mean amoxicillin MICs were less affected than faropenem by the presence of either inactivated human serum or albumin. Faropenem and amoxicillin exhibited similar time-dependent kinetics. Faropenem was bacteriostatic on Moraxella catarrhalis, Haemophilus influenzae and group A streptococci, and bactericidal for Streptococcus pneumoniae (after 4 h with concentrations equivalent to 5 x and 10 x MIC) in Iso-Sensitest broth. In 70% inactivated human serum faropenem was slowly bactericidal against M. catarrhalis, H. influenzae (one strain) and S. pneumoniae (one strain) but not group A streptococci and the other S. pneumoniae strain. A significant inoculum effect was observed with all strains except S. pneumoniae. Both faropenem and amoxicillin appeared more active in 70% inactivated human serum than in Iso-Sensitest broth. PMID:12356797

  18. The factor H binding protein of Neisseria meningitidis interacts with xenosiderophores in vitro.

    PubMed

    Veggi, Daniele; Gentile, Maria A; Cantini, Francesca; Lo Surdo, Paola; Nardi-Dei, Vincenzo; Seib, Kate L; Pizza, Mariagrazia; Rappuoli, Rino; Banci, Lucia; Savino, Silvana; Scarselli, Maria

    2012-11-20

    The factor H binding protein (fHbp) is a key virulence factor of Neisseria meningitidis that confers to the bacterium the ability to resist killing by human serum. The determination of its three-dimensional structure revealed that the carboxyl terminus of the protein folds into an eight-stranded β barrel. The structural similarity of this part of the protein to lipocalins provided the rationale for exploring the ability of fHbp to bind siderophores. We found that fHbp was able to bind in vitro siderophores belonging to the cathecolate family and mapped the interaction site by nuclear magnetic resonance. Our results indicated that the enterobactin binding site was distinct from the site involved in binding to human factor H and stimulates new hypotheses about possible multiple activities of fHbp. PMID:23121397

  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. Competitive protein binding assay for piritrexim

    SciTech Connect

    Woolley, J.L. Jr.; Ringstad, J.L.; Sigel, C.W. )

    1989-09-01

    A competitive protein binding assay for piritrexim (PTX, 1) that makes use of a commercially available radioassay kit for methotrexate has been developed. After it is selectively extracted from plasma, PTX competes with ({sup 125}I)methotrexate for binding to dihydrofolate reductase isolated from Lactobacillus casei. Free drug is separated from bound drug by adsorption to dextran-coated charcoal. Piritrexim is measurable over a range of 0.01 to 10.0 micrograms/mL in plasma with a coefficient of variation less than 15%. The limit of sensitivity of the assay is approximately 2 ng/mL. An excellent correlation between this assay and a previously published HPLC method was found.

  1. In vitro protein binding of liraglutide in human plasma determined by reiterated stepwise equilibrium dialysis

    PubMed Central

    Plum, Anne; Jensen, Lisbeth Bjerring; Kristensen, Jesper Bøggild

    2013-01-01

    Liraglutide is a human glucagon-like peptide-1 (GLP-1) analogue approved for the treatment of type 2 diabetes. It is based on human GLP-1 with the addition of a 16-carbon fatty acid, which facilitates binding to plasma proteins, thus prolonging the elimination half-life and allowing once-daily administration. It has not been possible to quantify liraglutide protein binding by ultrafiltration (the usual method of choice), as the lipophilic molecule becomes trapped in the filter membrane. Therefore, the aim of this study was to develop a methodology that could determine the extent of liraglutide binding to plasma proteins in vitro. We report here the details of a novel reiterated stepwise equilibrium dialysis assay that has successfully been used to quantify liraglutide plasma protein binding. The assay allowed quantification of liraglutide binding to proteins in purified plasma protein solutions and human plasma samples and was effective at plasma dilutions as low as 5%. At a clinically relevant liraglutide concentration (104 pM), greater than 98.9% of liraglutide was bound to protein. Specific binding to human serum albumin and α1-acid glycoprotein was 99.4% and 99.3%, respectively. The novel methodology described herein could have an application in the quantification of plasma protein binding of other highly lipophilic drug molecules. PMID:23853127

  2. Is serum sex hormone-binding globulin a dominant risk factor for metabolic syndrome?

    PubMed Central

    Yang, Yi-Hong; Zhao, Ming-Jia; Zhou, Shan-Jie; Lu, Wen-Hong; Liang, Xiao-Wei; Xiong, Cheng-Liang; Wan, Chang-Chun; Shang, Xue-Jun; Gu, Yi-Qun

    2015-01-01

    This multi-center, cross-sectional study investigated the association between serum testosterone (T) levels, serum sex hormone-binding globulin (SHBG) levels, and the risk of metabolic syndrome (MS) in 3332 adult Chinese men. The prevalence of MS was 34.7%, and men with MS had lower serum levels of total T (TT) and SHBG than those without MS (P < 0.001). There was no significant difference in serum free T (FT) levels between subjects with and without MS (P = 0.627). In logistic regression analysis, the association between MS and serum SHBG levels persisted after adjusting for age, body mass index (BMI), smoking and drinking status, and serum TT (odds ratio [OR] 0.962, 95% confidence interval [95% CI] 0.954−0.969, P< 0.01). However, the association between serum TT level and the risk of MS was weak after adjusting for age, BMI, SHBG level, and smoking and drinking status (OR 0.981, 95% CI 0.960−1.007). Our study reveals that both serum TT and SHBG levels, but not serum FT, are inversely associated with the prevalence of MS and that serum SHBG is an independent and dominant risk factor for MS. PMID:25851658

  3. Identification of diagnostic serum protein profiles of glioblastoma patients.

    PubMed

    Elstner, Anja; Stockhammer, Florian; Nguyen-Dobinsky, Trong-Nghia; Nguyen, Quang Long; Pilgermann, Ingo; Gill, Amanjit; Guhr, Anke; Zhang, Tingguo; von Eckardstein, Kajetan; Picht, Thomas; Veelken, Julian; Martuza, Robert L; von Deimling, Andreas; Kurtz, Andreas

    2011-03-01

    Diagnosis of a glioblastoma (GBM) is triggered by the onset of symptoms and is based on cerebral imaging and histological examination. Serum-based biomarkers may support detection of GBM. Here, we explored serum protein concentrations of GBM patients and used data mining to explore profiles of biomarkers and determine whether these are associated with the clinical status of the patients. Gene and protein expression data for astrocytoma and GBM were used to identify secreted proteins differently expressed in tumors and in normal brain tissues. Tumor expression and serum concentrations of 14 candidate proteins were analyzed for 23 GBM patients and nine healthy subjects. Data-mining methods involving all 14 proteins were used as an initial evaluation step to find clinically informative profiles. Data mining identified a serum protein profile formed by BMP2, HSP70, and CXCL10 that enabled correct assignment to the GBM group with specificity and sensitivity of 89 and 96%, respectively (p < 0.0001, Fischer's exact test). Survival for more than 15 months after tumor resection was associated with a profile formed by TSP1, HSP70, and IGFBP3, enabling correct assignment in all cases (p < 0.0001, Fischer's exact test). No correlation was found with tumor size or age of the patient. This study shows that robust serum profiles for GBM may be identified by data mining on the basis of a relatively small study cohort. Profiles of more than one biomarker enable more specific assignment to the GBM and survival group than those based on single proteins, confirming earlier attempts to correlate single markers with cancer. These conceptual findings will be a basis for validation in a larger sample size. PMID:20617365

  4. Serum protein polymorphism in Bali (Indonesia).

    PubMed

    Constans, J; Gouaillard, C; Breguet, G

    1986-01-01

    Serum samples from Bali, obtained in three different ethnic groups and in one isolated village were tested by isoelectric focusing electrophoresis for Gc, Pi, Tf and Hp subtyping. In addition to the three common alleles Gc1F, Gc1S and Gc2, two variants Gc1A1 and Gc1A8 were observed. In the Pi system, five alleles were present: PiM1, PiM2, PiM3, PiM4 and PiX. The Tf variability was exceptional with the presence of eight alleles: TfB1, TfC1, TfC2, TfC3, TfC4, TfC8, TfD1 and TfDchi. For Hp, there were two common alleles Hp1S and Hp1FS and two rare ones: Hp1F and Hp2SS. As expected, the genetic polymorphism is reduced in the isolated community. The anthropological significance of these genetic data is discussed. PMID:3493727

  5. Spectroscopic characterization of effective components anthraquinones in Chinese medicinal herbs binding with serum albumins

    NASA Astrophysics Data System (ADS)

    Bi, Shuyun; Song, Daqian; Kan, Yuhe; Xu, Dong; Tian, Yuan; Zhou, Xin; Zhang, Hanqi

    2005-11-01

    The interactions of serum albumins such as human serum albumin (HSA) and bovine serum albumin (BSA) with emodin, rhein, aloe-emodin and aloin were assessed employing fluorescence quenching and absorption spectroscopic techniques. The results obtained revealed that there are relatively strong binding affinity for the four anthraquinones with HSA and BSA and the binding constants for the interactions of anthraquinones with HSA or BSA at 20 °C were obtained. Anthraquinone-albumin interactions were studied at different temperatures and in the presence of some metal ions. And the competition binding of anthraquinones with serum albumins was also discussed. The Stern-Volmer curves suggested that the quenching occurring in the reactions was the static quenching process. The binding distances and transfer efficiencies for each binding reactions were calculated according to the Föster theory of non-radiation energy transfer. Using thermodynamic equations, the main action forces of these reactions were also obtained. The reasons of the different binding affinities for different anthraquinone-albumin reactions were probed from the point of view of molecular structures.

  6. Acute hantavirus infection induces galectin-3-binding protein.

    PubMed

    Hepojoki, Jussi; Strandin, Tomas; Hetzel, Udo; Sironen, Tarja; Klingström, Jonas; Sane, Jussi; Mäkelä, Satu; Mustonen, Jukka; Meri, Seppo; Lundkvist, Ake; Vapalahti, Olli; Lankinen, Hilkka; Vaheri, Antti

    2014-11-01

    Hantaviruses are zoonotic viruses that cause life-threatening diseases when transmitted to humans. Severe hantavirus infection is manifested by impairment of renal function, pulmonary oedema and capillary leakage. Both innate and adaptive immune responses contribute to the pathogenesis, but the underlying mechanisms are not fully understood. Here, we showed that galectin-3-binding protein (Gal-3BP) was upregulated as a result of hantavirus infection both in vitro and in vivo. Gal-3BP is a secreted glycoprotein found in human serum, and increased Gal-3BP levels have been reported in chronic viral infections and in several types of cancer. Our in vitro experiments showed that, whilst Vero E6 cells (an African green monkey kidney cell line) constitutively expressed and secreted Gal-3BP, this protein was detected in primary human cells only as a result of hantavirus infection. Analysis of Gal-3BP levels in serum samples of cynomolgus macaques infected experimentally with hantavirus indicated that hantavirus infection induced Gal-3BP also in vivo. Finally, analysis of plasma samples collected from patients hospitalized because of acute hantavirus infection showed higher Gal-3BP levels during the acute than the convalescent phase. Furthermore, the Gal-3BP levels in patients with haemorrhagic fever with renal syndrome correlated with increased complement activation and with clinical variables reflecting the severity of acute hantavirus infection. PMID:25013204

  7. Landscape of protein-small ligand binding modes.

    PubMed

    Kasahara, Kota; Kinoshita, Kengo

    2016-09-01

    Elucidating the mechanisms of specific small-molecule (ligand) recognition by proteins is a long-standing conundrum. While the structures of these molecules, proteins and ligands, have been extensively studied, protein-ligand interactions, or binding modes, have not been comprehensively analyzed. Although methods for assessing similarities of binding site structures have been extensively developed, the methods for the computational treatment of binding modes have not been well established. Here, we developed a computational method for encoding the information about binding modes as graphs, and assessing their similarities. An all-against-all comparison of 20,040 protein-ligand complexes provided the landscape of the protein-ligand binding modes and its relationships with protein- and chemical spaces. While similar proteins in the same SCOP Family tend to bind relatively similar ligands with similar binding modes, the correlation between ligand and binding similarities was not very high (R(2)  = 0.443). We found many pairs with novel relationships, in which two evolutionally distant proteins recognize dissimilar ligands by similar binding modes (757,474 pairs out of 200,790,780 pairs were categorized into this relationship, in our dataset). In addition, there were an abundance of pairs of homologous proteins binding to similar ligands with different binding modes (68,217 pairs). Our results showed that many interesting relationships between protein-ligand complexes are still hidden in the structure database, and our new method for assessing binding mode similarities is effective to find them. PMID:27327045

  8. Review: modifications of human serum albumin and their binding effect.

    PubMed

    Lee, Philbert; Wu, Xiaoyang

    2015-01-01

    Human serum albumin (HSA) regulates the transport and availability of numerous chemical compounds and molecules in the blood vascular system. While previous HSA research has found that HSA interacts with specific varieties of ligands, new research efforts aim to expand HSA's ability to interact with more different drugs in order to improve the delivery of various pharmacological drugs. This review will cover fatty acid chain and posttranslational modifications of HSA that potentially modulate how HSA interacts with various pharmacological drugs, including glycation, cysteinylation, S-nitrosylation, S-transnitrosation and S-guanylation. PMID:25732553

  9. Review: Modifications of Human Serum Albumin and Their Binding Effect

    PubMed Central

    Lee, Philbert; Wu, Xiaoyang

    2015-01-01

    Human serum albumin (HSA) regulates the transport and availability of numerous chemical compounds and molecules in the blood vascular system. While previous HSA research has found that HSA interacts with specific varieties of ligands, new research efforts aim to expand HSA’s ability to interact with more different drugs in order to improve the delivery of various pharmacological drugs. This review will cover fatty acid chain and post-translational modifications of HSA that potentially modulate how HSA interacts with various pharmacological drugs, including glycation, cysteinylation, S-nitrosylation, S-transnitrosation and S-guanylation. PMID:25732553

  10. Regulation of Pluripotency by RNA Binding Proteins

    PubMed Central

    Ye, Julia; Blelloch, Robert

    2015-01-01

    Establishment, maintenance, and exit from pluripotency require precise coordination of a cell’s molecular machinery. Substantial headway has been made in deciphering many aspects of this elaborate system, particularly with respect to epigenetics, transcription, and noncoding RNAs. Less attention has been paid to posttranscriptional regulatory processes such as alternative splicing, RNA processing and modification, nuclear export, regulation of transcript stability, and translation. Here, we introduce the RNA binding proteins that enable the posttranscriptional regulation of gene expression, summarizing current and ongoing research on their roles at different regulatory points and discussing how they help script the fate of pluripotent stem cells. PMID:25192462

  11. Gene encoding herbicide safener binding protein

    SciTech Connect

    Walton, Jonathan D.; Scott-Craig, John S.

    1999-01-01

    The cDNA encoding safener binding protein (SafBP), also referred to as SBP1, is set forth in FIG. 5 and SEQ ID No. 1. The deduced amino acid sequence is provided in FIG. 5 and SEQ ID No. 2. Methods of making and using SBP1 and SafBP to alter a plant's sensitivity to certain herbicides or a plant's responsiveness to certain safeners are also provided, as well as expression vectors, transgenic plants or other organisms transfected with said vectors and seeds from said plants.

  12. The influence of fatty acids on theophylline binding to human serum albumin. Comparative fluorescence study

    NASA Astrophysics Data System (ADS)

    Maciążek-Jurczyk, M.; Sułkowska, A.; Bojko, B.; Równicka-Zubik, J.; Szkudlarek-Haśnik, A.; Zubik-Skupień, I.; Góra, A.; Dubas, M.; Korzonek-Szlacheta, I.; Wielkoszyński, T.; Żurawiński, W.; Sosada, K.

    2012-04-01

    Theophylline, popular diuretic, is used to treat asthma and bronchospasm. In blood it forms complexes with albumin, which is also the main transporter of fatty acids. The aim of the present study was to describe the influence of fatty acids (FA) on binding of theophylline (Th) to human serum albumin (HSA) in the high affinity binding sites. Binding parameters have been obtained on the basis of the fluorescence analysis. The data obtained for the complex of Th and natural human serum albumin (nHSA) obtained from blood of obese patients qualified for surgical removal of stomach was compared with our previous studies on the influence of FA on the complex of Th and commercially available defatted human serum albumin (dHSA).

  13. Computational Design of DNA-Binding Proteins.

    PubMed

    Thyme, Summer; Song, Yifan

    2016-01-01

    Predicting the outcome of engineered and naturally occurring sequence perturbations to protein-DNA interfaces requires accurate computational modeling technologies. It has been well established that computational design to accommodate small numbers of DNA target site substitutions is possible. This chapter details the basic method of design used in the Rosetta macromolecular modeling program that has been successfully used to modulate the specificity of DNA-binding proteins. More recently, combining computational design and directed evolution has become a common approach for increasing the success rate of protein engineering projects. The power of such high-throughput screening depends on computational methods producing multiple potential solutions. Therefore, this chapter describes several protocols for increasing the diversity of designed output. Lastly, we describe an approach for building comparative models of protein-DNA complexes in order to utilize information from homologous sequences. These models can be used to explore how nature modulates specificity of protein-DNA interfaces and potentially can even be used as starting templates for further engineering. PMID:27094297

  14. Alpha-amylase inhibitor, CS-1036 binds to serum amylase in a concentration-dependent and saturable manner.

    PubMed

    Honda, Tomohiro; Kaneno-Urasaki, Yoko; Ito, Takashi; Kimura, Takako; Matsushima, Nobuko; Okabe, Hiromi; Yamasaki, Atsushi; Izumi, Takashi

    2014-03-01

    (2R,3R,4R)-4-hydroxy-2-(hydroxymethyl)pyrrolidin-3-yl 4-O-(6-deoxy-β-D-glucopyranosyl)-α-D-glucopyranoside (CS-1036), which is an α-amylase inhibitor, exhibited biphasic and sustained elimination with a long t1/2 (18.4-30.0 hours) in rats and monkeys, but exhibited a short t1/2 (3.7-7.9 hours) in humans. To clarify the species differences in the t1/2, the plasma protein binding of CS-1036 was evaluated by ultrafiltration. A concentration-dependent and saturable plasma protein binding of CS-1036 was observed in rats and monkeys with the dissociation rate constant (KD) of 8.95 and 27.2 nM, and maximal binding capacity (Bmax) of 52.8 and 22.1 nM, respectively. By the assessments of the recombinant amylase and immunoprecipitation, the major binding protein of CS-1036 in rats was identified as salivary amylase (KD 5.64 nM). CS-1036 also showed concentration-dependent and saturable binding to human salivary and pancreatic amylase, with similar binding affinity in rats. However, the protein binding of CS-1036 was constant in human plasma (≤10.2%) due to the lower serum amylase level compared with rats and monkeys. From the calculation of the unbound fraction (fu) in plasma based on in vitro KD and Bmax, the dose-dependent increase in fu after oral administration is speculated to lead to a dose-dependent increase in total body clearance and a high area under the curve/dose at lower doses, such as 0.3 mg/kg in rats. PMID:24319124

  15. Probing the micellar properties of Quinacrine 2HCl and its binding with surfactants and Human Serum Albumin

    NASA Astrophysics Data System (ADS)

    Usman, Muhammad; Siddiq, Mohammad

    2013-09-01

    This manuscript reports physicochemical behavior of an antimalarial drug Quinacrine 2HCl (QUN) drug as well as its interaction with surfactant and Human Serum Albumin (HSA). Surface tension and specific conductivity were employed to detect the critical micelle concentration (CMC) and thus its surface and thermodynamic parameters were calculated. Solublization of this drug within micelles of anionic surfactant sodium dodecyl sulfate (SDS) has also been studied. UV/Visible spectroscopy was used to calculate partition coefficient (Kx), free energy of partition and number of drug molecules per micelle. The complexation of drug with HSA at physiological conditions (pH 7.4) has been analyzed by using UV/Visible and fluorescence spectroscopy. In this way the values of drug-protein binding constant, number of binding sites and free energy of binding were calculated.

  16. Measuring Binding Affinity of Protein-Ligand Interaction Using Spectrophotometry: Binding of Neutral Red to Riboflavin-Binding Protein

    ERIC Educational Resources Information Center

    Chenprakhon, Pirom; Sucharitakul, Jeerus; Panijpan, Bhinyo; Chaiyen, Pimchai

    2010-01-01

    The dissociation constant, K[subscript d], of the binding of riboflavin-binding protein (RP) with neutral red (NR) can be determined by titrating RP to a fixed concentration of NR. Upon adding RP to the NR solution, the maximum absorption peak of NR shifts to 545 nm from 450 nm for the free NR. The change of the absorption can be used to determine…

  17. C1q binding and activation of the complement classical pathway by Klebsiella pneumoniae outer membrane proteins.

    PubMed Central

    Albertí, S; Marqués, G; Camprubí, S; Merino, S; Tomás, J M; Vivanco, F; Benedí, V J

    1993-01-01

    The mechanisms of killing of Klebsiella pneumoniae serum-sensitive strains in nonimmune serum by the complement classical pathway have been studied. The bacterial cell surface components that bind C1q more efficiently were identified as two major outer membrane proteins, presumably the porins of this bacterial species. These two outer membrane proteins were isolated from a representative serum-sensitive strain. We have demonstrated that in their purified form, they bind C1q and activate the classical pathway in an antibody-independent manner, with the subsequent consumption of C4 and reduction of the serum total hemolytic activity. Activation of the classical pathway has been observed in human nonimmune serum and agammaglobulinemic serum (both depleted in factor D). Binding of C1q to other components of the bacterial outer membrane, in particular the rough lipopolysaccharide, could not be demonstrated. Activation of the classical pathway by this lipopolysaccharide was also much less efficient than activation by the two outer membrane proteins. The antibody-independent binding of C1q to serum-sensitive strains was independent of the presence of capsular polysaccharide, while strains possessing lipopolysaccharide O antigen bind less C1q and are resistant to complement-mediated killing. Images PMID:8432605

  18. Chemical labelling of active serum thioester proteins for quantification.

    PubMed

    Holm, Lotta; Ackland, Gareth L; Edwards, Mark R; Breckenridge, Ross A; Sim, Robert B; Offer, John

    2012-02-01

    The complement serum proteins C3 and C4 and the protease inhibitor α-2 macroglobulin are all members of the C3/α-2M thioester protein family, an evolutionarily ancient and conserved family that contains an intrachain thioester bond. The chemistry of the thioester bond is a key to the function of the thioester proteins. All these proteins function by covalently linking to their target by acyl transfer of the protein via the thioester moiety. We show that the signature thioester bond can be targeted with nucleophiles linked to a bioreporter molecule, site-specifically modifying the whole, intact thioester protein. Conditions were optimised to label selectively and efficiently pull-down unprocessed thioester-containing proteins from serum. We demonstrated pull-down of full-length C3, α-2M and C4 from sera in high salt, using a biotinylated nucleophile and streptavidin-coated resin, confirmed by MALDI-TOF MS identification of the gel bands. The potential for the development of a quantitative method for measuring active C3 in serum was investigated in patient sera pre and post operation. Quantifying active C3 in clinical assays using current methods is difficult. Methods based on antibody detection (e.g. nephelometry) do not distinguish between active C3 and inactive breakdown products. C3-specific haemolytic assays can be used, but these require use of relatively unstable reagents. The current work represents a promising robust, enzyme- and antibody-free chemical method for detecting active thioester proteins in blood, plasma or serum. PMID:21852021

  19. Changes in serum protein profiles of chickens with tibial dyschondroplasia

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Differences in serum protein profiles were analyzed to identify biomarkers associated with a poultry leg problem named tibial dyschondroplasia (TD) that can cause lameness. We used a bead-based affinity matrix containing a combinatorial library of hexapeptides (ProteoMinerTM) to deplete high abundan...

  20. Serum protein-expression profiling using the ProteinChip biomarker system.

    PubMed

    Gilbert, Kate; Figueredo, Sharel; Meng, Xiao-Ying; Yip, Christine; Fung, Eric T

    2004-01-01

    Protein-expression profiling of serum is a common approach to the discovery of potential diagnostic and therapeutic markers of disease. Like any other proteome, the serum proteome is characterized by protein expression across a large dynamic range. This single facet requires the employment of fractionation procedures prior to detection of protein. The authors use a combination of conventional column chromatography with array-based chromatography to simplify the serum proteome into subproteomes, thus providing a greater representation of the serum proteome. Robotics is employed to increase the throughput of sample processing. These procedures result in large amounts of data that are analyzed through a series of preprocessing and postprocessing steps. A well-designed serum profiling project can therefore result in the discovery of statistically sound, clinically meaningful protein biomarkers. PMID:15020796

  1. The alternative complement pathway control protein H binds to immune complexes and serves their detection

    SciTech Connect

    Nydegger, U.E.; Corvetta, A.; Spaeth, P.J.; Spycher, M.

    1983-01-01

    During solubilization of immune complexes C3b becomes fixed to the immunoglobulin part and serves as a receptor for the alternative complement pathway control protein H. The H-C3b immune complex interaction can be made detectable using 4% polyethyleneglycol to separate free from bound /sup 125/I-H. Tetanus toxoid (Te)/anti-Te complexes kept soluble with fresh serum and containing 125 IU of specific antibody bound 18% of /sup 125/I-H; when fresh serum was chelated with 10 mM EDTA, /sup 125/I-H binding was only 5%. On sucrose density gradients, the H-binding material sedimented in the range of 12 to 30 S. In 36 serum samples from rheumatoid arthritis (RA) patients and in 12 serum samples from patients with systemic lupus erythematosus (SLE), /sup 125/I-H binding was significantly elevated to 9.5 +/- 4.7% (mean +/- 1 SD) and 13.3 +/- 5.6%, respectively, while /sup 125/I-H binding by 36 normal human sera was 4 +/- 2%. RA samples (17/36, 47%) and SLE samples (9/12, 75%) had H-binding values increased by more than 2 SD above the normal mean. The serum samples were also assessed for conglutinin- and C1q-binding activities; a significant correlation between H and C1q binding was observed (P less than 0.001); there was no correlation between H and conglutinin binding. Although binding to immune complexes through its interaction with C3b, H clearly detects a population of complexes other than conglutinin, thus expanding the possibilities of further characterizing pathological complexes.

  2. Cloning, expression and purification of the factor H binding protein and its interaction with factor H

    PubMed Central

    Yarian, Fatemeh; Bandehpour, Mojgan; Seyed, Negar; Kazemi, Bahram

    2016-01-01

    Background and Objective: Neisseria meningitidis is a leading cause of meningitis and sepsis worldwide. The factor H binding protein (fHBP) is a key virulence factor of Neisseria meningitidis that is able to selectively bind to human factor H, the key regulator of the alternative complement pathway, which it has important implications for meningococcal pathogenesis and vaccine design. The aims of present research were cloning, expression, purification of fHbp and confirmation of the interaction between serum factor H (fH) and produced factor H binding protein. Materials and Methods: A 820 base pairs fhbp gene fragment was amplified by PCR and cloned into expression vector pET28a (+) in Bam HI and SalI restriction enzymes sites. Recombinant DNA was expressed in BL21 (DE3) cell. fHBP protein was purified by Ni-NTA agarose resin. Coupling of recombinant protein into CNBr activated Sepharose 4B resin was carried out for application in serum fH protein purification. (fH-fHBP) interaction was confirmed by SDS-PAGE and far-western blotting. Results and Conclusions: SDS-PAGE results showed a 35 kDa protein band. 150 kDa fH protein was purified by designed Sepharose 4B resin. Far-western blotting confirmed (fH-fHBP) interaction and proper folding of factor H binding protein. PMID:27092222

  3. Staphylococcus aureus extracellular adherence protein contributes to biofilm formation in the presence of serum

    PubMed Central

    Thompson, Karl M.; Abraham, Nabil; Jefferson, Kimberly K.

    2010-01-01

    Staphylococcus aureus extracellular adherence protein (EAP) is secreted, but it can redock on the bacterial cell surface via neutral phosphatase (Nptase). EAP binds to certain blood proteins and to itself, and through these affinities, it contributes to adherence and aggregation. It has been demonstrated previously that EAP expression is iron regulated and it contributes to biofilm formation under iron-deplete conditions. In this study, we found that EAP and Nptase also play a role in biofilm formation under iron-replete conditions in the presence of human serum. PMID:20199571

  4. Ligand configurational entropy and protein binding.

    PubMed

    Chang, Chia-en A; Chen, Wei; Gilson, Michael K

    2007-01-30

    The restriction of a small molecule's motion on binding to a protein causes a loss of configurational entropy, and thus a penalty in binding affinity. Some energy models used in computer-aided ligand design neglect this entropic penalty, whereas others account for it based on an expected drop in the number of accessible rotamers upon binding. However, the validity of the physical assumptions underlying the various approaches is largely unexamined. The present study addresses this issue by using Mining Minima calculations to analyze the association of amprenavir with HIV protease. The computed loss in ligand configurational entropy is large, contributing approximately 25 kcal/mol (4.184 kJ/kcal) to DeltaG degrees. Most of this loss results from narrower energy wells in the bound state, rather than a drop in the number of accessible rotamers. Coupling among rotation/translation and internal degrees of freedom complicates the decomposition of the entropy change into additive terms. The results highlight the potential to gain affinity by designing conformationally restricted ligands and have implications for the formulation of energy models for ligand scoring. PMID:17242351

  5. Ligand configurational entropy and protein binding

    PubMed Central

    Chang, Chia-en A.; Chen, Wei; Gilson, Michael K.

    2007-01-01

    The restriction of a small molecule's motion on binding to a protein causes a loss of configurational entropy, and thus a penalty in binding affinity. Some energy models used in computer-aided ligand design neglect this entropic penalty, whereas others account for it based on an expected drop in the number of accessible rotamers upon binding. However, the validity of the physical assumptions underlying the various approaches is largely unexamined. The present study addresses this issue by using Mining Minima calculations to analyze the association of amprenavir with HIV protease. The computed loss in ligand configurational entropy is large, contributing ∼25 kcal/mol (4.184 kJ/kcal) to ΔG°. Most of this loss results from narrower energy wells in the bound state, rather than a drop in the number of accessible rotamers. Coupling among rotation/translation and internal degrees of freedom complicates the decomposition of the entropy change into additive terms. The results highlight the potential to gain affinity by designing conformationally restricted ligands and have implications for the formulation of energy models for ligand scoring. PMID:17242351

  6. Alternative polyadenylation and RNA-binding proteins.

    PubMed

    Erson-Bensan, Ayse Elif

    2016-08-01

    Our understanding of the extent of microRNA-based gene regulation has expanded in an impressive pace over the past decade. Now, we are beginning to better appreciate the role of 3'-UTR (untranslated region) cis-elements which harbor not only microRNA but also RNA-binding protein (RBP) binding sites that have significant effect on the stability and translational rate of mRNAs. To add further complexity, alternative polyadenylation (APA) emerges as a widespread mechanism to regulate gene expression by producing shorter or longer mRNA isoforms that differ in the length of their 3'-UTRs or even coding sequences. Resulting shorter mRNA isoforms generally lack cis-elements where trans-acting factors bind, and hence are differentially regulated compared with the longer isoforms. This review focuses on the RBPs involved in APA regulation and their action mechanisms on APA-generated isoforms. A better understanding of the complex interactions between APA and RBPs is promising for mechanistic and clinical implications including biomarker discovery and new therapeutic approaches. PMID:27208003

  7. Technical advance: identification of plant actin-binding proteins by F-actin affinity chromatography

    NASA Technical Reports Server (NTRS)

    Hu, S.; Brady, S. R.; Kovar, D. R.; Staiger, C. J.; Clark, G. B.; Roux, S. J.; Muday, G. K.

    2000-01-01

    Proteins that interact with the actin cytoskeleton often modulate the dynamics or organization of the cytoskeleton or use the cytoskeleton to control their localization. In plants, very few actin-binding proteins have been identified and most are thought to modulate cytoskeleton function. To identify actin-binding proteins that are unique to plants, the development of new biochemical procedures will be critical. Affinity columns using actin monomers (globular actin, G-actin) or actin filaments (filamentous actin, F-actin) have been used to identify actin-binding proteins from a wide variety of organisms. Monomeric actin from zucchini (Cucurbita pepo L.) hypocotyl tissue was purified to electrophoretic homogeneity and shown to be native and competent for polymerization to actin filaments. G-actin, F-actin and bovine serum albumin affinity columns were prepared and used to separate samples enriched in either soluble or membrane-associated actin-binding proteins. Extracts of soluble actin-binding proteins yield distinct patterns when eluted from the G-actin and F-actin columns, respectively, leading to the identification of a putative F-actin-binding protein of approximately 40 kDa. When plasma membrane-associated proteins were applied to these columns, two abundant polypeptides eluted selectively from the F-actin column and cross-reacted with antiserum against pea annexins. Additionally, a protein that binds auxin transport inhibitors, the naphthylphthalamic acid binding protein, which has been previously suggested to associate with the actin cytoskeleton, was eluted in a single peak from the F-actin column. These experiments provide a new approach that may help to identify novel actin-binding proteins from plants.

  8. The modular architecture of protein-protein binding interfaces.

    PubMed

    Reichmann, D; Rahat, O; Albeck, S; Meged, R; Dym, O; Schreiber, G

    2005-01-01

    Protein-protein interactions are essential for life. Yet, our understanding of the general principles governing binding is not complete. In the present study, we show that the interface between proteins is built in a modular fashion; each module is comprised of a number of closely interacting residues, with few interactions between the modules. The boundaries between modules are defined by clustering the contact map of the interface. We show that mutations in one module do not affect residues located in a neighboring module. As a result, the structural and energetic consequences of the deletion of entire modules are surprisingly small. To the contrary, within their module, mutations cause complex energetic and structural consequences. Experimentally, this phenomenon is shown on the interaction between TEM1-beta-lactamase and beta-lactamase inhibitor protein (BLIP) by using multiple-mutant analysis and x-ray crystallography. Replacing an entire module of five interface residues with Ala created a large cavity in the interface, with no effect on the detailed structure of the remaining interface. The modular architecture of binding sites, which resembles human engineering design, greatly simplifies the design of new protein interactions and provides a feasible view of how these interactions evolved. PMID:15618400

  9. Distinct binding and immunogenic properties of the gonococcal homologue of meningococcal factor h binding protein.

    PubMed

    Jongerius, Ilse; Lavender, Hayley; Tan, Lionel; Ruivo, Nicola; Exley, Rachel M; Caesar, Joseph J E; Lea, Susan M; Johnson, Steven; Tang, Christoph M

    2013-01-01

    Neisseria meningitidis is a leading cause of sepsis and meningitis. The bacterium recruits factor H (fH), a negative regulator of the complement system, to its surface via fH binding protein (fHbp), providing a mechanism to avoid complement-mediated killing. fHbp is an important antigen that elicits protective immunity against the meningococcus and has been divided into three different variant groups, V1, V2 and V3, or families A and B. However, immunisation with fHbp V1 does not result in cross-protection against V2 and V3 and vice versa. Furthermore, high affinity binding of fH could impair immune responses against fHbp. Here, we investigate a homologue of fHbp in Neisseria gonorrhoeae, designated as Gonococcal homologue of fHbp (Ghfp) which we show is a promising vaccine candidate for N. meningitidis. We demonstrate that Gfhp is not expressed on the surface of the gonococcus and, despite its high level of identity with fHbp, does not bind fH. Substitution of only two amino acids in Ghfp is sufficient to confer fH binding, while the corresponding residues in V3 fHbp are essential for high affinity fH binding. Furthermore, immune responses against Ghfp recognise V1, V2 and V3 fHbps expressed by a range of clinical isolates, and have serum bactericidal activity against N. meningitidis expressing fHbps from all variant groups. PMID:23935503

  10. Comparison of the Folding Mechanism of Highly Homologous Proteins in the Lipid-binding Protein Family

    EPA Science Inventory

    The folding mechanism of two closely related proteins in the intracellular lipid binding protein family, human bile acid binding protein (hBABP) and rat bile acid binding protein (rBABP) were examined. These proteins are 77% identical (93% similar) in sequence Both of these singl...

  11. Serum levels, ontogeny and heritability of chicken mannan-binding lectin (MBL).

    PubMed Central

    Laursen, S B; Hedemand, J E; Nielsen, O L; Thiel, S; Koch, C; Jensenius, J C

    1998-01-01

    Mannan-binding lectin (MBL) is a serum lectin found in mammals and recently also in birds. It is thought to play an important role in the innate immune defence through binding to surface carbohydrates on micro-organisms followed by complement activation via the MBL pathway. This results in opsonization or direct complement-mediated killing. To gain further knowledge about the physiology and function of the protein, we developed an enzyme-linked immunosorbent assay for chicken MBL and used this to investigate the level of MBL in different chicken strains during embryogenesis, early and adult life. The MBL concentrations in 308 chickens, representing 14 different strains, showed a non-Gaussian, unimodal distribution profile with a mean concentration of 5.8 micrograms/ml (range 0.4-37.8 micrograms/ml). No difference between the strains could be demonstrated and no chickens were found deficient in MBL. Ontogenetic studies showed that MBL is already detectable in embryos at a gestational age of 10 days (11 days before hatching). At hatching, the level is comparable to the level found in adult chickens. This level is fairly stable during the first weeks of life, but a deficiency state develops at 4 weeks of age, whereafter the level is normalized again at 5 weeks of age. Chickens with relatively low or high MBL levels were bred with cockerels having similar MBL levels and this resulted in F1 generations with significantly different MBL levels, suggesting that the protein level is genetically influenced. PMID:9767449

  12. Bovine serum albumin binding, antioxidant and anticancer properties of an oxidovanadium(IV) complex with luteolin.

    PubMed

    Naso, Luciana G; Lezama, Luis; Valcarcel, María; Salado, Clarisa; Villacé, Patricia; Kortazar, Danel; Ferrer, Evelina G; Williams, Patricia A M

    2016-04-01

    Chemotherapy using metal coordination compounds for cancer treatment is the work of the ongoing research. Continuing our research on the improvement of the anticancer activity of natural flavonoids by metal complexation, a coordination compound of the natural antioxidant flavone luteolin (lut) and the oxidovanadium(IV) cation has been synthesized and characterized. Using different physicochemical measurements some structural aspects of [VO(lut)(H2O)2]Na·3H2O (VOlut) were determined. The metal coordinated to two cis-deprotonated oxygen atoms (ArO(-)) of the ligand and two H2O molecules. Magnetic measurements in solid state indicated the presence of an effective exchange pathway between adjacent vanadium ions. VOlut improved the antioxidant capacity of luteolin only against hydroxyl radical. The antitumoral effects were evaluated on MDAMB231 breast cancer and A549 lung cancer cell lines. VOlut exhibited higher viability inhibition (IC50=17 μM) than the ligand on MDAMB231 cells but they have the same behavior on A549 cells (ca. IC50=60 μM). At least oxidative stress processes were active during cancer cell-killing. When metals chelated through the carbonyl group and one adjacent OH group of the flavonoid an effective improvement of the biological properties has been observed. In VOlut the different coordination may be the cause of the small improvement of some of the tested properties of the flavonoid. Luteolin and VOlut could be distributed and transported in vivo. Luteolin interacted in the microenvironment of the tryptophan group of the serum binding protein, BSA, by means of electrostatic forces and its complex bind the protein by H bonding and van der Waals interactions. PMID:26828287

  13. Isolation of a Thiamine-binding Protein from Rice Germ and Distribution of Similar Proteins.

    PubMed

    Shimizu, M; Yoshida, T; Toda, T; Iwashima, A; Mitsunaga, T

    1996-01-01

    A thiamine-binding protein was purified from rice germ (Oryza sativa L.) by extraction, salting-out with ammonium sulfate, and column chromatography. From the results of molecular mass, Kd and Bmax values for thiamine-binding, binding specificity for thiamine phosphates and analog, the protein was suggested to be identical to the thiamine-binding protein in rice bran. The thiamine-binding protein w as more efficiently purified from rice germ than from rice bran. The protein was rich in glutamic acid (and/or glutamine) and glycine. The protein did not show immunological similarity to thiamine-binding proteins in buckwheat and sesame seeds. However proteins similar to the thiamine-binding protein from rice germ existed in gramineous seeds. They were suggested to have thiamine-binding activity and to be of the same molecular mass as the thiamine-binding protein. PMID:27299548

  14. Binding of cationic surfactants to DNA, protein and DNA-protein mixtures.

    PubMed

    Gani, S A; Chattoraj, D K; Mukherjee, D C

    1999-06-01

    Extent of binding (gamma 2(1)) of cationic surfactants cetyltrimethyl ammonium bromide (CTAB), myristyltrimethyl ammonium bromide (MTAB) and dodecyl trimethyl ammonium bromide (DTAB) to calf-thymus DNA, bovine serum albumin (BSA) and to their binary mixture respectively have been measured as function of bulk concentration of the surfactant by using equilibrium dialysis technique. Binding of CTAB has been studied at different pH, ionic strength (mu), temperature and biopolymer composition and with native and denatured states of the biopolymers. The chain-length of different long chain amines plays a significant role in the extent of binding under identical solution condition. The binding ratios for CTAB to collagen, gelatin, DNA-collagen and DNA-gelatin mixtures respectively have also been determined. The conformational structures of different biopolymers are observed to play significant role in macromolecular interactions between protein and DNA in the presence of CTAB. From the experimental values of the maximum binding ratio (gamma 2m) at the saturation level for each individual biopolymer, ideal values (gamma 2m)id have been theoretically calculated for binary mixtures of biopolymers using additivity rule. The protein-DNA-CTAB interaction in mixture has been explained in terms of the deviation (delta) of (gamma 2m) from (gamma 2m)id in the presence of a surfactant in bulk. The binding of surfactants to biopolymers and to their binary mixtures are compared more precisely in terms of the Gibbs' free energy decrease (-delta G degree) for the saturation of the binding sites in the biopolymers or biopolymer mixtures with the change of the bulk surfactant activity from zero to unity in the rational mole fraction scale. PMID:10650715

  15. Interaction between curcumin and human serum albumin in the presence of excipients and the effect of binding on curcumin photostability.

    PubMed

    Vukićević, Milica; Tønnesen, Hanne Hjorth

    2016-06-01

    Curcumin (Cur) is known to bind to human serum albumin (HSA) which may lead to a reduced phototoxic effect of the compound in the presence of serum or saliva. The influence of excipients on the Cur-HSA binding was studied by HSA florescence quenching and Cur absorption and emission spectroscopy in the presence and absence of the selected excipients. Photostabilty of Cur in the presence of HSA was evaluated, as well as the effect of excipients on HSA bound Cur photodegradation. Cyclodextrins (CDs) (2-hydroxypropyl-β-cyclodextrin and 2-hydroxypropyl-γ-cyclodextrin) and polymers (polyethylene glycol 400, PEG 400 and Pluronic F-127, PF-127) were selected for the study. CDs and PF-127 seem to decrease Cur binding to HSA, probably through competitive binding. Cur was still bound to HSA in polyethylene glycol (PEG) solutions at the highest investigated concentration (5% w/v). However, high PEG concentration appears to have effect on the protein conformation, as shown by the fluorescence quenching study. Low Cur photostability in the presence of HSA could be improved by the addition of hydroxylpropyl-γ-cyclodextrin (HPγCD) to the samples, whereas PEG and PF-127 showed no effect. PMID:25716057

  16. Limitations of protein-coated charcoal in the separation of free from bound vitamin B12 in serum.

    PubMed Central

    Jacob, E; Wong, K T

    1983-01-01

    The effect of haemoglobin and albumin-coated charcoal on the concentration of vitamin B12 binding proteins in serum has been investigated. As commonly employed, coated charcoal removes a significant amount of transcobalamin II (TCII) from serum, but does not affect transcobalamin I and III (TCI and III). Increasing the protein coat up to about 10 mg of protein per 25 mg of charcoal reduces the adsorption of TCII, but increasing the protein concentration beyond this has little added effect. The amount of adsorption is proportional to the amount of coated charcoal employed, but even small amounts adsorb some TCII. These results indicate that protein-coated charcoal is not the ideal way of separating free from bound vitamin B12 in serum; it cannot reliably be used for the measurement of the concentration of apo-TCII but can be employed for the measurement of apo-TCI and III. PMID:6886019

  17. Alpha 1-acid glycoprotein concentration and molecular heterogeneity: relationship to oxprenolol binding in serum from healthy volunteers and patients with lung carcinoma or cirrhosis.

    PubMed Central

    Fraeyman, N F; Dello, C D; Belpaire, F M

    1988-01-01

    1. alpha 1-acid glycoprotein (AAG) concentration and molecular heterogeneity, and oxprenolol protein binding were studied in serum of 15 healthy volunteers, 14 patients with lung carcinoma and 17 patients with liver cirrhosis. 2. The AAG serum concentration was increased to 180.7% in patients with lung cancer and decreased to 73.4% in cirrhotic patients as compared with controls (P less than 0.05). 3. The concanavalin A (conA) dependent heterogeneity of serum AAG was very similar in controls and patients with lung cancer: a ratio of 9/9/2 was obtained for the conA nonreactive, the conA weakly reactive and the conA strongly reactive subfraction respectively; in cirrhotic patients, the ratio shifted to 11/7/1. 4. The heterogeneity in electric charge, demonstrated by isoelectric focusing, was similar in the three groups of subjects: 70-80% of the focussed bands were found in the main three bands. 5. The binding of oxprenolol to serum proteins was increased in lung tumour patients and decreased in liver cirrhotic patients as compared with controls (P less than 0.05). There was no change in binding affinity and oxprenolol binding was significantly correlated to total AAG serum concentration and to the concentration of each of the conA dependent subtypes, in controls as well as in both patients groups. Images Figure 1 PMID:3203044

  18. Polypyrimidine-tract-binding protein: a multifunctional RNA-binding protein.

    PubMed

    Sawicka, Kirsty; Bushell, Martin; Spriggs, Keith A; Willis, Anne E

    2008-08-01

    PTB (polypyrimidine-tract-binding protein) is a ubiquitous RNA-binding protein. It was originally identified as a protein with a role in splicing but it is now known to function in a large number of diverse cellular processes including polyadenylation, mRNA stability and translation initiation. Specificity of PTB function is achieved by a combination of changes in the cellular localization of this protein (its ability to shuttle from the nucleus to the cytoplasm is tightly controlled) and its interaction with additional proteins. These differences in location and trans-acting factor requirements account for the fact that PTB acts both as a suppressor of splicing and an activator of translation. In the latter case, the role of PTB in translation has been studied extensively and it appears that this protein is required for an alternative form of translation initiation that is mediated by a large RNA structural element termed an IRES (internal ribosome entry site) that allows the synthesis of picornaviral proteins and cellular proteins that function to control cell growth and cell death. In the present review, we discuss how PTB regulates these disparate processes. PMID:18631133

  19. Characterization of additional vitamin D binding protein variants.

    PubMed

    Fu, Lei; Borges, Chad R; Rehder, Douglas S; Wong, Betty Y L; Williams, Rashida; Carpenter, Thomas O; Cole, David E C

    2016-05-01

    The gene (GC) for the vitamin D binding protein (DBP) shows significant genetic variation. Two missense variants, p.D432E and p.T436K, are common polymorphisms and both may influence vitamin D metabolism. However, less common variants, identified biochemically, have been reported previously. This study aimed to identify the underlying mutations by molecular screening and to characterize the mutant proteins by mass spectrometry. Denaturing high performance liquid chromatography (DHPLC) was used for screening genetic variants in GC exons and exon/intron boundaries of genomic DNA samples. Sanger sequencing identified the specific mutations. An immuno-capture coupled mass spectrometry method was used to characterize protein variants in serum samples. Initial molecular screening identified 10 samples (out of 761) containing an alanine deletion at codon 246 in exon 7 (p.A246del, c.737_739delCTG), and 1 sample (out of 97) containing a cysteine to phenylalanine substitution at codon 311 in exon 8 (p.C311F, c.932G>T). The mutant allele proteins and posttranslational modified products were distinguishable from the wild-type proteins by mass spectrum profiling. Loss of a disulfide bond due to loss of cysteine-311 was accompanied by the appearance of a novel mixed disulfide species, consistent with S-cysteinylation of the remaining unpaired cysteine-299 in the mutant protein. We confirm earlier biochemical studies indicating that there are additional deleterious GC mutations, some of which may be low-frequency variants. The major findings of this study indicate that additional mutant proteins are secreted and can be identified in the circulation. By combining molecular screening and mass spectrometric methods, mutant DBP species can be identified and characterized. PMID:26924582

  20. New potential role of serum apolipoprotein E mediated by its binding to clumping factor A during Staphylococcus aureus invasive infections to humans

    PubMed Central

    Hair, Pamela S.; Nyalwidhe, Julius O.; Cunnion, Kenji M.

    2015-01-01

    Staphylococcus aureus is a crucial human pathogen expressing various immune-evasion proteins that interact with the host-cell molecules. Clumping factor A (ClfA) is a microbial surface protein that promotes S. aureus binding to fibrinogen, and is associated with septic arthritis and infective endocarditis. In order to identify the major human serum proteins that bind the ClfA, we utilized recombinant ClfA region A in a plate-based assay. SDS-PAGE analysis of the bound proteins yielded five prominent bands, which were analysed by MS yielding apolipoprotein E (ApoE) as the predominant protein. ClfA-sufficient S. aureus bound purified ApoE by more than one log greater than an isogenic ClfA-deficient mutant. An immunodot-blot assay yielded a linearity model for ClfA binding to human ApoE with a stoichiometric-binding ratio of 1.702 at maximal Pearson's correlation coefficient (0.927). These data suggest that ApoE could be a major and novel binding target for the S. aureus virulence factor ClfA. Thus, ClfA recruitment of serum ApoE to the S. aureus surface may sequester ApoE and blunt its host defence function against S. aureus-invasive infections to humans. In this context, compounds that can block or suppress ClfA binding to ApoE might be utilized as prophylactic or therapeutic agents. PMID:25878259

  1. New potential role of serum apolipoprotein E mediated by its binding to clumping factor A during Staphylococcus aureus invasive infections to humans.

    PubMed

    Elkhatib, Walid F; Hair, Pamela S; Nyalwidhe, Julius O; Cunnion, Kenji M

    2015-04-01

    Staphylococcus aureus is a crucial human pathogen expressing various immune-evasion proteins that interact with the host-cell molecules. Clumping factor A (ClfA) is a microbial surface protein that promotes S. aureus binding to fibrinogen, and is associated with septic arthritis and infective endocarditis. In order to identify the major human serum proteins that bind the ClfA, we utilized recombinant ClfA region A in a plate-based assay. SDS-PAGE analysis of the bound proteins yielded five prominent bands, which were analysed by MS yielding apolipoprotein E (ApoE) as the predominant protein. ClfA-sufficient S. aureus bound purified ApoE by more than one log greater than an isogenic ClfA-deficient mutant. An immunodot-blot assay yielded a linearity model for ClfA binding to human ApoE with a stoichiometric-binding ratio of 1.702 at maximal Pearson's correlation coefficient (0.927). These data suggest that ApoE could be a major and novel binding target for the S. aureus virulence factor ClfA. Thus, ClfA recruitment of serum ApoE to the S. aureus surface may sequester ApoE and blunt its host defence function against S. aureus-invasive infections to humans. In this context, compounds that can block or suppress ClfA binding to ApoE might be utilized as prophylactic or therapeutic agents. PMID:25878259

  2. Absolute quantitation of NAPQI-modified rat serum albumin by LC-MS/MS: monitoring acetaminophen covalent binding in vivo.

    PubMed

    LeBlanc, André; Shiao, Tze Chieh; Roy, René; Sleno, Lekha

    2014-09-15

    Acetaminophen is known to cause hepatoxicity via the formation of a reactive metabolite, N-acetyl p-benzoquinone imine (NAPQI), as a result of covalent binding to liver proteins. Serum albumin (SA) is known to be covalently modified by NAPQI and is present at high concentrations in the bloodstream and is therefore a potential biomarker to assess the levels of protein modification by NAPQI. A newly developed method for the absolute quantitation of serum albumin containing NAPQI covalently bound to its active site cysteine (Cys34) is described. This optimized assay represents the first absolute quantitation of a modified protein, with very low stoichiometric abundance, using a protein-level standard combined with isotope dilution. The LC-MS/MS assay is based on a protein standard modified with a custom-designed reagent, yielding a surrogate peptide (following digestion) that is a positional isomer to the target peptide modified by NAPQI. To illustrate the potential of this approach, the method was applied to quantify NAPQI-modified SA in plasma from rats dosed with acetaminophen. The resulting method is highly sensitive (capable of quantifying down to 0.0006% of total RSA in its NAPQI-modified form) and yields excellent precision and accuracy statistics. A time-course pharmacokinetic study was performed to test the usefulness of this method for following acetaminophen-induced covalent binding at four dosing levels (75-600 mg/kg IP), showing the viability of this approach to directly monitor in vivo samples. This approach can reliably quantify NAPQI-modified albumin, allowing direct monitoring of acetaminophen-related covalent binding. PMID:25168029

  3. High-Performance Affinity Chromatography: Applications in Drug-Protein Binding Studies and Personalized Medicine.

    PubMed

    Li, Zhao; Beeram, Sandya R; Bi, Cong; Suresh, D; Zheng, Xiwei; Hage, David S

    2016-01-01

    The binding of drugs with proteins and other agents in serum is of interest in personalized medicine because this process can affect the dosage and action of drugs. The extent of this binding may also vary with a given disease state. These interactions may involve serum proteins, such as human serum albumin or α1-acid glycoprotein, or other agents, such as lipoproteins. High-performance affinity chromatography (HPAC) is a tool that has received increasing interest as a means for studying these interactions. This review discusses the general principles of HPAC and the various approaches that have been used in this technique to examine drug-protein binding and in work related to personalized medicine. These approaches include frontal analysis and zonal elution, as well as peak decay analysis, ultrafast affinity extraction, and chromatographic immunoassays. The operation of each method is described and examples of applications for these techniques are provided. The type of information that can be obtained by these methods is also discussed, as related to the analysis of drug-protein binding and the study of clinical or pharmaceutical samples. PMID:26827600

  4. Structural neighboring property for identifying protein-protein binding sites

    PubMed Central

    2015-01-01

    Background The protein-protein interaction plays a key role in the control of many biological functions, such as drug design and functional analysis. Determination of binding sites is widely applied in molecular biology research. Therefore, many efficient methods have been developed for identifying binding sites. In this paper, we calculate structural neighboring property through Voronoi diagram. Using 6,438 complexes, we study local biases of structural neighboring property on interface. Results We propose a novel statistical method to extract interacting residues, and interacting patches can be clustered as predicted interface residues. In addition, structural neighboring property can be adopted to construct a new energy function, for evaluating docking solutions. It includes new statistical property as well as existing energy items. Comparing to existing methods, our approach improves overall Fnat value by at least 3%. On Benchmark v4.0, our method has average Irmsd value of 3.31Å and overall Fnat value of 63%, which improves upon Irmsd of 3.89 Å and Fnat of 49% for ZRANK, and Irmsd of 3.99Å and Fnat of 46% for ClusPro. On the CAPRI targets, our method has average Irmsd value of 3.46 Å and overall Fnat value of 45%, which improves upon Irmsd of 4.18 Å and Fnat of 40% for ZRANK, and Irmsd of 5.12 Å and Fnat of 32% for ClusPro. Conclusions Experiments show that our method achieves better results than some state-of-the-art methods for identifying protein-protein binding sites, with the prediction quality improved in terms of CAPRI evaluation criteria. PMID:26356630

  5. Serum proteins are extracted along with monolayer cells in plasticware and interfere with protein analysis

    PubMed Central

    Hong, Xin; Meng, Yuling; Kalkanis, Steven N.

    2016-01-01

    Washing and lysing monolayer cells directly from cell culture plasticware is a commonly used method for protein extraction. We found that multiple protein bands were enriched in samples with low cell numbers from the 6-well plate cultures. These proteins contributed to the overestimation of cell proteins and led to the uneven protein loading in Western blotting analysis. In Coomassie blue stained SDS-PAGE gels, the main enriched protein band is about 69 kDa and it makes up 13.6% of total protein from 104 U251n cells. Analyzed by mass spectrometry, we identified two of the enriched proteins: bovine serum albumin and bovine serum transferrin. We further observed that serum proteins could be extracted from other cell culture plates, dishes and flasks even after washing the cells 3 times with PBS. A total of 2.3 mg of protein was collected from a single well of the 6-well plate. A trace amount of the protein band was still visible after washing the cells 5 times with PBS. Thus, serum proteins should be considered if extracting proteins from plasticware, especially for samples with low cell numbers.

  6. RNA Bind-n-Seq: Measuring the Binding Affinity Landscape of RNA-Binding Proteins.

    PubMed

    Lambert, Nicole J; Robertson, Alex D; Burge, Christopher B

    2015-01-01

    RNA-binding proteins (RBPs) coordinate post-transcriptional control of gene expression, often through sequence-specific recognition of primary transcripts or mature messenger RNAs. Hundreds of RBPs are encoded in the human genome, most with undefined or incompletely defined biological roles. Understanding the function of these factors will require the identification of each RBP's distinct RNA binding specificity. RNA Bind-n-Seq (RBNS) is a high-throughput, cost-effective in vitro method capable of resolving sequence and secondary structure preferences of RBPs. Dissociation constants can also be inferred from RBNS data when provided with additional experimental information. Here, we describe the experimental procedures to perform RBNS and discuss important parameters of the method and ways that the experiment can be tailored to the specific RBP under study. Additionally, we present the conceptual framework and execution of the freely available RBNS computational pipeline and describe the outputs of the pipeline. Different approaches to quantify binding specificity, quality control metrics, and estimation of binding constants are also covered. PMID:26068750

  7. Role of arg-410 and tyr-411 in human serum albumin for ligand binding and esterase-like activity.

    PubMed Central

    Watanabe, H; Tanase, S; Nakajou, K; Maruyama, T; Kragh-Hansen, U; Otagiri, M

    2000-01-01

    Recombinant wild-type human serum albumin (rHSA), the single-residue mutants R410A, Y411A, Y411S and Y411F and the double mutant R410A/Y411A were produced using a yeast expression system. The recombinant proteins were correctly folded, as they had the same stability towards guanidine hydrochloride and the same CD spectrum as HSA isolated from serum (native HSA). Thus the global structures of the recombinant proteins are probably very similar to that of native HSA. We investigated, by ultrafiltration and CD, the high-affinity binding of two representative site II ligands, namely ketoprofen and diazepam. According to the crystal structure of HSA, the residues Arg-410 and Tyr-411 protrude into the centre of site II (in subdomain 3A), and the binding results showed that the guanidino moiety of Arg-410, the phenolic oxygen and the aromatic ring of Tyr-411 are important for ketoprofen binding. The guanidino moiety probably interacts electrostatically with the carboxy group of ketoprofen, the phenolic oxygen could make a hydrogen-bond with the keto group of the ligand, and the aromatic ring may participate in a specific stacking interaction with one of or both of the aromatic rings of ketoprofen. By contrast, Arg-410 is not important for diazepam binding. The two parts of Tyr-411 interact favourably with diazepam, and probably do so in the same way as with ketoprofen. In addition to its unique ligand binding properties, HSA also possesses an esterase-like activity, and studies with p-nitrophenyl acetate as a substrate showed that, although Arg-410 is important, the enzymic activity of HSA is much more dependent on the presence of Tyr-411. A minor activity could be registered when serine, but not alanine or phenylalanine, was present at position 411. PMID:10903143

  8. Role of arg-410 and tyr-411 in human serum albumin for ligand binding and esterase-like activity.

    PubMed

    Watanabe, H; Tanase, S; Nakajou, K; Maruyama, T; Kragh-Hansen, U; Otagiri, M

    2000-08-01

    Recombinant wild-type human serum albumin (rHSA), the single-residue mutants R410A, Y411A, Y411S and Y411F and the double mutant R410A/Y411A were produced using a yeast expression system. The recombinant proteins were correctly folded, as they had the same stability towards guanidine hydrochloride and the same CD spectrum as HSA isolated from serum (native HSA). Thus the global structures of the recombinant proteins are probably very similar to that of native HSA. We investigated, by ultrafiltration and CD, the high-affinity binding of two representative site II ligands, namely ketoprofen and diazepam. According to the crystal structure of HSA, the residues Arg-410 and Tyr-411 protrude into the centre of site II (in subdomain 3A), and the binding results showed that the guanidino moiety of Arg-410, the phenolic oxygen and the aromatic ring of Tyr-411 are important for ketoprofen binding. The guanidino moiety probably interacts electrostatically with the carboxy group of ketoprofen, the phenolic oxygen could make a hydrogen-bond with the keto group of the ligand, and the aromatic ring may participate in a specific stacking interaction with one of or both of the aromatic rings of ketoprofen. By contrast, Arg-410 is not important for diazepam binding. The two parts of Tyr-411 interact favourably with diazepam, and probably do so in the same way as with ketoprofen. In addition to its unique ligand binding properties, HSA also possesses an esterase-like activity, and studies with p-nitrophenyl acetate as a substrate showed that, although Arg-410 is important, the enzymic activity of HSA is much more dependent on the presence of Tyr-411. A minor activity could be registered when serine, but not alanine or phenylalanine, was present at position 411. PMID:10903143

  9. Calcyclin Binding Protein/Siah-1 Interacting Protein Is a Hsp90 Binding Chaperone

    PubMed Central

    Góral, Agnieszka; Bieganowski, Paweł; Prus, Wiktor; Krzemień-Ojak, Łucja; Kądziołka, Beata; Fabczak, Hanna; Filipek, Anna

    2016-01-01

    The Hsp90 chaperone activity is tightly regulated by interaction with many co-chaperones. Since CacyBP/SIP shares some sequence homology with a known Hsp90 co-chaperone, Sgt1, in this work we performed a set of experiments in order to verify whether CacyBP/SIP can interact with Hsp90. By applying the immunoprecipitation assay we have found that CacyBP/SIP binds to Hsp90 and that the middle (M) domain of Hsp90 is responsible for this binding. Furthermore, the proximity ligation assay (PLA) performed on HEp-2 cells has shown that the CacyBP/SIP-Hsp90 complexes are mainly localized in the cytoplasm of these cells. Using purified proteins and applying an ELISA we have shown that Hsp90 interacts directly with CacyBP/SIP and that the latter protein does not compete with Sgt1 for the binding to Hsp90. Moreover, inhibitors of Hsp90 do not perturb CacyBP/SIP-Hsp90 binding. Luciferase renaturation assay and citrate synthase aggregation assay with the use of recombinant proteins have revealed that CacyBP/SIP exhibits chaperone properties. Also, CacyBP/SIP-3xFLAG expression in HEp-2 cells results in the appearance of more basic Hsp90 forms in 2D electrophoresis, which may indicate that CacyBP/SIP dephosphorylates Hsp90. Altogether, the obtained results suggest that CacyBP/SIP is involved in regulation of the Hsp90 chaperone machinery. PMID:27249023

  10. Glycan Masking of Plasmodium vivax Duffy Binding Protein for Probing Protein Binding Function and Vaccine Development

    PubMed Central

    Janes, Joel; Gurumoorthy, Sairam; Gibson, Claire; Melcher, Martin; Chitnis, Chetan E.; Wang, Ruobing; Schief, William R.; Smith, Joseph D.

    2013-01-01

    Glycan masking is an emerging vaccine design strategy to focus antibody responses to specific epitopes, but it has mostly been evaluated on the already heavily glycosylated HIV gp120 envelope glycoprotein. Here this approach was used to investigate the binding interaction of Plasmodium vivax Duffy Binding Protein (PvDBP) and the Duffy Antigen Receptor for Chemokines (DARC) and to evaluate if glycan-masked PvDBPII immunogens would focus the antibody response on key interaction surfaces. Four variants of PVDBPII were generated and probed for function and immunogenicity. Whereas two PvDBPII glycosylation variants with increased glycan surface coverage distant from predicted interaction sites had equivalent binding activity to wild-type protein, one of them elicited slightly better DARC-binding-inhibitory activity than wild-type immunogen. Conversely, the addition of an N-glycosylation site adjacent to a predicted PvDBP interaction site both abolished its interaction with DARC and resulted in weaker inhibitory antibody responses. PvDBP is composed of three subdomains and is thought to function as a dimer; a meta-analysis of published PvDBP mutants and the new DBPII glycosylation variants indicates that critical DARC binding residues are concentrated at the dimer interface and along a relatively flat surface spanning portions of two subdomains. Our findings suggest that DARC-binding-inhibitory antibody epitope(s) lie close to the predicted DARC interaction site, and that addition of N-glycan sites distant from this site may augment inhibitory antibodies. Thus, glycan resurfacing is an attractive and feasible tool to investigate protein structure-function, and glycan-masked PvDBPII immunogens might contribute to P. vivax vaccine development. PMID:23853575

  11. Increased (/sup 125/I)trypsin-binding in serum from cystic fibrosis patients

    SciTech Connect

    Cox, K.L.; Frates, R.C. Jr.; Sheikholislam, B.M.; Iwahashi-Hosoda, C.K.

    1982-01-01

    The capacities of normal and cystic fibrosis (CF) sera to bind to exogenous human (/sup 125/I)trypsin were compared. Sera from eight older CF patients bound significantly more exogenous human (/sup 125/I)trypsin than did sera from eight normal subjects (p less than 0.001). Disregarding the increased trypsin-binding (TB) of CF sera, serum immunoreactive trypsinogen (SIRT) levels were not detectable in these eight older CF patients. However, when SIRT levels were corrected for TB, four CF patients had normal SIRT concentrations and four had low but detectable SIRT levels. As compared to five normal newborns' sera, serum from a newborn with CF had normal TB and the SIRT levels were very high. In conclusion, increased TB in CF serum lowers results of SIRT assays. Therefore, unless SIRT levels are corrected for TB, results obtained from currently available SIRT kits may be invalid.

  12. Identification and isoprenylation of plant GTP-binding proteins.

    PubMed

    Biermann, B; Randall, S K; Crowell, D N

    1996-08-01

    To identify isoprenylated plant GTP-binding proteins, Arabidopsis thaliana and Nicotiana tabacum cDNA expression libraries were screened for cDNA-encoded proteins capable of binding [32P]GTP in vitro. ATGB2, an Arabidopsis homologue of the GTP-binding protein Rab2, was found to bind GTP in vitro and to be a substrate for a geranylgeranyl:protein transferase (GGTase) present in plant extracts. The carboxyl terminus of this protein contains a -GCCG sequence, which has not previously been shown to be recognized by any prenyl:protein transferase (PTase), but which most closely resembles that isoprenylated by the type II GGTase (-XXCC, -XCXC, or -CCXX). In vitro geranylgeranylation of an Arabidopsis Rab1 protein containing a carboxyl-terminal-CCGQ sequence confirmed the presence of a type II GGTase-like activity in plant extracts. Several other proteins were also identified by in vitro GTP binding, including Arabidopsis and tobacco homologues of Rab11, ARF (ADP-ribosylation factor) and Sar proteins, as well as a novel 22 kDa Arabidopsis protein (ATG81). This 22 kDa protein had consensus GTP-binding motifs and bound GTP with high specificity, but its structure was not closely related to that of any known GTP-binding protein (it most resembled proteins within the ARF/Sar and G protein alpha-subunit superfamilies). PMID:8843944

  13. Protein Function Annotation By Local Binding Site Surface Similarity

    PubMed Central

    Spitzer, Russell; Cleves, Ann E.; Varela, Rocco; Jain, Ajay N.

    2013-01-01

    Hundreds of protein crystal structures exist for proteins whose function cannot be confidently determined from sequence similarity. Surflex-PSIM, a previously reported surface-based protein similarity algorithm, provides an alternative method for hypothesizing function for such proteins. The method now supports fully automatic binding site detection and is fast enough to screen comprehensive databases of protein binding sites. The binding site detection methodology was validated on apo/holo cognate protein pairs, correctly identifying 91% of ligand binding sites in holo structures and 88% in apo structures where corresponding sites existed. For correctly detected apo binding sites, the cognate holo site was the most similar binding site 87% of the time. PSIM was used to screen a set of proteins that had poorly characterized functions at the time of crystallization, but were later biochemically annotated. Using a fully automated protocol, this set of 8 proteins was screened against approximately 60,000 ligand binding sites from the PDB. PSIM correctly identified functional matches that pre-dated query protein biochemical annotation for five out of the eight query proteins. A panel of twelve currently unannotated proteins was also screened, resulting in a large number of statistically significant binding site matches, some of which suggest likely functions for the poorly characterized proteins. PMID:24166661

  14. RNA-binding protein nucleolin in disease.

    PubMed

    Abdelmohsen, Kotb; Gorospe, Myriam

    2012-06-01

    Nucleolin is a multifunctional protein localized primarily in the nucleolus, but also found in the nucleoplasm, cytoplasm and cell membrane. It is involved in several aspects of DNA metabolism, and participates extensively in RNA regulatory mechanisms, including transcription, ribosome assembly, mRNA stability and translation, and microRNA processing. Nucleolin's implication in disease is linked to its ability to associate with target RNAs via its four RNA-binding domains and its arginine/glycin-rich domain. By modulating the post-transcriptional fate of target mRNAs, which typically bear AU-rich and/or G-rich elements, nucleolin has been linked to cellular events that influence disease, notably cell proliferation and protection against apoptotic death. Through its diverse RNA functions, nucleolin is increasingly implicated in pathological processes, particularly cancer and viral infection. Here, we review the RNA-binding activities of nucleolin, its influence on gene expression patterns, and its impact upon diseases. We also discuss the rising interest in targeting nucleolin therapeutically. PMID:22617883

  15. RNA-binding protein nucleolin in disease

    PubMed Central

    Abdelmohsen, Kotb; Gorospe, Myriam

    2012-01-01

    Nucleolin is a multifunctional protein localized primarily in the nucleolus, but also found in the nucleoplasm, cytoplasm and cell membrane. It is involved in several aspects of DNA metabolism, and participates extensively in RNA regulatory mechanisms, including transcription, ribosome assembly, mRNA stability and translation, and microRNA processing. Nucleolin’s implication in disease is linked to its ability to associate with target RNAs via its four RNA-binding domains and its arginine/glycin-rich domain. By modulating the post-transcriptional fate of target mRNAs, which typically bear AU-rich and/or G-rich elements, nucleolin has been linked to cellular events that influence disease, notably cell proliferation and protection against apoptotic death. Through its diverse RNA functions, nucleolin is increasingly implicated in pathological processes, particularly cancer and viral infection. Here, we review the RNA-binding activities of nucleolin, its influence on gene expression patterns, and its impact upon diseases. We also discuss the rising interest in targeting nucleolin therapeutically. PMID:22617883

  16. Latent TGF-β-binding proteins

    PubMed Central

    Robertson, Ian B.; Horiguchi, Masahito; Zilberberg, Lior; Dabovic, Branka; Hadjiolova, Krassimira; Rifkin, Daniel B.

    2016-01-01

    The LTBPs (or latent transforming growth factor β binding proteins) are important components of the extracellular matrix (ECM) that interact with fibrillin microfibrils and have a number of different roles in microfibril biology. There are four LTBPs isoforms in the human genome (LTBP-1, -2, -3, and -4), all of which appear to associate with fibrillin and the biology of each isoform is reviewed here. The LTBPs were first identified as forming latent complexes with TGFβ by covalently binding the TGFβ propeptide (LAP) via disulfide bonds in the endoplasmic reticulum. LAP in turn is cleaved from the mature TGFβ precursor in the trans golgi network but LAP and TGFβ remain strongly bound through non-covalent interactions. LAP, TGFβ, and LTBP together form the large latent complex (LLC). LTBPs were originally thought to primarily play a role in maintaining TGFβ latency and targeting the latent growth factor to the extracellular matrix (ECM), but it has also been shown that LTBP-1 participates in TGFβ activation by integrins and may also regulate activation by proteases and other factors. LTBP-3 appears to have a role in skeletal formation including tooth development. As well as having important functions in TGFβ regulation, TGFβ-independent activities have recently been identified for LTBP-2 and LTBP-4 in stabilizing microfibril bundles and regulating elastic fiber assembly. PMID:25960419

  17. Characterizing the morphology of protein binding patches.

    PubMed

    Malod-Dognin, Noël; Bansal, Achin; Cazals, Frédéric

    2012-12-01

    Let the patch of a partner in a protein complex be the collection of atoms accounting for the interaction. To improve our understanding of the structure-function relationship, we present a patch model decoupling the topological and geometric properties. While the geometry is classically encoded by the atomic positions, the topology is recorded in a graph encoding the relative position of concentric shells partitioning the interface atoms. The topological-geometric duality provides the basis of a generic dynamic programming-based algorithm comparing patches at the shell level, which may favor topological or geometric features. On the biological side, we address four questions, using 249 cocrystallized heterodimers organized in biological families. First, we dissect the morphology of binding patches and show that Nature enjoyed the topological and geometric degrees of freedom independently while retaining a finite set of qualitatively distinct topological signatures. Second, we argue that our shell-based comparison is effective to perform atomic-level comparisons and show that topological similarity is a less stringent than geometric similarity. We also use the topological versus geometric duality to exhibit topo-rigid patches, whose topology (but not geometry) remains stable upon docking. Third, we use our comparison algorithms to infer specificity-related information amidst a database of complexes. Finally, we exhibit a descriptor outperforming its contenders to predict the binding affinities of the affinity benchmark. The softwares developed with this article are availablefrom http://team.inria.fr/abs/vorpatch_compatch/. PMID:22806945

  18. CD4 peptide-protein conjugates, but not recombinant human CD4, bind to recombinant gp120 from the human immunodeficiency virus in the presence of serum from AIDS patients.

    PubMed Central

    Ghetie, V; Slaughter, C; Wheeler, H T; Uhr, J W; Vitetta, E S

    1991-01-01

    Sera from human immunodeficiency virus-positive (HIV+; Walter Reed stage 6) individuals inhibit the interaction between recombinant human CD4 and recombinant gp120 from HIV (rCD4 and rgp120, respectively), thereby interfering with the ability of soluble rCD4 to block infection with HIV or rCD4-toxin conjugates to kill HIV-infected cells. In this report we demonstrate that the inhibitory activity of such sera is caused primarily by anti-gp120 antibodies that do not recognize the CD4 interaction site on gp120. To circumvent the problem of inhibition, we have generated a construct containing a peptide of CD4 (residues 41-84) conjugated to ovalbumin (three to five peptides per molecule). This multivalent conjugate binds to rgp120 and binding is not inhibited by antibodies in HIV+ sera. PMID:2062847

  19. Interaction of cucurbitacins with human serum albumin: Thermodynamic characteristics and influence on the binding of site specific ligands.

    PubMed

    Abou-Khalil, Rony; Jraij, Alia; Magdalou, Jacques; Ouaini, Naïm; Tome, Daniel; Greige-Gerges, Hélène

    2009-06-01

    Cucurbitacins (Cuc) are cytotoxic oxygenated triterpenes. Their binding to albumin may control their diffusion and consequently their biological effects. The specific binding site of Cuc to albumin is important to be defined as it could determine some of the drug interactions of the compounds. This paper deals with the interaction between human serum albumin and a series of four cucurbitacins (B, D, E and I) measured by fluorescence and circular dichroism spectroscopies. Cuc B and E at C25, are the acetylated forms of Cuc D and I. The binding parameters (K(a) and n) of Cuc B, D and E to albumin were determined at 288, 293, 298 and 303K. Cuc B possesses the higher binding constant (K(a)) values followed by Cuc E and D. The thermodynamic parameters DeltaH, DeltaG and DeltaS were calculated. They indicated hydrophobic and electrostatic interactions for Cuc B, hydrophobic interaction for Cuc E, hydrophobic and hydrogen bond interactions for Cuc D. In addition to bilirubin, Cuc B, D, and E increased the binding constant values for warfarin to albumin, whereas they did not affect the binding of other ligands of site I such as chloroform and salicylate. The increase of the K(a) values of warfarin and bilirubin was associated with an increase of the binding constant value of cucurbitacin to albumin. Cuc I did not bind to albumin and could be considered less capable to affect the interaction of ligands to albumin than Cuc B, D and E. CD spectra indicated that Cuc binding to HSA was not associated with substantial structural changes of the protein. PMID:19380237

  20. Manifold significance of serum eosinophil cationic protein in asthmatic children.

    PubMed

    Zubović, Ivan; Rozmanić, Vojko; Ahel, Vladimir; Banac, Srdan

    2002-01-01

    Asthma is the result of complex interaction between different cells, mediators and nervous system that leads to an inflammatory response accompanied by increased bronchial hyperactivity. Its clinical manifestations include recurrent cough, wheezing and difficult breathing. The purpose of this study was to establish the possibility of diagnosing inflammation in asthmatic patients based on the assessment of serum eosinophil cationic protein (ECP), and of following the efficacy of asthmatic treatment by the levels of inflammation mediators. In a prospective study, 134 children aged 1 to 18 (mean 8) years underwent serum ECP assessment. Experimental group included 87 patients with asthma, 56 boys and 31 girls, mean age 9.1 (range 2-17) years. Control group included patients with recurrent non-allergic disorders, 27 boys and 20 girls aged 1-16 (mean 6.1) years. Serum ECP was assessed using the Pharmacia CAP system ECP-FEIA method, i.e. fluoroimmunoassay test for quantitative assessment of serum ECP levels. Serum values of ECP were significantly higher in asthmatics than in controls (p = 0.001). Our results showed that increased levels of serum ECP to significantly correlate with increased eosinophil (p = 0.018) and immunoglobulin E (p = 0.003) levels. Increased ECP levels reflect the degree of inflammation and correlate with the clinical picture severity in asthmatic patients. Assessment of serum ECP levels can reveal eosinophilic activity, and indirectly detect immunologic inflammation in asthmatics. It is possible to follow the dynamics of immunologic inflammation during the course of treatment as well as treatment efficacy. PMID:12596625

  1. Cellular retinol-binding protein and retinoic acid-binding protein in rat testes: effect of retinol depletion.

    PubMed

    Ong, D E; Tsai, C H; Chytil, F

    1976-02-01

    Testes of rats contain two cellular binding proteins of interest in vitamin A metabolism. One protein binds retinoic acid with high specificity; the other binds retinol with high specificity. When the cellular retinol-binding protein was partially purified from rat testes, it exhibited fluorescence excitation and emission spectra similar to that of all-trans-retinol in hexane. Exposure of this preparation to UV light destroyed this fluorescence but spectra identical to the original were obtained after addition of retinol. Hexane extracts of the binding protein had fluorescence spectra identical to all-trans-retinol, suggesting that this compound is bound to the protein in vivo. Extracts of testes from retinol depleted rats were submitted to gel filtration but failed to show a retinol-like fluorescence at the elution position of retinol binding protein. This fluorescence was observed in the preparations from pair fed control animals. However, after addition of all-trans-retinol to the extracts from the depleted rats, fluorescence at that elution position was observed. This indicates that in testes of retinol depleted rats the cellular retinol binding protein is present but without bound retinol, in contrast to the non-depleted rats where 30-43% of the binding protein had bound retinol. The amounts of cellular retinol binding protein and retinoic acid binding protein in testes, as determined by sucrose gradient centrifugation, were found to be similar for retinol depleted and pair fed control rats. PMID:942996

  2. How Does Confinement Change Ligand-Receptor Binding Equilibrium? Protein Binding in Nanopores and Nanochannels.

    PubMed

    Tagliazucchi, Mario; Szleifer, Igal

    2015-10-01

    We present systematic studies for the binding of small model proteins to ligands attached to the inner walls of long nanochannels and short nanopores by polymeric tethers. Binding of proteins to specific ligands inside nanometric channels and pores leads to changes in their ionic conductance, which have been exploited in sensors that quantify the concentration of the proteins in solution. The theoretical predictions presented in this work are aimed to provide a fundamental understanding of protein binding under geometrically confined environments and to guide the design of this kind of nanochannel-based sensors. The theory predicts that the fraction of the channel volume filled by bound proteins is a nonmonotonic function of the channel radius, the length of the tethers, the surface density of the ligands and the size of the proteins. Notably, increasing the density of ligands, decreasing the size of the channel or increasing the size of the protein may lead to a decrease of the fraction of the channel volume filled by bound proteins. These results are explained from the incomplete binding of proteins to the ligands due to repulsive protein-protein and protein-ligand steric interactions. Our work suggests strategies to optimize the change in conductance due to protein binding, for example: (i) proteins much smaller than the radius of the channel may effectively block the channel if tethers of appropriate length are used, and (ii) a large decrease in conductance upon protein binding can be achieved if the channel and the protein are oppositely charged. PMID:26368839

  3. Detection of secondary binding sites in proteins using fragment screening

    PubMed Central

    Ludlow, R. Frederick; Verdonk, Marcel L.; Saini, Harpreet K.; Tickle, Ian J.; Jhoti, Harren

    2015-01-01

    Proteins need to be tightly regulated as they control biological processes in most normal cellular functions. The precise mechanisms of regulation are rarely completely understood but can involve binding of endogenous ligands and/or partner proteins at specific locations on a protein that can modulate function. Often, these additional secondary binding sites appear separate to the primary binding site, which, for example for an enzyme, may bind a substrate. In previous work, we have uncovered several examples in which secondary binding sites were discovered on proteins using fragment screening approaches. In each case, we were able to establish that the newly identified secondary binding site was biologically relevant as it was able to modulate function by the binding of a small molecule. In this study, we investigate how often secondary binding sites are located on proteins by analyzing 24 protein targets for which we have performed a fragment screen using X-ray crystallography. Our analysis shows that, surprisingly, the majority of proteins contain secondary binding sites based on their ability to bind fragments. Furthermore, sequence analysis of these previously unknown sites indicate high conservation, which suggests that they may have a biological function, perhaps via an allosteric mechanism. Comparing the physicochemical properties of the secondary sites with known primary ligand binding sites also shows broad similarities indicating that many of the secondary sites may be druggable in nature with small molecules that could provide new opportunities to modulate potential therapeutic targets. PMID:26655740

  4. Analysing saturable antibody binding based on serum data and pharmacokinetic modelling

    NASA Astrophysics Data System (ADS)

    Kletting, Peter; Kiryakos, Hady; Reske, Sven N.; Glatting, Gerhard

    2011-01-01

    In radioimmunotherapy, organ dose calculations are frequently based on pretherapeutic biodistribution measurements, assuming equivalence between pretherapeutic and therapeutic biodistribution. However, when saturation of antibody binding sites is important, this assumption might not be justified. Residual antibody and different amounts of administered antibody may lead to a considerably altered therapeutic biodistribution. In this study we developed a method based on serum activity measurements to investigate this effect in radioimmunotherapy with 90Y-labelled anti-CD66 antibody. Pretherapeutic and therapeutic serum activity data of ten patients with acute leukaemia were fitted to a set of four parsimonious pharmacokinetic models. All models included the key mechanisms of antibody binding, immunoreactivity and degradation; however, they differed with respect to linear or nonlinear binding and global or individual fitting of the model parameters. The empirically most supported model was chosen according to the corrected Akaike information criterion. The nonlinear models were most supported by the data (sum of probabilities ≈100%). Using the presented method, we identified relevant saturable binding for radioimmunotherapy with 90Y-labelled anti-CD66 antibody solely based on serum data. This general method may also be applicable to investigate other systems where saturation of binding sites might be important.

  5. Improving Binding Affinity and Selectivity of Computationally Designed Ligand-Binding Proteins Using Experiments.

    PubMed

    Tinberg, Christine E; Khare, Sagar D

    2016-01-01

    The ability to de novo design proteins that can bind small molecules has wide implications for synthetic biology and medicine. Combining computational protein design with the high-throughput screening of mutagenic libraries of computationally designed proteins is emerging as a general approach for creating binding proteins with programmable binding modes, affinities, and selectivities. The computational step enables the creation of a binding site in a protein that otherwise does not (measurably) bind the intended ligand, and targeted mutagenic screening allows for validation and refinement of the computational model as well as provides orders-of-magnitude increases in the binding affinity. Deep sequencing of mutagenic libraries can provide insights into the mutagenic binding landscape and enable further affinity improvements. Moreover, in such a combined computational-experimental approach where the binding mode is preprogrammed and iteratively refined, selectivity can be achieved (and modulated) by the placement of specified amino acid side chain groups around the ligand in defined orientations. Here, we describe the experimental aspects of a combined computational-experimental approach for designing-using the software suite Rosetta-proteins that bind a small molecule of choice and engineering, using fluorescence-activated cell sorting and high-throughput yeast surface display, high affinity and ligand selectivity. We illustrated the utility of this approach by performing the design of a selective digoxigenin (DIG)-binding protein that, after affinity maturation, binds DIG with picomolar affinity and high selectivity over structurally related steroids. PMID:27094290

  6. Simultaneous analysis of serum immunoglobulins in patients with M protein using cellulose acetate membrane isoelectric focusing.

    PubMed

    Iijima, S; Shiba, K; Kurihara, Y; Kamei, S; Kimura, S; Kimura, M; Fukumura, Y; Kobayashi, I

    1999-01-01

    We developed a method for the simultaneous analysis of microheterogeneity of human serum IgG, IgA, IgM, IgD, and IgE, and serum protein pattern using cellulose acetate membrane isoelectric focusing, and analyzed in 11 healthy subjects and 67 patients with M protein (17 cases of multiple myeloma [MM] and 50 cases of monoclonal gammopathy of undetermined significance [MGUS]). Using this method, bands indicating the microheterogeneity of each immunoglobulin could clearly be detected.Among healthy subjects, the detected IgG, IgA, and IgM bands did not vary, but the detected IgE and IgD bands did vary. Therefore, IgA, IgM, and IgG were selected for comparison of serum immunoglobulins in MM and in MGUS. In the IgA-type M protein group, normal IgM and IgG bands were decreased in MM patients compared to MGUS patients, while the M band and other bands were increased in MM patients compared to MGUS patients, but the differences between the two groups were not significant. In the IgG-type M protein group, normal IgM, IgA, and IgG were significantly decreased in MM patients compared to MGUS patients. We examined the changes in electrophoretic pattern in six MM patients and eight MGUS patients with IgA-type M protein after neuraminidase treatment. The width of the M band in MM patients with IgA-type M protein decreased with neuraminidase treatment. On the other hand, the width of the M band in MGUS patients with IgA-type M protein increased with neuraminidase treatment. We concluded that the decrease of the normal immunoglobulins in MM patients with IgG type M protein could be detected by this method, and IgA type of M protein binding sugar chain were different between MM and MGUS patients. PMID:10414593

  7. Minimalistic predictor of protein binding energy: contribution of solvation factor to protein binding.

    PubMed

    Choi, Jeong-Mo; Serohijos, Adrian W R; Murphy, Sean; Lucarelli, Dennis; Lofranco, Leo L; Feldman, Andrew; Shakhnovich, Eugene I

    2015-02-17

    It has long been known that solvation plays an important role in protein-protein interactions. Here, we use a minimalistic solvation-based model for predicting protein binding energy to estimate quantitatively the contribution of the solvation factor in protein binding. The factor is described by a simple linear combination of buried surface areas according to amino-acid types. Even without structural optimization, our minimalistic model demonstrates a predictive power comparable to more complex methods, making the proposed approach the basis for high throughput applications. Application of the model to a proteomic database shows that receptor-substrate complexes involved in signaling have lower affinities than enzyme-inhibitor and antibody-antigen complexes, and they differ by chemical compositions on interfaces. Also, we found that protein complexes with components that come from the same genes generally have lower affinities than complexes formed by proteins from different genes, but in this case the difference originates from different interface areas. The model was implemented in the software PYTHON, and the source code can be found on the Shakhnovich group webpage: http://faculty.chemistry.harvard.edu/shakhnovich/software. PMID:25692584

  8. Photoaffinity labeling of retinoic acid-binding proteins.

    PubMed Central

    Bernstein, P S; Choi, S Y; Ho, Y C; Rando, R R

    1995-01-01

    Retinoid-binding proteins are essential mediators of vitamin A function in vertebrate organisms. They solubilize and stabilize retinoids, and they direct the intercellular and intracellular trafficking, transport, and metabolic function of vitamin A compounds in vision and in growth and development. Although many soluble retinoid-binding proteins and receptors have been purified and extensively characterized, relatively few membrane-associated enzymes and other proteins that interact with retinoids have been isolated and studied, due primarily to their inherent instabilities during purification. In an effort to identify and purify previously uncharacterized retinoid-binding proteins, it is shown that radioactively labeled all-trans-retinoic acid can be used as a photoaffinity labeling reagent to specifically tag two known retinoic acid-binding proteins, cellular retinoic acid-binding protein and albumin, in complex mixtures of cytosolic proteins. Additionally, a number of other soluble and membrane-associated proteins that bind all-trans-[11,12-3H]retinoic acid with high specificity are labeled utilizing the same photoaffinity techniques. Most of these labeled proteins have molecular weights that do not correspond to any known retinoid-binding proteins. Thus, photoaffinity labeling with all-trans-retinoic acid and related photoactivatable retinoids is a method that should prove extremely useful in the identification and purification of novel soluble and membrane-associated retinoid-binding proteins from ocular and nonocular tissues. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:7846032

  9. Neurospecific proteins in the serum of patients with brain tumors.

    PubMed

    Lyubimova, N V; Toms, M G; Popova, E E; Bondarenko, Y V; Krat, V B; Kushlinskii, N E

    2011-04-01

    Neurospecific proteins S-100 and GFAP were measured in the serum of 145 patients with neural tumors and 69 healthy individuals. In patients with glyoblastomas, the concentrations of S-100 and GFAP were significantly higher than in patients with anaplastic astrocytomas, benign meningiomas, and brain metastases and in healthy individuals. Serum S-100 concentrations in patients with anaplastic astrocytomas, benign meningiomas, and brain metastases were similar; significant difference from the control was found only for patients with cerebral metastases. A specific feature of GFAP was high incidence of its detection in patients with glioblastomas (83%) compared to other groups of patients with neural tumors and healthy volunteers who demonstrated practically zero level of this protein. These findings attest to the possibility of using S-100 as an additional biochemical criterion of brain involvement in tumor patients and GFAP as a glioblastoma marker. PMID:22235430

  10. RNA-Binding Proteins in Trichomonas vaginalis: Atypical Multifunctional Proteins.

    PubMed

    Figueroa-Angulo, Elisa E; Calla-Choque, Jaeson S; Mancilla-Olea, Maria Inocente; Arroyo, Rossana

    2015-01-01

    Iron homeostasis is highly regulated in vertebrates through a regulatory system mediated by RNA-protein interactions between the iron regulatory proteins (IRPs) that interact with an iron responsive element (IRE) located in certain mRNAs, dubbed the IRE-IRP regulatory system. Trichomonas vaginalis, the causal agent of trichomoniasis, presents high iron dependency to regulate its growth, metabolism, and virulence properties. Although T. vaginalis lacks IRPs or proteins with aconitase activity, possesses gene expression mechanisms of iron regulation at the transcriptional and posttranscriptional levels. However, only one gene with iron regulation at the transcriptional level has been described. Recently, our research group described an iron posttranscriptional regulatory mechanism in the T. vaginalis tvcp4 and tvcp12 cysteine proteinase mRNAs. The tvcp4 and tvcp12 mRNAs have a stem-loop structure in the 5'-coding region or in the 3'-UTR, respectively that interacts with T. vaginalis multifunctional proteins HSP70, α-Actinin, and Actin under iron starvation condition, causing translation inhibition or mRNA stabilization similar to the previously characterized IRE-IRP system in eukaryotes. Herein, we summarize recent progress and shed some light on atypical RNA-binding proteins that may participate in the iron posttranscriptional regulation in T. vaginalis. PMID:26703754

  11. Isolation and partial characterization of a fatty acid binding protein in rat liver plasma membranes.

    PubMed Central

    Stremmel, W; Strohmeyer, G; Borchard, F; Kochwa, S; Berk, P D

    1985-01-01

    When [14C]oleate-bovine serum albumin complexes were incubated in vitro with rat liver plasma membranes (LPM), specific, saturable binding of oleate to the membranes was observed. Maximal heat-sensitive (i.e., specific) binding was 3.2 nmol/mg of membrane protein. Oleate-agarose affinity chromatography of Triton X-100-solubilized LPM was used to isolate a single 40-kDa protein with high affinity for oleate. On gel filtration, the protein comigrated with various fatty acids but not with [14C]bilirubin, [35S]sulfobromophthalein, [14C]taurocholate, [14C]phosphatidylcholine, or [14C]cholesteryloleate. A rabbit antibody to this membrane fatty acid-binding protein gave a single precipitin line with the antigen but no reactivity with concentrated cytosolic proteins, LPM bilirubin/sulfobromophthalein-binding protein, or rat albumin or other rat plasma proteins. The antibody selectively inhibited heat-sensitive binding of [14C]oleate to LPM. Immunofluorescence studies localized the antigen in liver-cell plasma membranes as well as in other major sites of fatty acid transport. These data are compatible with the hypothesis that this protein may act as a receptor in a hepatocellular uptake mechanism for fatty acids. Images PMID:3881757

  12. DNA Shape versus Sequence Variations in the Protein Binding Process.

    PubMed

    Chen, Chuanying; Pettitt, B Montgomery

    2016-02-01

    The binding process of a protein with a DNA involves three stages: approach, encounter, and association. It has been known that the complexation of protein and DNA involves mutual conformational changes, especially for a specific sequence association. However, it is still unclear how the conformation and the information in the DNA sequences affects the binding process. What is the extent to which the DNA structure adopted in the complex is induced by protein binding, or is instead intrinsic to the DNA sequence? In this study, we used the multiscale simulation method to explore the binding process of a protein with DNA in terms of DNA sequence, conformation, and interactions. We found that in the approach stage the protein can bind both the major and minor groove of the DNA, but uses different features to locate the binding site. The intrinsic conformational properties of the DNA play a significant role in this binding stage. By comparing the specific DNA with the nonspecific in unbound, intermediate, and associated states, we found that for a specific DNA sequence, ∼40% of the bending in the association forms is intrinsic and that ∼60% is induced by the protein. The protein does not induce appreciable bending of nonspecific DNA. In addition, we proposed that the DNA shape variations induced by protein binding are required in the early stage of the binding process, so that the protein is able to approach, encounter, and form an intermediate at the correct site on DNA. PMID:26840719

  13. Probing the binding of an endocrine disrupting compound-Bisphenol F to human serum albumin: Insights into the interactions of harmful chemicals with functional biomacromolecules

    NASA Astrophysics Data System (ADS)

    Pan, Fang; Xu, Tianci; Yang, Lijun; Jiang, Xiaoqing; Zhang, Lei

    2014-11-01

    Bisphenol F (BPF) as an endocrine disrupting compounds (EDCs) pollutant in the environment poses a great threat to human health. To evaluate the toxicity of BPF at the protein level, the effects of BPF on human serum albumin (HSA) were investigated at three temperatures 283, 298, and 308 K by multiple spectroscopic techniques. The experimental results showed that BPF effectively quenched the intrinsic fluorescence of HSA via static quenching. The number of binding sites, the binding constant, the thermodynamic parameters and the binding subdomain were measured, and indicated that BPF could spontaneously bind with HSA on subdomain IIA through H-bond and van der Waals interactions. Furthermore, the conformation of HSA was demonstrably changed in the presence of BPF. The work provides accurate and full basic data for clarifying the binding mechanisms of BPF with HSA in vivo and is helpful for understanding its effect on protein function during its transportation and distribution in blood.

  14. Serum albumin binding of structurally diverse neutral organic compounds: data and models.

    PubMed

    Endo, Satoshi; Goss, Kai-Uwe

    2011-12-19

    Binding to serum albumin has a strong influence on freely dissolved, unbound concentrations of chemicals in vivo and in vitro. For neutral organic solutes, previous studies have suggested a log-log correlation between the albumin-water partition coefficient and the octanol-water partition coefficient (K(ow)) and postulated highly nonspecific binding that is mechanistically analogous to dissolution into solvents. These relationships and concepts were further explored in this study. Bovine serum albumin (BSA)-water partition coefficients (K(BSA/w)) were measured for 83 structurally diverse neutral organic chemicals in consistent experimental conditions. The correlation between log K(BSA/w) and log K(ow) was moderate, with R(2) = 0.76 and SD = 0.43. The log K(BSA/w) of low-polarity compounds including a series of chlorobenzenes and polycyclic aromatic hydrocarbons increased with log K(ow) linearly up to log K(ow) = 4-5, but then the linear relationship apparently broke off, and the increase became gradual. The fitting of polyparameter linear free energy relationship models with five solute descriptors was just comparable to that of the log K(ow) model (R(2) = 0.78-0.79, SD = 0.41-0.42); the relatively high SD obtained suggests that solvent dissolution models are not capable of modeling albumin binding accurately. A size limitation of the binding site(s) of albumin is suggested as a possible reason for the high SD. An equilibrium distribution model indicates that serum albumin generally has high contributions to the binding in the serum of polar compounds and relatively small low-polarity compounds, whereas albumin binding for large low-polarity compounds is outcompeted by the strong partitioning into lipids due to low relative affinity of albumin for these compounds. PMID:22070391

  15. Serum protein changes in ponies on different parasite control programmes.

    PubMed

    Herd, R P; Kent, J E

    1986-11-01

    Serum protein responses were examined in 52 ponies divided into five groups and subjected to various control strategies that resulted in pasture infectivity ranging from 706 to 18,486 infective third stage, cyathostome and Trichostrongylus axei larvae per kilogram of herbage (L3/kg) by 17 September 1984. Major protein changes occurred only in young ponies (Groups 4 and 5) and were observed before exposure to maximum numbers of pasture larvae (Group 4; 10,210 L3/kg, Group 5: 10,042 L3/kg) on 17 September. It appeared that a primary infection of T axei was a greater stimulus to serum beta-globulin and immunoglobulin (Ig)G(T) responses that provided by continued infection with cyathostome (small strongyle) worms. The large strongyles (Strongylus vulgaris, S edentatus and S equinus) were not detected in any larval cultures or on pastures grazed by the young ponies. A fall in beta-globulin and IgG(T) concentrations of Group 5 ponies one month after treatment with ivermectin indicated a larvicidal action against T axei and/or the cyathostomes. A subsequent rise in serum albumin concentrations of Group 5 ponies suggested that a protein-losing gastroenteropathy had been alleviated by the larvicidal action of ivermectin. Mature control ponies (Group 1) showed little beta-globulin response and only a modest IgG(T) response in six of the 10 ponies after exposure to heavily infected lawns (18,486 L3/kg) in September 1984. It was concluded that serum protein and IgG(T) responses were of limited value as an aid to diagnosis of parasitism because of numerous difficulties of interpretation. PMID:3803358

  16. Serum C-reactive protein in dairy herds

    PubMed Central

    Lee, Wen-Chuan; Hsiao, Huo-Cheng; Wu, Ying-Ling; Lin, Jyh-Hung; Lee, Yen-Pai; Fung, Hang-Poung; Chen, Hsin-Hsin; Chen, Yu-Hsin; Chu, Rea-Min

    2003-01-01

    The purpose of this study was to determine the relationship between the serum level of C-reactive protein (CRP) and lactation and health status. Blood samples were collected every 2 wk for 12 mo from 29 randomly selected dairy cattle on 3 farms. At the time the blood samples were collected, the stage of pregnancy, lactation status, breeding records, general health condition, reproductive status, and body condition score were recorded for each cow. Serum CRP was detected with sodium dodecyl sulfate polyacrylamide gel electrophoresis and western immunoblotting. C-reactive protein levels were measured with a densitometer and expressed as an optimal dose value. C-reactive protein levels were correlated with the body condition score, lactation status, and animal health (P < 0.05), but not with ambient temperature, animal age, or parity. C-reactive protein levels increased with milk production, peaking during high lactation (2 to 4 mo of pregnancy), and decreased when lactation ceased. In addition, the CRP level was highest during naturally occurring infections, such as mastitis and other tissue inflammation. Thus, the CRP level can confirm the presence of inflammation. The stress effect of taking blood samples as measured by the CRP level, was also examined. The CRP level became rapidly elevated 12 h after the blood samples were taken but returned to normal 36 h later. In conclusion, the stresses resulting from overall poor health, heavy lactation, and blood sampling caused the elevation of serum CRP. C-reactive protein is a marker or tool for evaluating the health status of a herd. C-reactive protein should also be considered as a useful criteria to assess the stress levels and may be useful in early surveillance of disease conditions in a dairy herd. PMID:12760474

  17. Partial characterization of GTP-binding proteins in Neurospora

    SciTech Connect

    Hasunuma, K.; Miyamoto-Shinohara, Y.; Furukawa, K.

    1987-08-14

    Six fractions of GTP-binding proteins separated by gel filtration of a mycelial extract containing membrane components of Neurospora crassa were partially characterized. (/sup 35/S)GTP gamma S bound to GTP-binding protein was assayed by repeated treatments with a Norit solution and centrifugation. The binding of (/sup 35/S)GTP gamma S to GTP-binding proteins was competitively prevented in the presence of 0.1 to 1 mM GTP but not in the presence of ATP. These GTP-binding proteins fractionated by the gel column had Km values of 20, 7, 4, 4, 80 and 2 nM. All six fractions of these GTP-binding proteins showed the capacity to be ADP-ribosylated by pertussis toxin.

  18. Dot-blot assay for heparin-binding proteins

    SciTech Connect

    Hirose, N.; Krivanek, M.; Jackson, R.L.; Cardin, A.D.

    1986-08-01

    A method for the detection and quantitation of picomole amounts of heparin-binding proteins is described. Proteins are first spotted on nitrocellulose and then incubated with /sup 125/I-heparin. Binding of heparin to the proteins is detected by radioautography and quantitated by scanning densitometry; proteins are quantitated by densitometric analysis of the amido black stained nitrocellulose. Heparin-binding was time-dependent and sensitive to the presence of metal ions, urea, and detergents (anionic, nonionic, and zwitterionic). The divalent cations Ca/sup 2 +/ and Mg/sup 2 +/ and the zwitterionic detergent 3-((3-cholamidopropyl)dimethylammonio)-1-propanesulfonate increased heparin binding whereas NaCl, urea, sodium dodecylsulfate, and La3+ decreased binding. This assay is applicable to the identification and characterization of a variety of heparin-binding proteins.

  19. Bacterial periplasmic sialic acid-binding proteins exhibit a conserved binding site

    SciTech Connect

    Gangi Setty, Thanuja; Cho, Christine; Govindappa, Sowmya; Apicella, Michael A.; Ramaswamy, S.

    2014-07-01

    Structure–function studies of sialic acid-binding proteins from F. nucleatum, P. multocida, V. cholerae and H. influenzae reveal a conserved network of hydrogen bonds involved in conformational change on ligand binding. Sialic acids are a family of related nine-carbon sugar acids that play important roles in both eukaryotes and prokaryotes. These sialic acids are incorporated/decorated onto lipooligosaccharides as terminal sugars in multiple bacteria to evade the host immune system. Many pathogenic bacteria scavenge sialic acids from their host and use them for molecular mimicry. The first step of this process is the transport of sialic acid to the cytoplasm, which often takes place using a tripartite ATP-independent transport system consisting of a periplasmic binding protein and a membrane transporter. In this paper, the structural characterization of periplasmic binding proteins from the pathogenic bacteria Fusobacterium nucleatum, Pasteurella multocida and Vibrio cholerae and their thermodynamic characterization are reported. The binding affinities of several mutations in the Neu5Ac binding site of the Haemophilus influenzae protein are also reported. The structure and the thermodynamics of the binding of sugars suggest that all of these proteins have a very well conserved binding pocket and similar binding affinities. A significant conformational change occurs when these proteins bind the sugar. While the C1 carboxylate has been identified as the primary binding site, a second conserved hydrogen-bonding network is involved in the initiation and stabilization of the conformational states.

  20. Binding and hydrolysis of soman by human serum albumin.

    PubMed

    Li, Bin; Nachon, Florian; Froment, Marie-Thérèse; Verdier, Laurent; Debouzy, Jean-Claude; Brasme, Bernardo; Gillon, Emilie; Schopfer, Lawrence M; Lockridge, Oksana; Masson, Patrick

    2008-02-01

    Human plasma and fatty acid free human albumin were incubated with soman at pH 8.0 and 25 degrees C. Four methods were used to monitor the reaction of albumin with soman: progressive inhibition of the aryl acylamidase activity of albumin, the release of fluoride ion from soman, 31P NMR, and mass spectrometry. Inhibition (phosphonylation) was slow with a bimolecular rate constant of 15 +/- 3 M(-1) min (-1). MALDI-TOF and tandem mass spectrometry of the soman-albumin adduct showed that albumin was phosphonylated on tyrosine 411. No secondary dealkylation of the adduct (aging) occurred. Covalent docking simulations and 31P NMR experiments showed that albumin has no enantiomeric preference for the four stereoisomers of soman. Spontaneous reactivation at pH 8.0 and 25 degrees C, measured as regaining of aryl acylamidase activity and decrease of covalent adduct (pinacolyl methylphosphonylated albumin) by NMR, occurred at a rate of 0.0044 h (-1), indicating that the adduct is quite stable ( t1/2 = 6.5 days). At pH 7.4 and 22 degrees C, the covalent soman-albumin adduct, measured by MALDI-TOF mass spectrometry, was more stable ( t1/2 = 20 days). Though the concentration of albumin in plasma is very high (about 0.6 mM), its reactivity with soman (phosphonylation and phosphotriesterase activity) is too slow to play a major role in detoxification of the highly toxic organophosphorus compound soman. Increasing the bimolecular rate constant of albumin for organophosphates is a protein engineering challenge that could lead to a new class of bioscavengers to be used against poisoning by nerve agents. Soman-albumin adducts detected by mass spectrometry could be useful for the diagnosis of soman exposure. PMID:18163544

  1. Binding study of tetracyclines to human serum albumin using difference spectrophotometry.

    PubMed

    Zia, H; Price, J C

    1976-02-01

    The binding of several tetracyclines to human serum albumin was studied using difference spectrophotometry and a spectrophotometric probe, 2-(4'-hydroxybenzeneazo)benzoic acid. Difference spectra observed for the interaction between the probe and human serum albumin were similar to probe-bovine serum albumin spectra but were less intense for a given concentration of probe and did not reach saturation as quickly. Difference spectra for the tetracyclines were dependent on the characteristics of the ring substituents. More hydrophobic substituents on the D and C rings tended to give more intense difference spectra, but charge-transfer complexing may also have been involved since methacycline with a methylene group in the 6-position showed the most intense spectra of the compounds studied. Solvent perturbation, pH, and urea studies tended to confirm that something other than hydrophobic binding of the tetracyclines was involved. Drug-probe displacement studies showed that methacycline gave the greatest probe displacement followed by doxycycline, chlortetracycline, oxytetracycline, and tetracycline. This order of displacement of the anionic probe indicates that both hydrophobic and charge-transfer binding are involved. Experiments with calcium ion and ethylenediaminetetraacetic acid showed that the difference spectra obtained with the tetracyclines and human serum albumin were not the result of metallic bridge-chelate formation. PMID:3641

  2. In vivo and in vitro studies of hafnium-binding to rat serum transferrin.

    PubMed

    Then, G M; Appel, H; Duffield, J; Taylor, D M; Thies, W G

    1986-08-01

    The binding of hafnium to rat serum transferrin was studied using the time differential perturbed angular correlation (TDPAC) technique. Hafnium is interesting as a toxic metal binding to transferrin because it behaves metabolically similarly to plutonium. The isotope 181Hf offers favorable access to the TDPAC-method. Samples were prepared in vivo by intravenous injection of Hf-NTA, Hf-citrate, and Hf-oxalate solutions, respectively, into Sprague-Dawley rats and in vitro by adding Hf-NTA solution to fresh rat serum. In both cases two specific electric quadrupole interactions were observed, which correspond to two well-defined binding configurations. They may be attributed to the N-terminal and the C-terminal binding site in the transferrin molecule. The 181Hf-distribution between these two binding states depends on pH, salt and hafnium concentrations, temperature, and incubation time. With a fast TDPAC-setup of four BaF2-detectors a time resolution of about 600 ps could be achieved. The specific binding configurations of 181Hf and the comparatively slow relaxation times lead to spectra of considerable accuracy. PMID:3746294

  3. In vivo and in vitro studies of hafnium-binding to rat serum transferrin

    SciTech Connect

    Then, G.M.; Appel, H.; Duffield, J.; Taylor, D.M.; Thies, W.G.

    1986-08-01

    The binding of hafnium to rat serum transferrin was studied using the time differential perturbed angular correlation (TDPAC) technique. Hafnium is interesting as a toxic metal binding to transferrin because it behaves metabolically similarly to plutonium. The isotope 181Hf offers favorable access to the TDPAC-method. Samples were prepared in vivo by intravenous injection of Hf-NTA, Hf-citrate, and Hf-oxalate solutions, respectively, into Sprague-Dawley rats and in vitro by adding Hf-NTA solution to fresh rat serum. In both cases two specific electric quadrupole interactions were observed, which correspond to two well-defined binding configurations. They may be attributed to the N-terminal and the C-terminal binding site in the transferrin molecule. The 181Hf-distribution between these two binding states depends on pH, salt and hafnium concentrations, temperature, and incubation time. With a fast TDPAC-setup of four BaF2-detectors a time resolution of about 600 ps could be achieved. The specific binding configurations of 181Hf and the comparatively slow relaxation times lead to spectra of considerable accuracy.

  4. Interaction of lipid vesicle with silver nanoparticle-serum albumin protein corona

    NASA Astrophysics Data System (ADS)

    Chen, Ran; Choudhary, Poonam; Schurr, Ryan N.; Bhattacharya, Priyanka; Brown, Jared M.; Chun Ke, Pu

    2012-01-01

    The physical interaction between a lipid vesicle and a silver nanoparticle (AgNP)-human serum albumin (HSA) protein "corona" has been examined. Specifically, the binding of AgNPs and HSA was analyzed by spectrophotometry, and the induced conformational changes of the HSA were inferred from circular dichroism spectroscopy. The fluidity of the vesicle, a model system for mimicking cell membrane, was found to increase with the increased exposure to AgNP-HSA corona, though less pronounced compared to that induced by AgNPs alone. This study offers additional information for understanding the role of physical forces in nanoparticle-cell interaction and has implications for nanomedicine and nanotoxicology.

  5. Elucidation of binding mechanism and identification of binding site for an anti HIV drug, stavudine on human blood proteins.

    PubMed

    Sandhya, B; Hegde, Ashwini H; Seetharamappa, J

    2013-05-01

    The binding of stavudine (STV) to two human blood proteins [human hemoglobin (HHb) and human serum albumin (HSA)] was studied in vitro under simulated physiological conditions by spectroscopic methods viz., fluorescence, UV absorption, resonance light scattering, synchronous fluorescence, circular dichroism (CD) and three-dimensional fluorescence. The binding parameters of STV-blood protein were determined from fluorescence quenching studies. Stern-Volmer plots indicated the presence of static quenching mechanism in the interaction of STV with blood proteins. The values of n close to unity indicated that one molecule of STV bound to one molecule of blood protein. The binding process was found to be spontaneous. Analysis of thermodynamic parameters revealed the presence of hydrogen bond and van der Waals forces between protein and STV. Displacement experiments indicated the binding of STV to Sudlow's site I on HSA. Secondary structures of blood proteins have undergone changes upon interaction with STV as evident from the reduction of α-helices (from 46.11% in free HHb to 38.34% in STV-HHb, and from 66.44% in free HSA to 52.26% in STV-HSA). Further, the alterations in secondary structures of proteins in the presence of STV were confirmed by synchronous and 3D-fluorescence spectral data. The distance between the blood protein (donor) and acceptor (STV) was found to be 5.211 and 5.402 nm for STV-HHb and STV-HSA, respectively based on Föster's non-radiative energy transfer theory. Effect of some metal ions was also investigated. The fraction of STV bound to HSA was found to be 87.8%. PMID:23275205

  6. Serum antibody immunoreactivity to equine zona protein after SpayVac vaccination.

    PubMed

    Mask, Tracy A; Schoenecker, Kathryn A; Kane, Albert J; Ransom, Jason I; Bruemmer, Jason E

    2015-07-15

    Immunocontraception with porcine ZP (pZP) can be an effective means of fertility control in feral horses. Previous studies suggest that antibodies produced after pZP vaccination may both inhibit fertilization and cause follicular dysgenesis. Zonastat-H, PZP-22, and SpayVac are three pZP vaccines proposed for use in horses. Although all these vaccines contain the pZP antigen, variations in antigen preparation and vaccine formulation lead to differences in antigenic properties among them. Likewise, despite numerous efficacy and safety studies of Zonastat-H and PZP-22, the contraceptive mechanisms of SpayVac remain unclear. The preparation of pZP for SpayVac is thought to include more nonzona proteins, making it less pure than the other two vaccines. This may result in increased antigenicity of the vaccine. We therefore investigated the immunoreactivity of serum antibodies from SpayVac-vaccinated mares to equine zona protein. Western blot analyses revealed an immunoreactivity of these antibodies to protein isolated from mature equine oocytes, ZP, follicular tissues, and ovarian tissues. Immunohistochemical analyses were used to locate the binding of serum antibodies to the ZP of immature oocytes in ovarian stromal tissue. We also found serum antibodies from SpayVac-treated mares to be predominantly specific for zona protein 3. Collectively, our results suggest a model where serum antibodies produced in response to SpayVac vaccination are immunoreactive to equine zona protein in vitro. Our study lends insight into the contraceptive mechanisms underlying the infertility observed after SpayVac vaccination. PMID:25922172

  7. Protein-Polyelectrolyte Coacervation: Morphology Diagram, Binding Affinity, and Protein Separation

    NASA Astrophysics Data System (ADS)

    Hoagland, David; Du, Xiaosong; Dubin, Paul

    2014-03-01

    For aqueous mixtures of negatively charged polysaccharide, hyaluronic acid (HA), and globular protein, either bovine serum albumin (BSA) or beta-lactoglobulin (BLG), a pH-ionic strength (I) morphology diagram, with regions of homogeneous solution, soluble complex, coacervation, precipitation, and redissolution, was developed by pH titrations performed at fixed I. The systems are models for coacervation, or liquid-liquid phase separation, between flexible and compact solutes of opposite charge. Protein charge here is tuned by pH, and titration keeps the mixtures close to equilibrium. At high I, only homogeneous solution is observed, as true at high and low pH. Diagrams for the proteins differ because HA affinity for BSA is higher than for BLG, traced to BSA's greater charge patchiness and higher net charge; isothermal solution titration calorimetry finds a factor of two difference in binding energy. Dependences of transition pH on protein charge Z and solution I offer additional insights into interactions underlying morphology transitions. At optimal conditions, the affinity disparity is sufficient to achieve highly selective BSA coacervation in a 1:1 protein mixture, suggesting coacervation to separate similar proteins under mild, non-denaturing conditions. Funding: NSF CBET-1133289, NSF (UMass MRSEC).

  8. Convergent evolution among immunoglobulin G-binding bacterial proteins.

    PubMed Central

    Frick, I M; Wikström, M; Forsén, S; Drakenberg, T; Gomi, H; Sjöbring, U; Björck, L

    1992-01-01

    Protein G, a bacterial cell-wall protein with high affinity for the constant region of IgG (IgGFc) antibodies, contains homologous repeats responsible for the interaction with IgGFc. A synthetic peptide corresponding to an 11-amino acid-long sequence in the COOH-terminal region of the repeats was found to bind to IgGFc and block the interaction with protein G. Moreover, two other IgGFc-binding bacterial proteins (proteins A and H), which do not contain any sequences homologous to the peptide, were also inhibited in their interactions with IgGFc by the peptide. Finally, a decapeptide based on a sequence in IgGFc blocked the binding of all three proteins to IgGFc. This unusually clear example of convergent evolution emphasizes the complexity of protein-protein interactions and suggests that bacterial surface-protein interaction with host protein adds selective advantages to the microorganism. Images PMID:1528858

  9. Unraveling the binding mechanism of asiatic acid with human serum albumin and its biological implications.

    PubMed

    Gokara, Mahesh; Malavath, Tirupathi; Kalangi, Suresh Kumar; Reddana, Pallu; Subramanyam, Rajagopal

    2014-01-01

    Asiatic acid (AsA), a naturally occurring pentacyclictriterpenoid found in Centella asiatica, plays a major role in neuroprotection, anticancer, antioxidant, and hepatoprotective activities. Human serum albumin (HSA), a blood plasma protein, participates in the regulation of plasma osmotic pressure and transports endogenous and exogenous substances. The study undertaken to analyze the drug-binding mechanisms of HSA is crucial in understanding the bioavailability of drugs. In this study, we analyzed the cytotoxic activity of AsA on HepG2 (human hepatocellular carcinoma) cell lines and its binding, conformational, docking, molecular simulation studies with HSA under physiological pH 7.2. These studies revealed a clear decrease in the viability of HepG2 cells upon exposure to AsA in a dose-dependent manner with an IC50 of 45 μM. Further studies showed the quenching of intrinsic fluorescence of HSA by AsA with a binding constant of KAsA = 3.86 ± 0.01 × 10(4) M(-1), which corresponds to the free energy of (ΔG) -6.3 kcal M(-1) at 25 °C. Circular dichroism (CD) studies revealed that there is a clear decrease in the α-helical content from 57.50 ± 2.4 to 50% ± 2.3 and an increase in the β-turns from 25 ± 0.65 to 29% ± 0.91 and random coils from 17.5% ± 0.95 to 21% ± 1.2, suggesting partial unfolding of HSA. Autodock studies revealed that the AsA is bound to the subdomain IIA with hydrophobic and hydrophilic interactions. From molecular dynamics, simulation data (RMSD, Rg and RMSF) emphasized the local conformational changes and rigidity of the residues of both HSA and HSA-AsA complexes. PMID:23844909

  10. Fused protein domains inhibit DNA binding by LexA.

    PubMed Central

    Golemis, E A; Brent, R

    1992-01-01

    Many studies of transcription activation employ fusions of activation domains to DNA binding domains derived from the bacterial repressor LexA and the yeast activator GAL4. Such studies often implicitly assume that DNA binding by the chimeric proteins is equivalent to that of the protein donating the DNA binding moiety. To directly investigate this issue, we compared operator binding by a series of LexA-derivative proteins to operator binding by native LexA, by using both in vivo and in vitro assays. We show that operator binding by many proteins such as LexA-Myc, LexA-Fos, and LexA-Bicoid is severely impaired, while binding of other LexA-derivative proteins, such as those that carry bacterially encoded acidic sequences ("acid blobs"), is not. Our results also show that DNA binding by LexA derivatives that contain the LexA carboxy-terminal dimerization domain (amino acids 88 to 202) is considerably stronger than binding by fusions that lack it and that heterologous dimerization motifs cannot substitute for the LexA88-202 function. These results suggest the need to reevaluate some previous studies of activation that employed LexA derivatives and modifications to recent experimental approaches that use LexA and GAL4 derivatives to detect and study protein-protein interactions. Images PMID:1620111

  11. Investigation of alginate binding to germanium and polystyrene substrata conditioned with mussel adhesive protein

    SciTech Connect

    Suci, P.A.; Geesey, G.G.

    1995-06-15

    Binding of alginate from Macrocystis pyrifera (kelp) to germanium and polystyrene substrata conditioned with mussel adhesive protein (MAP) from Mytilis edulis, to germanium substrata conditioned with bovine serum albumin (BSA) and polylysine, and to germanium substrata coated with aminopropyltriethoxysilane (APS) was investigated using attenuated total reflection Fourier transform infrared spectrometry. Binding of alginate to MAP appears to be proportional to surface coverage for levels tested. Distinct spectral features appear in the region associated with pyranose ring vibrations upon binding of alginate to MAP, polylysine, and APS, indicating that lysine residues play a prominent role in promoting irreversible adsorption with perturbation of pyranose ring atoms. BSA does not appear to enhance alginate adsorption over that observed on clean germanium and no new spectral features appear as a result of binding. The level of irreversible binding of alginate to germanium and polystyrene substrata conditioned with MAP is similar.

  12. Calmodulin Binding Proteins and Alzheimer’s Disease

    PubMed Central

    O’Day, Danton H.; Eshak, Kristeen; Myre, Michael A.

    2015-01-01

    Abstract The small, calcium-sensor protein, calmodulin, is ubiquitously expressed and central to cell function in all cell types. Here the literature linking calmodulin to Alzheimer’s disease is reviewed. Several experimentally-verified calmodulin-binding proteins are involved in the formation of amyloid-β plaques including amyloid-β protein precursor, β-secretase, presenilin-1, and ADAM10. Many others possess potential calmodulin-binding domains that remain to be verified. Three calmodulin binding proteins are associated with the formation of neurofibrillary tangles: two kinases (CaMKII, CDK5) and one protein phosphatase (PP2B or calcineurin). Many of the genes recently identified by genome wide association studies and other studies encode proteins that contain putative calmodulin-binding domains but only a couple (e.g., APOE, BIN1) have been experimentally confirmed as calmodulin binding proteins. At least two receptors involved in calcium metabolism and linked to Alzheimer’s disease (mAchR; NMDAR) have also been identified as calmodulin-binding proteins. In addition to this, many proteins that are involved in other cellular events intimately associated with Alzheimer’s disease including calcium channel function, cholesterol metabolism, neuroinflammation, endocytosis, cell cycle events, and apoptosis have been tentatively or experimentally verified as calmodulin binding proteins. The use of calmodulin as a potential biomarker and as a therapeutic target is discussed. PMID:25812852

  13. Odorant binding proteins: a biotechnological tool for odour control.

    PubMed

    Silva, Carla; Matamá, Teresa; Azoia, Nuno G; Mansilha, Catarina; Casal, Margarida; Cavaco-Paulo, Artur

    2014-04-01

    The application of an odorant binding protein for odour control and fragrance delayed release from a textile surface was first explored in this work. Pig OBP-1 gene was cloned and expressed in Escherichia coli, and the purified protein was biochemically characterized. The IC₅₀ values (concentrations of competitor that caused a decay of fluorescence to half-maximal intensity) were determined for four distinct fragrances, namely, citronellol, benzyl benzoate, citronellyl valerate and ethyl valerate. The results showed a strong binding of citronellyl valerate, citronellol and benzyl benzoate to the recombinant protein, while ethyl valerate displayed weaker binding. Cationized cotton substrates were coated with porcine odorant binding protein and tested for their capacity to retain citronellol and to mask the smell of cigarette smoke. The immobilized protein delayed the release of citronellol when compared to the untreated cotton. According to a blind evaluation of 30 assessors, the smell of cigarette smoke, trapped onto the fabrics' surface, was successfully attenuated by porcine odorant binding protein (more than 60 % identified the weakest smell intensity after protein exposure compared to β-cyclodextrin-treated and untreated cotton fabrics). This work demonstrated that porcine odorant binding protein can be an efficient solution to prevent and/or remove unpleasant odours trapped on the large surface of textiles. Its intrinsic properties make odorant binding proteins excellent candidates for controlled release systems which constitute a new application for this class of proteins. PMID:24092006

  14. Protein Binding: Do We Ever Learn?▿

    PubMed Central

    Zeitlinger, Markus A.; Derendorf, Hartmut; Mouton, Johan W.; Cars, Otto; Craig, William A.; Andes, David; Theuretzbacher, Ursula

    2011-01-01

    Although the influence of protein binding (PB) on antibacterial activity has been reported for many antibiotics and over many years, there is currently no standardization for pharmacodynamic models that account for the impact of protein binding of antimicrobial agents in vitro. This might explain the somewhat contradictory results obtained from different studies. Simple in vitro models which compare the MIC obtained in protein-free standard medium versus a protein-rich medium are prone to methodological pitfalls and may lead to flawed conclusions. Within in vitro test systems, a range of test conditions, including source of protein, concentration of the tested antibiotic, temperature, pH, electrolytes, and supplements may influence the impact of protein binding. As new antibiotics with a high degree of protein binding are in clinical development, attention and action directed toward the optimization and standardization of testing the impact of protein binding on the activity of antibiotics in vitro become even more urgent. In addition, the quantitative relationship between the effects of protein binding in vitro and in vivo needs to be established, since the physiological conditions differ. General recommendations for testing the impact of protein binding in vitro are suggested. PMID:21537013

  15. Identification of transferrin as the main binding site for protactinium in rat blood serum.

    PubMed

    Taylor, D M; Farrow, L C

    1987-01-01

    The distribution of 233Pa in rat serum at periods between 5 and 50 min after i.v. injection of a solution of protactinium chloride was studied by gel chromatography. Sequential analysis of sera on Sephacryl S-300 and DEAE-Sephadex showed that 233Pa was associated only with the transferrin fraction of the serum proteins. This finding was confirmed by iso-electric focusing electrophoresis. In the cytosol fractions prepared from the liver and kidneys of the 233Pa injected rats the nuclide was also shown to be protein bound. PMID:3583752

  16. The distribution of iron between the metal-binding sites of transferrin human serum.

    PubMed

    Williams, J; Moreton, K

    1980-02-01

    The Makey & Seal [(1976) Biochim. Biophys. Acta 453, 250--256] method of polyacrylamide-gel electrophoresis in buffer containing 6 M-urea was used to determine the distribution of iron between the N-terminal and C-terminal iron-binding sites of transferrin in human serum. In fresh serum the two sites are unequally occupied; there is preferential occupation of the N-terminal site. On incubation of the serum at 37 degrees C the preference of iron for the N-terminal site becomes more marked. On storage of serum at -15 degrees C the iron distribution changes so that there is a marked preference for the C-terminal site. Dialysis of serum against buffer at pH 7.4 also causes iron to be bound much more strongly by the C-terminal than by the N-terminal site. The original preference for the N-terminal site can be resroted to the dialysed serum by addition of the diffusible fraction. PMID:7396826

  17. The distribution of iron between the metal-binding sites of transferrin human serum.

    PubMed Central

    Williams, J; Moreton, K

    1980-01-01

    The Makey & Seal [(1976) Biochim. Biophys. Acta 453, 250--256] method of polyacrylamide-gel electrophoresis in buffer containing 6 M-urea was used to determine the distribution of iron between the N-terminal and C-terminal iron-binding sites of transferrin in human serum. In fresh serum the two sites are unequally occupied; there is preferential occupation of the N-terminal site. On incubation of the serum at 37 degrees C the preference of iron for the N-terminal site becomes more marked. On storage of serum at -15 degrees C the iron distribution changes so that there is a marked preference for the C-terminal site. Dialysis of serum against buffer at pH 7.4 also causes iron to be bound much more strongly by the C-terminal than by the N-terminal site. The original preference for the N-terminal site can be resroted to the dialysed serum by addition of the diffusible fraction. Images Fig. 1. PMID:7396826

  18. Genome-wide mapping of Myc binding and gene regulation in serum-stimulated fibroblasts

    PubMed Central

    Perna, D; Fagà, G; Verrecchia, A; Gorski, M M; Barozzi, I; Narang, V; Khng, J; Lim, K C; Sung, W-K; Sanges, R; Stupka, E; Oskarsson, T; Trumpp, A; Wei, C-L; Müller, H; Amati, B

    2012-01-01

    The transition from quiescence to proliferation is a key regulatory step that can be induced by serum stimulation in cultured fibroblasts. The transcription factor Myc is directly induced by serum mitogens and drives a secondary gene expression program that remains largely unknown. Using mRNA profiling, we identify close to 300 Myc-dependent serum response (MDSR) genes, which are induced by serum in a Myc-dependent manner in mouse fibroblasts. Mapping of genomic Myc-binding sites by ChIP-seq technology revealed that most MDSR genes were directly targeted by Myc, but represented a minor fraction (5.5%) of all Myc-bound promoters (which were 22.4% of all promoters). Other target loci were either induced by serum in a Myc-independent manner, were not significantly regulated or were negatively regulated. MDSR gene products were involved in a variety of processes, including nucleotide biosynthesis, ribosome biogenesis, DNA replication and RNA control. Of the 29 MDSR genes targeted by RNA interference, three showed a requirement for cell-cycle entry upon serum stimulation and 11 for long-term proliferation and/or survival. Hence, proper coordination of key regulatory and biosynthetic pathways following mitogenic stimulation relies upon the concerted regulation of multiple Myc-dependent genes. PMID:21860422

  19. Structural aspects of a protein-surfactant assembly: native and reduced States of human serum albumin.

    PubMed

    Anand, Uttam; Ray, Sutapa; Ghosh, Subhadip; Banerjee, Rajat; Mukherjee, Saptarshi

    2015-04-01

    The inherently present seventeen disulfide bonds of the circulatory protein, human serum albumin (HSA) provide the necessary structural stability. Various spectroscopic approaches were used to investigate the effect of reduction of these disulfide bonds and its binding with the anionic surfactant, sodium dodecyl sulfate (SDS). Based on several spectroscopic analyses, our investigations highlight the following interesting aspects: (1) HSA on reduction loses not only its tertiary structure but also a significant amount of secondary structure as well. However, the reduced state of the protein is not like the molten-globule, (2) this structural loss of the protein due to reduction is more prominent than that caused by higher SDS concentrations alone and can certainly be attributed to the role of disulfide bonds, (3) lower surfactant concentrations provide marginal structural rigidity to the native state of the protein, whereas, higher concentrations of SDS induces secondary structure to the reduced state of HSA, (4) the binding of SDS with both the native and reduced states of HSA, occurred in three distinct stages which was followed by a saturation stage. However, the nature of such binding is different for both the states as investigated by using the Stern-Volmer equations and estimating the thermodynamic parameters. Besides, in contrast to the native state, the reduced state of HSA shows that the lone tryptophan residue gets more buried. However, there occurs a sudden decrement in the lifetime of the tryptophan and the hydrodynamic diameter increases by twofold. PMID:25821118

  20. Analysis of serum proteins by LC-MS/MS.

    PubMed

    Tonack, Sarah; Neoptolemos, John P; Costello, Eithne

    2010-01-01

    Serum contains a vast array of proteins, some of which are specific to blood whilst others are secreted into blood from tissues and organs. The so-called tissue leakage factors reveal information about the tissue from which they originate and are therefore of great potential importance as disease biomarkers. There are already a number of blood-borne biomarkers in routine clinical use that aid in the diagnosis or management of cancer. However, there is a pressing need for additional markers, and new methods to find them are under development. Here we provide a protocol for serum protein profiling using liquid chromatography tandem mass spectrometry (LC-MS/MS). Included in this procedure, we detail the pre-processing steps of lipid and high-abundance protein removal. These procedures can also be employed up-stream of quantification methods such as isobaric tags for relative and absolute quantification (iTRAQ). Chapter 12 is devoted to the iTRAQ approach for quantifying proteins, and it is therefore not described in this chapter. PMID:20839111

  1. Therapeutic and analytical applications of arsenic binding to proteins.

    PubMed

    Chen, Beibei; Liu, Qingqing; Popowich, Aleksandra; Shen, Shengwen; Yan, Xiaowen; Zhang, Qi; Li, Xing-Fang; Weinfeld, Michael; Cullen, William R; Le, X Chris

    2015-01-01

    Arsenic binding to proteins plays a pivotal role in the health effects of arsenic. Further knowledge of arsenic binding to proteins will advance the development of bioanalytical techniques and therapeutic drugs. This review summarizes recent work on arsenic-based drugs, imaging of cellular events, capture and purification of arsenic-binding proteins, and biosensing of arsenic. Binding of arsenic to the promyelocytic leukemia fusion oncoprotein (PML-RARα) is a plausible mode of action leading to the successful treatment of acute promyelocytic leukemia (APL). Identification of other oncoproteins critical to other cancers and the development of various arsenicals and targeted delivery systems are promising approaches to the treatment of other types of cancers. Techniques for capture, purification, and identification of arsenic-binding proteins make use of specific binding between trivalent arsenicals and the thiols in proteins. Biarsenical probes, such as FlAsH-EDT2 and ReAsH-EDT2, coupled with tetracysteine tags that are genetically incorporated into the target proteins, are used for site-specific fluorescence labelling and imaging of the target proteins in living cells. These allow protein dynamics and protein-protein interactions to be studied. Arsenic affinity chromatography is useful for purification of thiol-containing proteins, and its combination with mass spectrometry provides a targeted proteomic approach for studying the interactions between arsenicals and proteins in cells. Arsenic biosensors evolved from the knowledge of arsenic resistance and arsenic binding to proteins in bacteria, and have now been developed into analytical techniques that are suitable for the detection of arsenic in the field. Examples in the four areas, arsenic-based drugs, imaging of cellular events, purification of specific proteins, and arsenic biosensors, demonstrate important therapeutic and analytical applications of arsenic protein binding. PMID:25356501

  2. Odorant-Binding Protein: Localization to Nasal Glands and Secretions

    NASA Astrophysics Data System (ADS)

    Pevsner, Jonathan; Sklar, Pamela B.; Snyder, Solomon H.

    1986-07-01

    An odorant-binding protein (OBP) was isolated from bovine olfactory and respiratory mucosa. We have produced polyclonal antisera to this protein and report its immunohistochemical localization to mucus-secreting glands of the olfactory and respiratory mucosa. Although OBP was originally isolated as a pyrazine binding protein, both rat and bovine OBP also bind the odorants [3H]methyldihydrojasmonate and 3,7-dimethyl-octan-1-ol as well as 2-isobutyl-3-[3H]methoxypyrazine. We detect substantial odorant-binding activity attributable to OBP in secreted rat nasal mucus and tears but not in saliva, suggesting a role for OBP in transporting or concentrating odorants.

  3. Dye-promoted precipitation of serum proteins. Mechanism and application.

    PubMed

    Birkenmeier, G; Kopperschläger, G

    1991-11-01

    Immobilized dyes have been used primarily for purification of nucleotide dependent enzymes and proteins from plasma and other sources. Due to their low costs, high protein binding capacity and resistance to degradation dyes bear the potential as ligand for affinity separation of proteins on a large scale. In this paper dyes have been used for precipitation of proteins. Using albumin, prealbumin, alpha 1-acid glycoprotein and immunoglobulin G as model proteins we could demonstrate that dye-promoted precipitation depends on several factors which include the structure of the dye, the pH of the solution, the dye/protein molar ratio and the intrinsic properties of the proteins. It revealed that most of the dyes tested were endowed with the precipitating potential. The efficacy of precipitation was found to increase with the complexity of the dye structure. However, the amount of a dye required for total precipitation was found to be different for a given protein. Electrostatic as well as hydrophobic forces are involved in the mechanism of precipitation. It was demonstrated that by optimizing the conditions, mixtures of proteins can be resolved by dye-promoted precipitation. The high sensitivity of the reaction offers the possibility of using this method for rapid concentration of very diluted protein solutions. PMID:1367693

  4. Actin binding proteins, spermatid transport and spermiation*

    PubMed Central

    Qian, Xiaojing; Mruk, Dolores D.; Cheng, Yan-Ho; Tang, Elizabeth I.; Han, Daishu; Lee, Will M.; Wong, Elissa W. P.; Cheng, C. Yan

    2014-01-01

    The transport of germ cells across the seminiferous epithelium is composed of a series of cellular events during the epithelial cycle essential to the completion of spermatogenesis. Without the timely transport of spermatids during spermiogenesis, spermatozoa that are transformed from step 19 spermatids in the rat testis fail to reach the luminal edge of the apical compartment and enter the tubule lumen at spermiation, thereby entering the epididymis for further maturation. Step 19 spermatids and/or sperms that remain in the epithelium will be removed by the Sertoli cell via phagocytosis to form phagosomes and be degraded by lysosomes, leading to subfertility and/or infertility. However, the biology of spermatid transport, in particular the final events that lead to spermiation remain elusive. Based on recent data in the field, we critically evaluate the biology of spermiation herein by focusing on the actin binding proteins (ABPs) that regulate the organization of actin microfilaments at the Sertoli-spermatid interface, which is crucial for spermatid transport during this event. The hypothesis we put forth herein also highlights some specific areas of research that can be pursued by investigators in the years to come. PMID:24735648

  5. Chemical characterization of binding properties of opacity-associated protein II from Neisseria gonorrhoeae.

    PubMed Central

    Bessen, D; Gotschlich, E C

    1987-01-01

    Binding of an opacity-associated protein II (PIIop) from Neisseria gonorrhoeae to eucaryotic macromolecules was studied. HeLa cell extracts were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose, and purified PIIop bound to approximately 50 distinct molecular species. The binding of PIIop to HeLa cell components was stable in high salt and nonionic detergent and was not inhibited by a variety of monosaccharides and polyionic substances. PIIop binding behavior was compared with that of two model carbohydrate-binding proteins, wheat germ agglutinin (WGA) and concanavalin A (ConA). Model glycoproteins (ovomucoid, fetuin, mucin, ovalbumin) inhibited binding by PIIop, WGA, and ConA to various degrees. HeLa cell glycopeptides, generated by pronase digestion of chloroform-methanol-extracted cells, were tested for their ability to inhibit binding by PIIop to Western blots of HeLa cell macromolecules. HeLa cell extracts inhibited PIIop binding before pronase treatment, but inhibitory activity was lost as a result of pronase digestion. Direct binding to defined glycosylated and nonglycosylated proteins revealed that ConA and WGA bound only glycoproteins, whereas PIIop bound to proteins lacking carbohydrate as well. PIIop binding to human and bovine serum albumins was of high affinity and required partial unfolding of albumin; native albumin was not bound by PIIop; however, both the denatured, reduced form of albumin and the compact, nonreduced form of carboxymethylated albumin were bound strongly by PIIop. Albumin-PIIop interaction did not involve covalent bond formation through sulfhydryl groups. The predominant binding interactions of PIIop found in this study were with protein rather than carbohydrate, and the chemical nature of the interactions is more complex than involvement of purely ionic or hydrophobic forces. Images PMID:3098683

  6. RNA binding protein and binding site useful for expression of recombinant molecules

    DOEpatents

    Mayfield, Stephen

    2000-01-01

    The present invention relates to a gene expression system in eukaryotic and prokaryotic cells, preferably plant cells and intact plants. In particular, the invention relates to an expression system having a RB47 binding site upstream of a translation initiation site for regulation of translation mediated by binding of RB47 protein, a member of the poly(A) binding protein family. Regulation is further effected by RB60, a protein disulfide isomerase. The expression system is capable of functioning in the nuclear/cytoplasm of cells and in the chloroplast of plants. Translation regulation of a desired molecule is enhanced approximately 100 fold over that obtained without RB47 binding site activation.

  7. RNA binding protein and binding site useful for expression of recombinant molecules

    DOEpatents

    Mayfield, Stephen P.

    2006-10-17

    The present invention relates to a gene expression system in eukaryotic and prokaryotic cells, preferably plant cells and intact plants. In particular, the invention relates to an expression system having a RB47 binding site upstream of a translation initiation site for regulation of translation mediated by binding of RB47 protein, a member of the poly(A) binding protein family. Regulation is further effected by RB60, a protein disulfide isomerase. The expression system is capable of functioning in the nuclear/cytoplasm of cells and in the chloroplast of plants. Translation regulation of a desired molecule is enhanced approximately 100 fold over that obtained without RB47 binding site activation.

  8. The Acute-Phase Proteins Serum Amyloid A and C Reactive Protein in Transudates and Exudates

    PubMed Central

    Okino, Alessandra M.; Bürger, Cristiani; Cardoso, Jefferson R.; Lavado, Edson L.; Lotufo, Paulo A.; Campa, Ana

    2006-01-01

    The distinction between exudates and transudates is very important in the patient management. Here we evaluate whether the acute-phase protein serum amyloid A (SAA), in comparison with C reactive protein (CRP) and total protein (TP), can be useful in this discrimination. CRP, SAA, and TP were determined in 36 exudate samples (27 pleural and 9 ascitic) and in 12 transudates (9 pleural and 3 ascitic). CRP, SAA, and TP were measured. SAA present in the exudate corresponded to 10% of the amount found in serum, that is, the exudate/serum ratio (E/S) was 0.10 ± 0.13. For comparison, the exudate/serum ratio for CRP and TP was 0.39 ± 0.37 and 0.68 ± 0.15, respectively. There was a strong positive correlation between serum and exudate SAA concentration (r = 0.764;p < 0.0001). The concentration of SAA in transudates was low and did not overlap with that found in exudates (0.02-0.21 versus 0.8–360.5 g/mL). SAA in pleural and ascitic exudates results mainly from leakage of the serum protein via the inflamed membrane. A comparison of the E/S ratio of SAA and CRP points SAA as a very good marker in discriminating between exudates and transudates. PMID:16864904

  9. The importance of selecting a proper biological milieu for protein corona analysis in vitro: Human plasma versus human serum.

    PubMed

    Mirshafiee, Vahid; Kim, Raehyun; Mahmoudi, Morteza; Kraft, Mary L

    2016-06-01

    Nanoparticle (NP) exposure to biological fluids in the body results in protein binding to the NP surface, which forms a protein coating that is called the "protein corona". To simplify studies of protein-NP interactions and protein corona formation, NPs are incubated with biological solutions, such as human serum or human plasma, and the effects of this exposure are characterized in vitro. Yet, how NP exposure to these two different biological milieus affects protein corona composition and cell response has not been investigated. Here, we explore the differences between the protein coronas that form when NPs are incubated in human serum versus human plasma. NP characterization indicated that NPs that were exposed to human plasma had higher amounts of proteins bound to their surfaces, and were slightly larger in size than those exposed to human serum. In addition, significant differences in corona composition were also detected with gel electrophoresis and liquid chromatography-mass spectrometry/mass spectrometry, where a higher fraction of coagulation proteins and complement factors were found on the plasma-exposed NPs. Flow cytometry and confocal microscopy showed that the uptake of plasma-exposed NPs was higher than that of serum-exposed NPs by RAW 264.7 macrophage immune cells, but not by NIH 3T3 fibroblast cells. This difference is likely due to the elevated amounts of opsonins, such as fibrinogen, on the surfaces of the NPs exposed to plasma, but not serum, because these components trigger NP internalization by immune cells. As the human plasma better mimics the composition of the in vivo environment, namely blood, in vitro protein corona studies should employ human plasma, and not human serum, so the biological phenomena that is observed is more similar to that occurring in vivo. PMID:26643610

  10. Selective polyamine-binding proteins. Spermine binding by an androgen-sensitive phosphoprotein.

    PubMed

    Liang, T; Mezzetti, G; Chen, C; Liao, S

    1978-09-01

    Rat ventral prostate contains an acidic protein which can bind spermine selectively. The relative binding affinities of various aliphatic amines for the protein are, in decreasing order, spermine greater than thermine greater than greater than putrecine greater than 1,10-diaminodecane, cadaverine and 1,12-diaminododecane. The binding protein has an isoelectric point at pH 4.3 and a sedimentation coefficient of 3 S. Its molecular weight is approx. 30 000. Histones and nuclear chromatin preparations of the prostate can interact with the binding protein. The spermine-binding activity of the purified prostate protein can be inactivated by treatment with intestinal alkaline phosphatases. The phosphatase treated preparation can then be reactivated by beef heart protein kinase in the presence of cyclic AMP and ATP. The spermine-binding activity of the prostate cytosol protein fraction decreases after castration, but increases very rapidly after the castrated rats are injected with 5alpha-dihydrotestosterone. This finding raises the possibility that, in the postate, certain androgen actions may be dependent on the androgen-induced increase in the acidic protein binding of polyamines and their translocation to a functional cellular site such as nuclear chromatin. In the prostate cytosol, spermine also binds to 4-S tRNAs and to a unique RNA which has a sedimentation coefficient of 1.5 S. PMID:28786

  11. In vitro, in silico and integrated strategies for the estimation of plasma protein binding. A review.

    PubMed

    Lambrinidis, George; Vallianatou, Theodosia; Tsantili-Kakoulidou, Anna

    2015-06-23

    Plasma protein binding (PPB) strongly affects drug distribution and pharmacokinetic behavior with consequences in overall pharmacological action. Extended plasma protein binding may be associated with drug safety issues and several adverse effects, like low clearance, low brain penetration, drug-drug interactions, loss of efficacy, while influencing the fate of enantiomers and diastereoisomers by stereoselective binding within the body. Therefore in holistic drug design approaches, where ADME(T) properties are considered in parallel with target affinity, considerable efforts are focused in early estimation of PPB mainly in regard to human serum albumin (HSA), which is the most abundant and most important plasma protein. The second critical serum protein α1-acid glycoprotein (AGP), although often underscored, plays also an important and complicated role in clinical therapy and thus the last years it has been studied thoroughly too. In the present review, after an overview of the principles of HSA and AGP binding as well as the structure topology of the proteins, the current trends and perspectives in the field of PPB predictions are presented and discussed considering both HSA and AGP binding. Since however for the latter protein systematic studies have started only the last years, the review focuses mainly to HSA. One part of the review highlights the challenge to develop rapid techniques for HSA and AGP binding simulation and their performance in assessment of PPB. The second part focuses on in silico approaches to predict HSA and AGP binding, analyzing and evaluating structure-based and ligand-based methods, as well as combination of both methods in the aim to exploit the different information and overcome the limitations of each individual approach. Ligand-based methods use the Quantitative Structure-Activity Relationships (QSAR) methodology to establish quantitate models for the prediction of binding constants from molecular descriptors, while they provide

  12. Genetic effects on serum testosterone and sex hormone-binding globulin in men: a Korean twin and family study

    PubMed Central

    Sung, Joohon; Song, Yun-Mi

    2016-01-01

    We conducted a community-based cross-sectional study to evaluate the role of genetics in determining the individual difference in total testosterone and sex hormone-binding globulin levels. Study participants comprised 730 Korean men consisting of 142 pairs of monozygotic twins, 191 pairs of siblings, and 259 father-offspring pairs from 270 families who participated in the Healthy Twin study. Serum concentration of total testosterone and sex hormone-binding globulin were measured by chemiluminescence immunoassay, and free testosterone and bioavailable testosterone were calculated using Vermeulen's method. Quantitative genetic analysis based on a variance decomposition model showed that the heritability of total testosterone, free testosterone, bioavailable testosterone, and sex hormone-binding globulin were 0.56, 0.45, 0.44, and 0.69, respectively after accounting for age and body mass index. Proportions of variance explained by age and body mass index varied across different traits, from 8% for total testosterone to 31% for sex hormone-binding globulin. Bivariate analysis showed a high degree of additive genetic correlation (ρG = 0.67) and a moderate degree of individual-specific environmental correlation (ρE = 0.42) between total testosterone and sex hormone-binding globulin. The findings confirmed the important role of genetics in determining the individually different levels of testosterone and sex hormone-binding globulin during adulthood in Korean men as found in non-Asian populations, which may suggest that common biologic control for determining testosterone level directly or indirectly through binding protein are largely shared among different populations. PMID:26486061

  13. Genetic effects on serum testosterone and sex hormone-binding globulin in men: a Korean twin and family study.

    PubMed

    Sung, Joohon; Song, Yun-Mi

    2016-01-01

    We conducted a community-based cross-sectional study to evaluate the role of genetics in determining the individual difference in total testosterone and sex hormone-binding globulin levels. Study participants comprised 730 Korean men consisting of 142 pairs of monozygotic twins, 191 pairs of siblings, and 259 father-offspring pairs from 270 families who participated in the Healthy Twin study. Serum concentration of total testosterone and sex hormone-binding globulin were measured by chemiluminescence immunoassay, and free testosterone and bioavailable testosterone were calculated using Vermeulen's method. Quantitative genetic analysis based on a variance decomposition model showed that the heritability of total testosterone, free testosterone, bioavailable testosterone, and sex hormone-binding globulin were 0.56, 0.45, 0.44, and 0.69, respectively after accounting for age and body mass index. Proportions of variance explained by age and body mass index varied across different traits, from 8% for total testosterone to 31% for sex hormone-binding globulin. Bivariate analysis showed a high degree of additive genetic correlation (ρG = 0.67) and a moderate degree of individual-specific environmental correlation (ρE = 0.42) between total testosterone and sex hormone-binding globulin. The findings confirmed the important role of genetics in determining the individually different levels of testosterone and sex hormone-binding globulin during adulthood in Korean men as found in non-Asian populations, which may suggest that common biologic control for determining testosterone level directly or indirectly through binding protein are largely shared among different populations. PMID:26486061

  14. Metallothionein gene expression is regulated by serum factors and activators of protein kinase C.

    PubMed Central

    Imbra, R J; Karin, M

    1987-01-01

    The exact physiological role of metallothionein (MT) is not clear. It has been suggested that these low-molecular-weight, highly inducible, heavy-metal-binding proteins serve in the regulation of intracellular Zn metabolism. Among the Zn-requiring systems are several enzymes involved in DNA replication and repair. Therefore, during periods of active DNA synthesis there is likely to be an increased demand for Zn, which could be met by elevated MT synthesis. For that reason, we examined whether stimulation of cellular proliferation leads to increased expression of MT. We report here that treatment of cultured mammalian cells with serum growth factors and activators of protein kinase C, all of which are known to have growth stimulatory activity, led to induction of MT mRNA. One of the required steps in the signal transduction pathways triggered by these agents, ending in MT induction, appears to be the activation of protein kinase C. Images PMID:3600629

  15. Steric and allosteric effects of fatty acids on the binding of warfarin to human serum albumin revealed by molecular dynamics and free energy calculations.

    PubMed

    Fujiwara, Shin-Ichi; Amisaki, Takashi

    2011-01-01

    Human serum albumin (HSA) binds with drugs and fatty acids (FAs). This study was initiated to elucidate the relationship between the warfarin binding affinity of HSA and the positions of bound FA molecules. Molecular dynamics simulations of 11 HSA-warfarin-myristate complexes were performed. HSA-warfarin binding free energy was then calculated for each of the complexes by the molecular mechanics-Poisson-Boltzmann surface area (MM-PBSA) method. The results indicated that the magnitude of the binding free energy was smaller in HSA-warfarin complexes that had 4 or more myristate molecules than in complexes with no myristate molecules. The unfavorable effect on the HSA-warfarin binding affinity was caused sterically by the binding of a myristate molecule to the FA binding site closest to the warfarin binding site. On the other hand, the magnitude of HSA-warfarin binding free energy was largest when 3 myristate molecules were bound to the high-affinity sites. The strongest HSA-warfarin binding was attributable to favorable entropic contribution related to larger atomic fluctuations of the amino acid residues at the warfarin binding site. In the binding of 2 myristate molecules to the sites with the highest and second-highest affinities, allosteric modulation that enhanced electrostatic interactions between warfarin and some of the amino acid residues around the warfarin binding site was observed. This study clarified the structural and energetic properties of steric/allosteric effects of FAs on the HSA-warfarin binding affinity and illustrated the approach to analyze protein-ligand interactions in situations such that multiple ligands bind to the other sites of the protein. PMID:21720037

  16. Characterization of the DNA binding properties of polyomavirus capsid protein

    NASA Technical Reports Server (NTRS)

    Chang, D.; Cai, X.; Consigli, R. A.; Spooner, B. S. (Principal Investigator)

    1993-01-01

    The DNA binding properties of the polyomavirus structural proteins VP1, VP2, and VP3 were studied by Southwestern analysis. The major viral structural protein VP1 and host-contributed histone proteins of polyomavirus virions were shown to exhibit DNA binding activity, but the minor capsid proteins VP2 and VP3 failed to bind DNA. The N-terminal first five amino acids (Ala-1 to Lys-5) were identified as the VP1 DNA binding domain by genetic and biochemical approaches. Wild-type VP1 expressed in Escherichia coli (RK1448) exhibited DNA binding activity, but the N-terminal truncated VP1 mutants (lacking Ala-1 to Lys-5 and Ala-1 to Cys-11) failed to bind DNA. The synthetic peptide (Ala-1 to Cys-11) was also shown to have an affinity for DNA binding. Site-directed mutagenesis of the VP1 gene showed that the point mutations at Pro-2, Lys-3, and Arg-4 on the VP1 molecule did not affect DNA binding properties but that the point mutation at Lys-5 drastically reduced DNA binding affinity. The N-terminal (Ala-1 to Lys-5) region of VP1 was found to be essential and specific for DNA binding, while the DNA appears to be non-sequence specific. The DNA binding domain and the nuclear localization signal are located in the same N-terminal region.

  17. AU-rich RNA binding proteins in hematopoiesis and leukemogenesis.

    PubMed

    Baou, Maria; Norton, John D; Murphy, John J

    2011-11-24

    Posttranscriptional mechanisms are now widely acknowledged to play a central role in orchestrating gene-regulatory networks in hematopoietic cell growth, differentiation, and tumorigenesis. Although much attention has focused on microRNAs as regulators of mRNA stability/translation, recent data have highlighted the role of several diverse classes of AU-rich RNA-binding protein in the regulation of mRNA decay/stabilization. AU-rich elements are found in the 3'-untranslated region of many mRNAs that encode regulators of cell growth and survival, such as cytokines and onco/tumor-suppressor proteins. These are targeted by a burgeoning number of different RNA-binding proteins. Three distinct types of AU-rich RNA binding protein (ARE poly-U-binding degradation factor-1/AUF1, Hu antigen/HuR/HuA/ELAVL1, and the tristetraprolin/ZFP36 family of proteins) are essential for normal hematopoiesis. Together with 2 further AU-rich RNA-binding proteins, nucleolin and KHSRP/KSRP, the functions of these proteins are intimately associated with pathways that are dysregulated in various hematopoietic malignancies. Significantly, all of these AU-rich RNA-binding proteins function via an interconnected network that is integrated with microRNA functions. Studies of these diverse types of RNA binding protein are providing novel insight into gene-regulatory mechanisms in hematopoiesis in addition to offering new opportunities for developing mechanism-based targeted therapeutics in leukemia and lymphoma. PMID:21917750

  18. Global discovery of protein kinases and other nucleotide-binding proteins by mass spectrometry.

    PubMed

    Xiao, Yongsheng; Wang, Yinsheng

    2016-09-01

    Nucleotide-binding proteins, such as protein kinases, ATPases and GTP-binding proteins, are among the most important families of proteins that are involved in a number of pivotal cellular processes. However, global study of the structure, function, and expression level of nucleotide-binding proteins as well as protein-nucleotide interactions can hardly be achieved with the use of conventional approaches owing to enormous diversity of the nucleotide-binding protein family. Recent advances in mass spectrometry (MS) instrumentation, coupled with a variety of nucleotide-binding protein enrichment methods, rendered MS-based proteomics a powerful tool for the comprehensive characterizations of the nucleotide-binding proteome, especially the kinome. Here, we review the recent developments in the use of mass spectrometry, together with general and widely used affinity enrichment approaches, for the proteome-wide capture, identification and quantification of nucleotide-binding proteins, including protein kinases, ATPases, GTPases, and other nucleotide-binding proteins. The working principles, advantages, and limitations of each enrichment platform in identifying nucleotide-binding proteins as well as profiling protein-nucleotide interactions are summarized. The perspectives in developing novel MS-based nucleotide-binding protein detection platform are also discussed. © 2014 Wiley Periodicals, Inc. Mass Spec Rev 35:601-619, 2016. PMID:25376990

  19. 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

  20. In Situ Quantification of Protein Binding to the Plasma Membrane

    PubMed Central

    Smith, Elizabeth M.; Hennen, Jared; Chen, Yan; Mueller, Joachim D.

    2015-01-01

    This study presents a fluorescence-based assay that allows for direct measurement of protein binding to the plasma membrane inside living cells. An axial scan through the cell generates a fluorescence intensity profile that is analyzed to determine the membrane-bound and cytoplasmic concentrations of a peripheral membrane protein labeled by the enhanced green fluorescent protein (EGFP). The membrane binding curve is constructed by mapping those concentrations for a population of cells with a wide range of protein expression levels, and a fit of the binding curve determines the number of binding sites and the dissociation coefficient. We experimentally verified the technique, using myosin-1C-EGFP as a model system and fit its binding curve. Furthermore, we studied the protein-lipid interactions of the membrane binding domains from lactadherin and phospholipase C-δ1 to evaluate the feasibility of using competition binding experiments to identify specific lipid-protein interactions in living cells. Finally, we applied the technique to determine the lipid specificity, the number of binding sites, and the dissociation coefficient of membrane binding for the Gag matrix domain of human T-lymphotropic virus type 1, which provides insight into early assembly steps of the retrovirus. PMID:26039166

  1. Suppression of serum iron-binding capacity and bone marrow cellularity in pigs fed aflatoxin

    SciTech Connect

    Harvey, R.B.; Clark, D.E.; Huff, W.E.; Kubena, L.F.; Corrier, D.E. ); Phillips, T.D. )

    1988-05-01

    Flavus-parasiticus species of the genus Aspergillus are recognized as the primary producers of aflatoxins B{sub 1}, B{sub 2}, G{sub 1}, and G{sub 2}, hereafter referred to as aflatoxin (AF). The effects of feeding AF-contaminated diets to growing and finishing pigs have been described with changes in clinical performance, serum biochemistry, histology, and hematology attributed to aflatoxicosis. However, most of these studies evaluated AF-induced changes for a single AF dosage at a given point in time. The present study was designed to characterize how various AF dosages influence bone marrow histology, hematology, prothrombin and activated thromboplastin times, serum amino acids, and serum iron binding capacity during aflatoxicosis in growing pigs.

  2. Suppression of serum iron-binding capacity and bone marrow cellularity in pigs fed aflatoxin

    SciTech Connect

    Harvey, R.B.; Clark, D.E.; Huff, W.E.; Kubena, L.F.; Corrier, D.E.; Phillips, I.D.

    1988-04-01

    Flavus-parasiticus species of the genus Aspergillus are recognized as the primary producers of aflatoxins B/sub 1/, B/sup 2/, G/sup 1/, and G/sup 2/, hereafter referred to as aflatoxin (AF). The effects of feeding AF-contaminated diets to growing and finishing pigs have been described with changes in clinical performance, serum biochemistry, histology, and hematology attributed to aflatoxicosis. However, most of these studies evaluated AF-induced changes for a single AF dosage at a given point in time. The present study was designed to characterize how various AF dosages influence bone marrow histology, hematology, prothrombin and activated thromboplastin times, serum amino acids, and serum iron binding capacity during aflatoxicosis in growing pigs.

  3. Protein-protein binding affinities by pulse proteolysis: application to TEM-1/BLIP protein complexes.

    PubMed

    Hanes, Melinda S; Ratcliff, Kathleen; Marqusee, Susan; Handel, Tracy M

    2010-10-01

    Efficient methods for quantifying dissociation constants have become increasingly important for high-throughput mutagenesis studies in the postgenomic era. However, experimentally determining binding affinity is often laborious, requires large amounts of purified protein, and utilizes specialized equipment. Recently, pulse proteolysis has been shown to be a robust and simple method to determine the dissociation constants for a protein-ligand pair based on the increase in thermodynamic stability upon ligand binding. Here, we extend this technique to determine binding affinities for a protein-protein complex involving the β-lactamase TEM-1 and various β-lactamase inhibitor protein (BLIP) mutants. Interaction with BLIP results in an increase in the denaturation curve midpoint, C(m), of TEM-1, which correlates with the rank order of binding affinities for several BLIP mutants. Hence, pulse proteolysis is a simple, effective method to assay for mutations that modulate binding affinity in protein-protein complexes. From a small set (n = 4) of TEM-1/BLIP mutant complexes, a linear relationship between energy of stabilization (dissociation constant) and ΔC(m) was observed. From this "calibration curve," accurate dissociation constants for two additional BLIP mutants were calculated directly from proteolysis-derived ΔC(m) values. Therefore, in addition to qualitative information, armed with knowledge of the dissociation constants from the WT protein and a limited number of mutants, accurate quantitation of binding affinities can be determined for additional mutants from pulse proteolysis. Minimal sample requirements and the suitability of impure protein preparations are important advantages that make pulse proteolysis a powerful tool for high-throughput mutagenesis binding studies. PMID:20669180

  4. 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

  5. Cu(II) Bis(thiosemicarbazone) Radiopharmaceutical Binding to Serum Albumin: Further Definition of Species-Dependence and Associated Substituent Effects

    PubMed Central

    Basken, Nathan E.; Green, Mark A.

    2009-01-01

    Introduction The Cu-PTSM (pyruvaldehyde bis(N4-methylthiosemicarbazonato)copper(II)) and Cu-ATSM (diacetyl bis(N4-methylthiosemicarbazonato)copper(II)) radiopharmaceuticals exhibit strong, species-dependent binding to the IIA site of human serum albumin (HSA), while the related Cu-ETS (ethylglyoxal bis(thiosemicarbazonato)copper(II)) radiopharmaceutical appears to only exhibit non-specific binding to human and animal serum albumins. Methods To further probe the structural basis for the species-dependence of this albumin binding interaction, protein binding of these three radiopharmaceuticals was examined in solutions of albumin and/or serum from a broader array of mammalian species (rat, sheep, donkey, rabbit, cow, pig, dog, baboon, mouse, cat, elephant). We also evaluated the albumin binding of several copper(II) bis(thiosemicarbazone) chelates offering more diverse substitution of the ligand backbone. Results Cu-PTSM and Cu-ATSM exhibit a strong interaction with HSA that is not apparent with the albumins of other species, while the binding of Cu-ETS to albumin is much less species-dependent. The strong interaction of Cu-PTSM with HSA does not appear to simply correlate with variation, relative to the animal albumins, of a single amino acid lining HSA's IIA site. Those agents that selectively interact with HSA share the common feature of only methyl or hydrogen substitution at the carbon atoms of the diimine fragment of the ligand backbone. Conclusions The interspecies variations in albumin binding of Cu-PTSM and Cu-ATSM are not simply explained by unique amino acid substitutions in the IIA binding pocket of the serum albumins. However, the specific affinity for this region of HSA is disrupted when substituents bulkier than a methyl group appear on the imine carbons of the copper bis(thiosemicarbazone) chelate. PMID:19520290

  6. Crystallographic analysis reveals the structural basis of the high-affinity binding of iophenoxic acid to human serum albumin

    PubMed Central

    2011-01-01

    Background Iophenoxic acid is an iodinated radiocontrast agent that was withdrawn from clinical use because of its exceptionally long half-life in the body, which was due in part to its high-affinity binding to human serum albumin (HSA). It was replaced by Iopanoic acid, which has an amino rather than a hydroxyl group at position 3 on the iodinated benzyl ring and, as a result, binds to albumin with lower affinity and is excreted more rapidly from the body. To understand how iophenoxic acid binds so tightly to albumin, we wanted to examine the structural basis of its interaction with HSA. Results We have determined the co-crystal structure of HSA in complex with iophenoxic acid at 2.75 Å resolution, revealing a total of four binding sites, two of which - in drugs sites 1 and 2 on the protein - are likely to be occupied at clinical doses. High-affinity binding of iophenoxic acid occurs at drug site 1. The structure reveals that polar and apolar groups on the compound are involved in its interactions with drug site 1. In particular, the 3-hydroxyl group makes three hydrogen bonds with the side-chains of Tyr 150 and Arg 257. The mode of binding to drug site 2 is similar except for the absence of a binding partner for the hydroxyl group on the benzyl ring of the compound. Conclusions The HSA-iophenoxic acid structure indicates that high-affinity binding to drug site 1 is likely to be due to extensive desolvation of the compound, coupled with the ability of the binding pocket to provide a full set of salt-bridging or hydrogen bonding partners for its polar groups. Consistent with this interpretation, the structure also suggests that the lower-affinity binding of iopanoic acid arises because replacement of the 3-hydroxyl by an amino group eliminates hydrogen bonding to Arg 257. This finding underscores the importance of polar interactions in high-affinity binding to albumin. PMID:21501503

  7. Odorant-binding proteins from a primitive termite.

    PubMed

    Ishida, Yuko; Chiang, Vicky P; Haverty, Michael I; Leal, Walter S

    2002-09-01

    Hitherto, odorant-binding proteins (OBPs) have been identified from insects belonging to more highly evolved insect orders (Lepidoptera, Coleoptera, Diptera, Hymenoptera, and Hemiptera), whereas only chemosensory proteins have been identified from more primitive species, such as orthopteran and phasmid species. Here, we report for the first time the isolation and cloning of odorant-binding proteins from a primitive termite species, the dampwood termite. Zootermopsis nevadensis nevadensis (Isoptera: Termopsidae). A major antennae-specific protein was detected by native PAGE along with four other minor proteins, which were also absent in the extract from control tissues (hindlegs). Multiple cDNA cloning led to the full characterization of the major antennae-specific protein (ZnevOBP1) and to the identification of two other antennae-specific cDNAs, encoding putative odorant-binding proteins (ZnevOBP2 and ZnevOBP3). N-terminal amino acid sequencing of the minor antennal bands and cDNA cloning showed that olfaction in Z. n. nevadensis may involve multiple odorant-binding proteins. Database searches suggest that the OBPs from this primitive termite are homologues of the pheromone-binding proteins from scarab beetles and antennal-binding proteins from moths. PMID:12449514

  8. The OspE-Related Proteins Inhibit Complement Deposition and Enhance Serum Resistance of Borrelia burgdorferi, the Lyme Disease Spirochete ▿

    PubMed Central

    Kenedy, Melisha R.; Akins, Darrin R.

    2011-01-01

    Borrelia burgdorferi, the Lyme disease spirochete, binds the host complement inhibitors factor H (FH) and FH-like protein 1 (FHL-1). Binding of FH/FHL-1 by the B. burgdorferi proteins CspA and the OspE-related proteins is thought to enhance resistance to serum-mediated killing. While previous reports have shown that CspA confers serum resistance in B. burgdorferi, it is unclear whether the OspE-related proteins are relevant in B. burgdorferi serum resistance when OspE is expressed on the borrelial surface. To assess the role of the OspE-related proteins, we overexpressed them in a serum-sensitive CspA mutant strain. OspE overexpression enhanced serum resistance of the CspA-deficient organisms. Furthermore, FH was more efficiently bound to the B. burgdorferi surface when OspE was overexpressed. Deposition of complement components C3 and C5b-9 (the membrane attack complex), however, was reduced on the surface of the OspE-overexpressing strain compared to that on the CspA mutant strain. These data demonstrate that OspE proteins expressed on the surface of B. burgdorferi bind FH and protect the organism from complement deposition and subsequent serum-mediated destruction. PMID:21282413

  9. A spectroscopic investigations of anticancer drugs binding to bovine serum albumin

    NASA Astrophysics Data System (ADS)

    Bakkialakshmi, S.; Chandrakala, D.

    2012-03-01

    The binding of anticancer drugs (i) Uracil (U), (ii) 5-Fluorouracil (5FU) and (iii) 5-Chlorouracil (5ClU), to bovine serum albumin (BSA) at two levels of temperature was studied by the fluorescence of quenching method. UV/Vis, time-resolved fluorescence, Fourier transform infrared spectroscopy (FTIR), proton nuclear magnetic resonance (1H NMR) and scanning electron microscope (SEM) analyses were also made. Binding constants (Ka) and binding sites (n) at various levels of temperature were calculated. The obtained binding sites were found to be equal to one for all the three quenchers (U, 5FU and 5ClU) at two different temperature levels. Thermodynamic parameters ΔH, ΔG and ΔS have been calculated and were presented in tables. Change in FTIR absorption intensity shows strong binding of anticancer drugs to BSA. Changes in chemical shifts of NMR and fluorescence lifetimes of the drugs indicate the presence of interaction and binding of BSA to anticancer drugs. 1H NMR spectra and SEM photographs also conform this binding.

  10. Studies on the synthesis, characterization, human serum albumin binding and biological activity of single chain surfactant-cobalt(III) complexes.

    PubMed

    Vignesh, G; Sugumar, K; Arunachalam, S; Vignesh, S; Arthur James, R; Arun, R; Premkumar, K

    2016-03-01

    The interaction of surfactant-cobalt(III) complexes [Co(bpy)(dien)TA](ClO4)3 · 3H2O (1) and [Co(dien)(phen)TA](ClO4)3 · 4H2O (2), where bpy = 2,2'-bipyridine, dien = diethylenetriamine, phen = 1,10-phenanthroline and TA = tetradecylamine with human serum albumin (HSA) under physiological conditions was analyzed using steady state, synchronous, 3D fluorescence, UV/visabsorption and circular dichroism spectroscopic techniques. The results show that these complexes cause the fluorescence quenching of HSA through a static mechanism. The binding constant (Kb ) and number of binding-sites (n) were obtained at different temperatures. The corresponding thermodynamic parameters (∆G°, ∆H° and ∆S°) and Ea were also obtained. According to Förster's non-radiation energy transfer theory, the binding distance (r) between the complexes and HSA were calculated. The results of synchronous and 3D fluorescence spectroscopy indicate that the binding process has changed considerably the polarity around the fluorophores, along with changes in the conformation of the protein. The antimicrobial and anticancer activities of the complexes were tested and the results show that the complexes have good activities against pathogenic microorganisms and cancer cells. PMID:26250655

  11. Induced circular dichroism as a tool to investigate the binding of drugs to carrier proteins: Classic approaches and new trends.

    PubMed

    Tedesco, Daniele; Bertucci, Carlo

    2015-09-10

    Induced circular dichroism (ICD) is a spectroscopic phenomenon that provides versatile and useful methods for characterizing the structural and dynamic properties of the binding of drugs to target proteins. The understanding of biorecognition processes at the molecular level is essential to discover and validate new pharmacological targets, and to design and develop new potent and selective drugs. The present article reviews the main applications of ICD to drug binding studies on serum carrier proteins, going from the classic approaches for the derivation of drug binding parameters and the identification of binding sites, to an overview of the emerging trends for the characterization of binding modes by means of quantum chemical (QC) techniques. The advantages and limits of the ICD methods for the determination of binding parameters are critically reviewed; the capability to investigate the binding interactions of drugs and metabolites to their target proteins is also underlined, as well as the possibility of characterizing the binding sites to obtain a complete picture of the binding mechanism and dynamics. The new applications of ICD methods to identify stereoselective binding modes of drug/protein complexes are then reviewed with relevant examples. The combined application of experimental ICD spectroscopy and QC calculations is shown to identify qualitatively the bound conformations of ligands to target proteins even in the absence of a detailed structure of the binding sites, either obtained from experimental X-ray crystallography and NMR measurements or from computational models of the complex. PMID:25769668

  12. Development of porcine ficolin-alpha monoclonal and polyclonal antibodies for determining the binding capacity of multiple GlcNAc-binding proteins to bacterial danger components.

    PubMed

    Nahid, M Abu; Ross, Steven J; Umiker, Benjamin R; Li, Huapeng; Sugii, Sunji; Bari, Latiful

    2016-02-01

    Ficolins are a group of oligomeric defense proteins assembled from collagen-like stalks and fibrinogen-like domains that have common biochemical specificity for N-acetyl-d-glucose amine (GlcNAc) and can function as opsonins. In this report, GlcNAc-binding protein (GBP) purified from porcine nonimmune serum was biochemically characterized as ficolin-α. Ficolin-α was used as an immunogen to generate both rabbit polyclonal and murine monoclonal anti-ficolin-α antibodies, which are not yet commercially available. GBPs have been shown to be present in many animals, including humans; however, their functions are largely unknown. GBPs from chicken, dog, horse, bovine, and human sera were isolated using various chromatography methods. Interestingly, anti-ficolin-α antibody showed cross-reaction with those animal sera GBPs. Furthermore, anti-ficolin-α antibody was reactive with the GlcNAc eluate of Escherichia coli O26-bound and Salmonella-bound porcine serum proteins. Functionally, GBPs and bacteria-reactive pig serum proteins were able to bind with pathogen-associated molecular patterns such as lipopolysaccharides and lipoteichoic acids. Our studies demonstrate that ficolin-α specific antibody was reactive with GBPs from many species as well as bacteria-reactive serum proteins. These proteins may play important roles in innate immunity by sensing danger components that can lead to antibacterial activity. PMID:26872626

  13. Thermodynamic characterization of drug binding to human serum albumin by isothermal titration microcalorimetry.

    PubMed

    Aki, H; Yamamoto, M

    1994-12-01

    Binding sites on human serum albumin (HSA) for anionic drugs and fatty acids have been thermodynamically characterized by microcalorimetry. The binding and the thermodynamic parameters were directly computed from the calorimetric titration data at 37 degrees C in a phosphate buffer (pH 7.4) using one- and two-class binding models. From compensation analyses plotting the molar enthalpy change (delta Hm,i) versus those of the molar free energy (delta Gm,i) and molar entropy (delta Sm,i) for each class of binding sites, HSA binding sites were classified into groups S1, S2, and S3. Group S1 included high-affinity binding sites for site II-bound drugs, such as ibuprofen, flufenamic acid, and ethacrynic acid, and short- or medium-length alkyl-chain fatty acids; group S2 included low-affinity binding sites of site II-bound drugs and long-length alkyl-chain fatty acids; and group S3 contained the high-affinity binding sites for site I-bound drugs, such as phenylbutazone, oxphenbutazone, and warfarin, and long-length alkyl-chain fatty acids. High- and low-affinity bindings sites for salicylic acid and acetylaslicylic acid agreed with the regions of groups S3 and S2, respectively. Groups S1 and S2 were characterized by large negative values of delta Hm,i and delta Sm,i, reflecting van der Waals interaction and hydrogen-bonding formation in low dielectric media, and the main force to stabilize the binding complex in group S3 was a hydrophobic interaction, characterized by a small negative delta Hm,i and minor or positive values of delta Sm,i (entropy-driven). PMID:7891299

  14. Population genetic studies in the Balkans. I. Serum proteins.

    PubMed

    Scheil, H G; Scheffrahn, W; Schmidt, H D; Huckenbeck, W; Efremovska, L; Xirotiris, N

    2001-09-01

    Within a study of the genetics of Southeastern European populations seven serum protein polymorphisms (AMY2, BF, C3, CP, GC, HPA, TF) were examined in three samples of Aromuns (Albania: the village of Andon Poci, province Gjirocaster, Republic of Macedonia: Stip region, Romania: the village Kogalniceanu, province Dobruja) and four reference samples (Albanians: Tirana, Romanians: Constanta and Ploiesti as well as Greeks (Northeastern Greece)). The Aromun samples from Albania and Romania form one separate cluster and the reference samples together with the Aromuns from Macedonia (Stip region) form a second one. PMID:11591047

  15. The actin binding protein adseverin regulates osteoclastogenesis.

    PubMed

    Hassanpour, Siavash; Jiang, Hongwei; Wang, Yongqiang; Kuiper, Johannes W P; Glogauer, Michael

    2014-01-01

    Adseverin (Ads), a member of the Gelsolin superfamily of actin binding proteins, regulates the actin cytoskeleton architecture by severing and capping existing filamentous actin (F-actin) strands and nucleating the assembly of new F-actin filaments. Ads has been implicated in cellular secretion, exocytosis and has also been shown to regulate chondrogenesis and megakaryoblastic leukemia cell differentiation. Here we report for the first time that Ads is involved in regulating osteoclastogenesis (OCG). Ads is induced during OCG downstream of RANK-ligand (RANKL) stimulation and is highly expressed in mature osteoclasts. The D5 isoform of Ads is not involved in regulating OCG, as its expression is not induced in response to RANKL. Three clonal Ads knockdown RAW264.7 (RAW) macrophage cell lines with varying degrees of Ads expression and OCG deficiency were generated. The most drastic OCG defect was noted in the clonal cell line with the greatest degree of Ads knockdown as indicated by a lack of TRAcP staining and multinucleation. RNAi mediated knockdown of Ads in osteoclast precursors resulted in distinct morphological changes characterized by altered F-actin distribution and increased filopodia formation. Ads knockdown precursor cells experienced enhanced migration while fusion of knockdown precursors cells was limited. Transient reintroduction of de novo Ads back into the knockdown system was capable of rescuing TRAcP expression but not osteoclast multinucleation most likely due to the transient nature of Ads expression. This preliminary study allows us to conclude that Ads is a RANKL induced early regulator of OCG with a potential role in pre-osteoclast differentiation and fusion. PMID:25275604

  16. The Actin Binding Protein Adseverin Regulates Osteoclastogenesis

    PubMed Central

    Wang, Yongqiang; Kuiper, Johannes W. P.; Glogauer, Michael

    2014-01-01

    Adseverin (Ads), a member of the Gelsolin superfamily of actin binding proteins, regulates the actin cytoskeleton architecture by severing and capping existing filamentous actin (F-actin) strands and nucleating the assembly of new F-actin filaments. Ads has been implicated in cellular secretion, exocytosis and has also been shown to regulate chondrogenesis and megakaryoblastic leukemia cell differentiation. Here we report for the first time that Ads is involved in regulating osteoclastogenesis (OCG). Ads is induced during OCG downstream of RANK-ligand (RANKL) stimulation and is highly expressed in mature osteoclasts. The D5 isoform of Ads is not involved in regulating OCG, as its expression is not induced in response to RANKL. Three clonal Ads knockdown RAW264.7 (RAW) macrophage cell lines with varying degrees of Ads expression and OCG deficiency were generated. The most drastic OCG defect was noted in the clonal cell line with the greatest degree of Ads knockdown as indicated by a lack of TRAcP staining and multinucleation. RNAi mediated knockdown of Ads in osteoclast precursors resulted in distinct morphological changes characterized by altered F-actin distribution and increased filopodia formation. Ads knockdown precursor cells experienced enhanced migration while fusion of knockdown precursors cells was limited. Transient reintroduction of de novo Ads back into the knockdown system was capable of rescuing TRAcP expression but not osteoclast multinucleation most likely due to the transient nature of Ads expression. This preliminary study allows us to conclude that Ads is a RANKL induced early regulator of OCG with a potential role in pre-osteoclast differentiation and fusion. PMID:25275604

  17. Concentration-dependent Cu(II) binding to prion protein

    NASA Astrophysics Data System (ADS)

    Hodak, Miroslav; Lu, Wenchang; Bernholc, Jerry

    2008-03-01

    The prion protein plays a causative role in several neurodegenerative diseases, including mad cow disease in cattle and Creutzfeldt-Jakob disease in humans. The normal function of the prion protein is unknown, but it has been linked to its ability to bind copper ions. Experimental evidence suggests that copper can be bound in three distinct modes depending on its concentration, but only one of those binding modes has been fully characterized experimentally. Using a newly developed hybrid DFT/DFT method [1], which combines Kohn-Sham DFT with orbital-free DFT, we have examined all the binding modes and obtained their detailed binding geometries and copper ion binding energies. Our results also provide explanation for experiments, which have found that when the copper concentration increases the copper binding mode changes, surprisingly, from a stronger to a weaker one. Overall, our results indicate that prion protein can function as a copper buffer. 1. Hodak, Lu, Bernholc, JCP, in press.