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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

    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 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

  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 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

  20. 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

  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 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

  20. 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

  1. 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

  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. 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

  11. 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

  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 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

  15. 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

  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 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

  2. 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

  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. 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

  15. 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

  16. 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

  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. 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.

  16. 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

  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. 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

  5. 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

  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. 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

  8. 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.

  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. 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.

  1. 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.

  2. 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

  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. 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

  7. 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

  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

    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 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

  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