Adsorption of BSA on strongly basic chitosan: Equilibria

Equilibrium isotherms for adsorption of bovine serum albumin (BSA) on a new adsorbent, a strongly basic crosslinked chitosan (Chitopearl 2503), which is hard and is not compressed by pressure in a column, have been presented and compared with diethylaminoethyl (DEAE) Sepharose Fast Flow (hard gel). In Chitopearl 2503, when only buffer existed in the BSA solution, the isotherm was not affected by the initial concentration of BSA but it was affected by pH considerably. The isotherm was favorable when pH >/= pl ( congruent with 4.8). When NaCl existed in the BSA solution, the amount of BSA absorbed on the resin decreased with increasing concentration of NaCl. When the concentration of NaCl was 200 mol/m(3), the resin did not adsorb BSA at all. The equilibrium data were correlated by the Langmuir equation reasonably well. The BSA may be adsorbed mainly by electrostatic attraction between negatively charged BSA and positively charged quanternary ammonium groups at pH > pl and by protonation reaction of the primary ammonium groups by weak acid groups of BSA at pH = pl. These are confirmed by measuring the amount of inorganic ion exchanged for BSA. In DEAE Sepharose Fast Flow, the isotherm was favorable when pH > pl but unfavorable ar pH = pl. The saturation capacity of BSA on Chitopearl 2503 is about 1.3 to 2.2 times larger than that on DEAE Sepharose Fast Flow. (c) 1994 John Wiley & Sons, Inc.     

残余牛血清白蛋白含量检测试剂盒抗干扰性研究

为了对目前使用的残余牛血清蛋白(BSA)含量检测试剂盒的抗干扰性进行评价,选用19个企业的12个品种,共计28份样品进行检测,包括冻干疫苗和液体疫苗两种剂型。分别检测15ng/ml BSA对照样品、二倍稀释的疫苗样品和添加15ng/ml BSA的疫苗样品。将添加BSA的疫苗样品的检测结果减去未添加BSA的疫苗样品的结果,其数值应当位于BSA对照样品均值的95%可信区间内。多数品种的疫苗添加BSA后回收率在85%和115%之间。个别制品的回收率在82%~83%之间。实验研究结果证明目前使用的BSA检测试剂盒具有较好的抗干扰作用。     

牛血清白蛋白在植物RAPD分析中的作用

以水杉、七子花DNA为模板,添加牛血清白蛋白（BSA）,观察其对植物RAPD扩增效果的改善情况。研究显示,在水杉及七子花RAPD扩增体系中,改善RAPD扩增反应的最佳的BSA浓度是不同的,分别为06μg/μl与1μg/μl。另外,BSA还可以封闭乙酰BSA对RAPD扩增反应的抑制作用,降低RAPD反应系统中Taq酶的用量。 Abstract:Using Metasequoia glyptostroboides and Heptacodium miconioides DNA as templates,the effect of bovine serum albumin(BSA) on RAPD in plants was studied.The results showed that suitable concentrations of BSA used in Metasequoia glyptostroboides and Heptacodium miconioides RAPD were different,which were 06μg/μl and 1μg/μl,respectively.The inhibition of acetylated BSA on the amplification of plant RAPD could be relieved by BSA.BSA could reduce the dosage of Taq DNA polymerase.     

Cleavage of BSA by a dipeptide seryl-histidine

A dipeptide seryl-histidine (Ser-His) was found to have theprotein cleavage activity. BSA was cleaved into smear at aroundpH 5.0–6.0, with a half-life around 15 hr at 60 °C.Phosphate could accelerate the reaction. This is a brand newprotein cleavage system. Since Ser and His are well-knowncatalytic residues at the active sites of many serine proteases,this results might provide clues to the possible roles of shortoligopeptides in the origins of modern enzymes.     

光谱及分子模拟法对HSA和BSA与酪氨酸激酶抑制剂Imatinib相互作用的比较分析

研究一种酪氨酸激酶抑制剂（tyrosine kinase inhibitor, TKI）伊马替尼（imatinib, IMA）与人血清清蛋白（HSA）及牛血清清蛋白（BSA）的相互作用,比较分析HSA和BSA与IMA相互作用机制的差异. 模拟生理条件下,计算机模拟技术结合荧光光谱和紫外光谱法,研究IMA与蛋白质的作用机制. 分子模建IMA与血清清蛋白的结合模型,表明伊马替尼与蛋白质的相互作用力为疏水作用力,兼有氢键作用. 光谱结果表明,IMA与HSA和BSA的相互作用表现为静态结合过程,结合强度较强,IMA与HSA和BSA分子的结合距离r值较小,说明发生了能量转移现象. IMA对HSA和BSA的结构域微区构象产生影响,使结合位域的疏水性发生改变. 荧光相图技术解析出IMA与HSA和BSA反应构象型态的变迁为“二态”模型. HSA与IMA相互作用的热力学参数表明,IMA与HSA之间是以疏水作用为主的分子间作用,而IMA与BSA之间的作用力为氢键和范德华力,兼有少量的疏水作用力. 光谱实验与计算机模拟结果基本一致,可为研究IMA与HSA和BSA相互作用本质提供一定参考.     

Cleavage of BSA by a dipeptide seryl-histidine

Summary A dipeptide seryl-histidine (Ser-His) was found to have the protein cleavage activity. BSA was cleaved into smear at around pH 5.0–6.0, with a half-life around 15 hr at 60 °C. Phosphate could accelerate the reaction. This is a brand new protein cleavage system. Since Ser and His are well-known catalytic residues at the active sites of many serine proteases, this results might provide clues to the possible roles of short oligopeptides in the origins of modern enzymes.     

Micro-assembly of functionalized particulate monolayer on C18-derivatized SiO2 surfaces

This work describes a simple approach to immobilize functionalized colloidal microstructures onto a C(18)-coated SiO(2) substrate via specific or non-specific bio-mediated interactions. Biotinylated bovine serum albumin pre-adsorbed onto a C(18) surface was used to mediate the surface assembly of streptavidin-coated microbeads (2.8 microm), while a bare C(18) surface was used to immobilize anti-Listeria antibody-coated microbeads (2.8 microm) through hydrophobic interactions. For a C(18) surface pre-adsorbed with bovine serum albumin, hydrophobic polystyrene microbeads (0.8 microm) and positively charged dimethylamino microbeads (0.8 microm) were allowed to self-assemble onto the surface. A monolayer with high surface coverage was observed for both polystyrene and dimethylamino microbeads. The adsorption characteristics of Escherichia coli and Listeria monocytogenes on these microbead-based surfaces were studied using fluorescence microscopy. Both streptavidin microbeads pre-adsorbed with biotinylated anti-Listeria antibody and anti-Listeria antibody-coated microbeads showed specific capture of L. monocytogenes, while polystyrene and dimethylamino microbeads captured both E. coli and L. monocytogenes non-specifically. The preparation of microbead-based surfaces for the construction of microfluidic devices for separation, detection, or analysis of specific biological species is discussed.     

Enhancement effects of BSA and linoleic acid on hybridoma cell growth and antibody production

The effects of linoleic acid and bovine serum albumin on hybridoma cell growth and antibody production were investigated. In dish cultivation, linoleic acid on its own promoted cell growth when used at concentrations below 50 mg L^–1, but strongly inhibited growth at a concentration of 100 mg L^–1 on more. However, linoleic acid bound to bovine serum albumin did not inhibit cell growth, even at a concentration as high as 100 mg L^–1. Also, linoleic acid did not affect the specific antibody production rate, with or without bovine serum albumin. In order to elucidate the enhancement of antibody production by bovine serum albumin, fractions were prepared by ultrafiltration (98% molecular weight cut-offs, 50,000 and 17,000) and the effects of the fractionation on antibody production were studied in batch cultivation. The high-molecular-weight fraction (50,000) promoted antibody production whereas the low-molecular-weight fraction (17,000) inhibited it. In continuous cultivation, the high-molecular-weight fraction was also found to enhance antibody production.     

Doxorubicin hinders DNA condensation promoted by the protein bovine serum albumin (BSA)

In this work, we have studied the interaction between the anticancer drug doxorubicin (doxo) and condensed DNA, using optical tweezers. To perform this task, we use the protein bovine serum albumin (BSA) in the working buffer to mimic two key conditions present in the real intracellular environment: the condensed state of the DNA and the abundant presence of charged macromolecules in the surrounding medium. In particular, we have found that, when doxo is previously intercalated in disperse DNA, the drug hinders the DNA condensation process upon the addition of BSA in the buffer. On the other hand, when bare DNA is firstly condensed by BSA, doxo is capable to intercalate and to unfold the DNA condensates at relatively high concentrations. In addition, a specific interaction between BSA and doxo was verified, which significantly changes the chemical equilibrium of the DNA–doxo interaction. Finally, the presence of BSA in the buffer stabilizes the double‐helix structure of the DNA–doxo complexes, preventing partial DNA denaturation induced by the stretching forces.     

Intermolecular interaction of fosinopril with bovine serum albumin (BSA): The multi‐spectroscopic and computational investigation

The intermolecular interaction of fosinopril, an angiotensin converting enzyme inhibitor with bovine serum albumin (BSA), has been investigated in physiological buffer (pH 7.4) by multi‐spectroscopic methods and molecular docking technique. The results obtained from fluorescence and UV absorption spectroscopy revealed that the fluorescence quenching mechanism of BSA induced by fosinopril was mediated by the combined dynamic and static quenching, and the static quenching was dominant in this system. The binding constant, K_b, value was found to lie between 2.69 × 10³ and 9.55 × 10³ M^?1 at experimental temperatures (293, 298, 303, and 308 K), implying the low or intermediate binding affinity between fosinopril and BSA. Competitive binding experiments with site markers (phenylbutazone and diazepam) suggested that fosinopril preferentially bound to the site I in sub‐domain IIA on BSA, as evidenced by molecular docking analysis. The negative sign for enthalpy change (ΔH⁰) and entropy change (ΔS⁰) indicated that van der Waals force and hydrogen bonds played important roles in the fosinopril‐BSA interaction, and 8‐anilino‐1‐naphthalenesulfonate binding assay experiments offered evidence of the involvements of hydrophobic interactions. Moreover, spectroscopic results (synchronous fluorescence, 3‐dimensional fluorescence, and Fourier transform infrared spectroscopy) indicated a slight conformational change in BSA upon fosinopril interaction.     

In vitro study on binding interaction of quinapril with bovine serum albumin (BSA) using multi-spectroscopic and molecular docking methods

The binding interaction between quinapril (QNPL) and bovine serum albumin (BSA) in vitro has been investigated using UV absorption spectroscopy, steady-state fluorescence spectroscopic, synchronous fluorescence spectroscopy, 3D fluorescence spectroscopy, Fourier transform infrared spectroscopy, circular dichroism, and molecular docking methods for obtaining the binding information of QNPL with BSA. The experimental results confirm that the quenching mechanism of the intrinsic fluorescence of BSA induced by QNPL is static quenching based on the decrease in the quenching constants of BSA in the presence of QNPL with the increase in temperature and the quenching rates of BSA larger than 10¹⁰ L mol^?1 s^?1, indicating forming QNPL–BSA complex through the intermolecular binding interaction. The binding constant for the QNPL–BSA complex is in the order of 10⁵ M^?1, indicating there is stronger binding interaction of QNPL with BSA. The analysis of thermodynamic parameters together with molecular docking study reveal that the main binding forces in the binding process of QNPL with BSA are van der Waal’s forces and hydrogen bonding interaction. And, the binding interaction of BSA with QNPL is an enthalpy-driven process. Based on Förster resonance energy transfer, the binding distance between QNPL and BSA is calculated to be 2.76 nm. The results of the competitive binding experiments and molecular docking confirm that QNPL binds to sub-domain IIA (site I) of BSA. It is confirmed there is a slight change in the conformation of BSA after binding QNPL, but BSA still retains its secondary structure α-helicity.     

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

To further understand the mechanism of action and pharmacokinetics of medroxyprogesterone acetate (MPA), the binding interaction of MPA with bovine serum albumin (BSA) under simulated physiological conditions (pH 7.4) was studied using fluorescence emission spectroscopy, synchronous fluorescence spectroscopy, circular dichroism and molecular docking methods. The experimental results reveal that the fluorescence of BSA quenches due to the formation of MPA–BSA complex. The number of binding sites (n) and the binding constant for MPA–BSA complex are ~1 and 4.6 × 10³ M^?1 at 310 K, respectively. However, it can be concluded that the binding process of MPA with BSA is spontaneous and the main interaction forces between MPA and BSA are van der Waals force and hydrogen bonding interaction due to the negative values of ΔG⁰, ΔH⁰ and ΔS⁰ in the binding process of MPA with BSA. MPA prefers binding on the hydrophobic cavity in subdomain IIIA (site II′′) of BSA resulting in a slight change in the conformation of BSA, but BSA retaining the α‐helix structure. Copyright © 2016 John Wiley & Sons, Ltd.     

Separation of growth-stimulating activity of BSA fraction V from the bulk of albumin using Heparin Sepharose Chromatography

Bovine serum albumin (BSA) is a potential source of biological contamination in cell culture medium. The aim of this work was to attempt to replace BSA in low serum and serum-free medium (SFM). BSA fraction V was subjected to a variety of processes in order to determine if the growth promoting activity observed for NRK cells could be extracted from the BSA molecule. These included solvent extractions, diafiltration, reverse phase HPLC and affinity chromatography using heparin sepharose. Solvent extraction and diafiltration failed to remove the activity from the BSA. Affinity chromatography using heparin sepharose indicated that all of the activity observed with BSA was retained in the 0.5 M NaCl fraction and was associated with less than 3% of the original protein. The major protein band in the 0.5 M NaCl fraction had the same apparent molecular weight as albumin (as seen by SDS-PAGE and analytical reverse phase HPLC). Unlike the untreated BSA, the 0.5 M NaCl fraction was partially susceptible to proteolytic digestion and to variations in pH.Abbreviations HS heparin sepharose - DHS donor horse serum - SFM serum free-medium     

Structural insights into the binding mode and conformational changes of BSA induced by bixin and crocin

Bixin and crocin are natural apocarotenoids utilized as food colorants and additives in food industries worldwide. For safety assessment, it is necessary to understand the biological interaction of food colorants. In our present study, we report the interaction of two apocarotenoids with bovine serum albumin (BSA) at physiological pH using spectroscopic techniques and in silico tools. The binding constant and the mode of binding sites have been studied. The enthalpic and entropic contribution to the intermolecular binding event was analyzed and it was found that the contribution of hydrogen bonding and hydrophobic interactions was dominant. The adverse temperature dependence in the unusual static quenching is found to be a reasonable consequence of the large activation energy requirement in the binding process, which is required to overcome the fundamental block and is a direct result of the unique microstructure of the binding sites. To confirm the experimental analysis, we investigated the binding patterns using different in silico tools. A combination of molecular docking, molecular dynamics, and toxicity analysis was performed, and the obtained results revealed that both the apocarotenoids had high binding affinity with a binding energy of ?5.44 and ?5.93 kcal/mol for bixin and crocin, respectively, with no toxic effects and are in accordance with our experimental analysis. The results directly revealed the flexibility of the protein toward bixin and crocin which has a great impact on the interaction. Thus bixin and crocin can guardedly be used as food colorants in food industries.     

Effects of homogeneous media, binary mixtures and microheterogeneous media on the fluorescence and fluorescence probe properties of some benzo[b][1,8]naphthyridiens with HSA and BSA

A rapid and efficient method for the synthesis of various poly‐substituted benzo[b][1,8]naphthyridines in high yield has been developed via the Friedländer condensation of 2‐aminoquinoline‐3‐carbaldehyde 1 with various alicyclic ketones in a base catalyst (aq. potassium hydroxide). A series of benzo[b][1,8]naphthyridines branched with various side‐chains and substituents were prepared with the aim of being investigated as a fluorescent agents. Electronic absorption and fluorescence properties of some representative benzonaphthyridines (3d, 5b and 21f) in homogeneous organic solvents, dioxane–water binary mixtures and in the microheterogeneous media (sodium dodecyl sulphate (SDS), cetyl trimethyl ammonium bromide (CTAB) and Triton‐X100 micelles) have been examined. A linear correlation between solvent polarity and fluorescence properties was observed. Further, the interaction of these benzonaphthyridines (3d, 5b and 21f) with human serum albumin (HSA) and bovine serum albumin (BSA) in phosphate buffer have been examined by UV‐vis absorption and fluorescence spectroscopy. The fluorescence intensity of 3d, 5b and 21f increases with the increasing HSA and BSA concentration. These benzonaphthyridines also quench the 345 nm fluorescence of BSA in phosphate buffer (λ_ex 280 nm). These compounds have potential for use as neutral and hydrophobic fluorescence probes for examining the microenvironments in proteins, polymers, micelles, etc. Copyright © 2011 John Wiley & Sons, Ltd.     

Impact of protein blocking on enzymatic saccharification of bagasse from sugarcane clones

Lignin plays an important functional and structural role in plants, but also contributes to the recalcitrance of lignocellulosic biomass to hydrolysis. This study addresses the influence of lignin in hydrolysis of sugarcane bagasse from conventional bred lines (UFV260 and UFV204) that were selected from 432 field-grown clones. In addition to higher sugar production, bagasse clone UFV204 had a small, but statistically significant, lower insoluble lignin content compared with clone UFV260 (15.5% vs, 16.6%) and also exhibited a significantly higher cellulose conversion to glucose (81.3% vs. 63.3%) at a cellulase loading of 5 (filter paper unit) FPU/g of glucan or 3 FPU/g total solids for liquid hot water pretreated bagasse (200°C, 10 min). The enzyme loading was further decreased by 50% to 2.5 FPU/g glucan and resulted in a similar glucan conversion (88.5%) for clone UFV204 when the bagasse was preincubated with bovine serum albumin at pH 4.8 and nonproductive binding of cellulase components was blocked. Comparison of Langmuir adsorption isotherms and differential adsorption of the three major cellulolytic enzyme components endoglucanase, cellobiohydrolase, and β-glucosidase help to explain differences due to lignin content.     

Protective effects of non-catalytic proteins on endoglucanase activity at air and lignin interfaces

The manner in which added non-catalytic proteins during enzymatic hydrolysis of lignocellulosic substrates enhances hydrolysis mechanisms is not completely understood. Prior research has indicated that a reduction in the non-specific adsorption of enzymes on lignin, and deactivation of enzymes exposed to air–liquid interface provide rationale. This work investigated root causes including effects of the air–liquid interface on non-catalytic proteins, and effects of lignin on endoglucanase. Three different experimental designs and three variables (air–liquid interfacial area, the types of lignin (acid or enzymatic lignin), and the presence of non-enzymatic protein (bovine serum albumin [BSA] or soy proteins ) were used. The results showed that acid isolated lignin adsorbed almost all endoglucanase activity initially present in supernatant, independent of air interface conditions (25 or 250 ml flasks) with the presence of BSA preventing this effect. Endoglucanase lost 30%–50% of its activity due to an air–liquid interface in the presence of lignin while addition of non-enzymatic protein helped to preserve this enzyme's activity. Langmuir and Freundlich models applied to experimental data indicated that the adsorption increases with increasing temperature for both endoglucanase and BSA. Adsorption of the enzyme and protein were endothermic with an increase in entropy. These results, combined, show that hydrophobicity plays a strong role in the adsorption of both endoglucanase and BSA on lignin.     

Optimization of bovine serum albumin partition coefficient in aqueous two-phase systems

Partitioning of proteins in aqueous two-phase systems has been shown to provide a powerful method for separating and purifying mixtures of biomolecules by extraction. These systems are composed of aqueous solutions of either two water-soluble polymers, usually polyethylene glycol (PEG) and dextran (Dx), or a polymer and a salt, usually PEG and phosphate or sulfate. There are many factors which influence the partition coefficient K, the ratio of biomolecule concentration in the top phase to that in the bottom phase, in aqueous two-phase systems. The value of the partition coefficient relies on the physico-chemical properties of the target biomolecule and other molecules and their interactions with those of the chosen system. In this work, the partition behavior of pure bovine serum albumin in aqueous two-phase systems was investigated in order to see the effects of changes in phase properties on the partition coefficient K. The concentration of NaCl and pH were considered to be the factors having influence on K. Optimal conditions of these factors were obtained using the Box-Wilson experimental design. The optimum value of K was found as 0.0126 when NaCl concentration and pH were 0.14 M and 9.8, respectively, for a phase system composed of 8% (w/w) polyethylene glycol 3,350 - 9 (% w/w) dextran 37,500 - 0.05 M phosphate at 20 °C.