The comparative interaction of human and bovine serum albumins with CYP2C9 in human liver microsomes

The effect of human serum albumin (HSA), in its endogenous, free fatty acid free (FAF) and globulin free (GF) form, on the activity of CYP2C9 was studied in human liver microsomes using tolbutamide as the substrate. The widely used BSA was included to assess the differential effect of BSA and HSA. CYP2C9 activity was expressed as CLint (Vmax/Km). HSA(FAF) and BSA showed a concentration-dependent and biphasic (activation and inhibition) interaction with CYP2C9 activity. HSA(GF) and HSA exhibited an inhibitory effect, with an inhibition constant, Ki, of 19.9 microM (0.13% albumin) and 42.2 microM (0.35% albumin), respectively. Enzyme-kinetics revealed that the activation is accompanied by a decrease in Km values, while with inhibition Km values increased. A simplified method to calculate clearance, utilizing a single slope (V/S) determination based on V over the lowest linear range of [S] (designated as CLone) was assessed. Virtually identical values were obtained for CLint and CLone. The free-drug hypothesis was tested by comparing ratios of relative CLint/unbound fraction (FDH Test ratio). The FDH Test ratio for HSA was about 1, indicating that HSA binding of tolbutamide reduced the CYP2C9 activity in accord with the free-drug hypothesis. The FDH Test ratios for BSA and HSA(FAF) were 3.7 and 3.0, revealing a monophasic activation of CYP2C9. For 2%HSA(GF) the ratio of 0.3 confirmed inhibition. As revealed by their removal, free fatty acids and globulins, significantly alter the interaction of HSA with CYP2C9. In addition, HSA and BSA showed different effects on the oxidation of tolbutamide by CYP2C9.     

Fat-free Albumin as a Novel Drug Delivery System

The search for effective drug delivery systems is one of the major challenges in drug formulation especially for biopharmaceuticals such as proteins, and peptide-based drugs and vaccines. A procedure has been developed whereby human serum albumin (HSA) can be used as a delivery vehicle for these biomolecules using its role as main fatty acid carrier. Using essentially fatty acid free HSA (HSAff) it is possible to form stable complexes with lipidic chain compounds (lipo-compounds). Two lipo-compounds have been used to develop this system, a novel antimicrobial lipopeptide and γ-amino-n-butyric acid, GABA, conjugated with an alkyl chain, lipo-GABA, in both cases C8 and C14 alkyl chain lengths were evaluated. The HAS–lipo compound complex had a mutual stabilizing effect on both the HSA and the lipo-compound. The protease enzyme study showed that the alkyl chains of these lipo-compounds bound to HSAff confer a similar if not greater biostability than caprylic acid shown by CD and importantly, the bound lipopeptide was stabilized by the HSA shown by mass spectrometry. Heat stability studies at 60°C over 10 h also confirmed that the lipo-HSA complexes confer stability and provide a method of preparing sterile formulation for therapeutic use. No further increased in stability of the lipo-compounds when HSA containing fatty acid (HSAfa) was used. With the antimicrobial lipopeptide, there was enhanced activity with HSAff formulation suggesting increased biostability and bioavailability of compounds. These finding allowed us to develop a simple and effective way of delivering lipo-compounds using fatty acid free HSA as the carrier.Australian Peptide Conference Issue.     

Albumin binding sites for etodolac enantiomers

Non-steroidal anti-inflammatory drugs (NSAIDs) are strongly bound to human serum albumin (HSA), mainly to sites I and II. The aim of this study was to characterize the binding site(s) of etodolac enantiomers under physiological conditions (580 μM HSA) using equilibrium dialysis. The protein binding of etodolac enantiomers, alone or in various ratios, was studied in order to evaluate the potential competition between them. Our results showed that (S)-etodolac was more strongly bound to HSA than (R)-etodolac. The displacement of one enantiomer by its antipode was observed only at high concentrations of the competitor, and was more pronounced for the (S)-form. Displacement studies of the enantiomers by specific probes of sites I and II of albumin, dansylamide, and dansylsarcosine, respectively, showed that (R)-etodolac was slightly displaced by both these probes whereas the free concentration of (S)-etodolac increased markedly in the presence of dansylsarcosine. Moreover, the binding of ligands to sites I and II is usually affected by alkaline pH, by chloride ions, and by fatty acids. For etodolac, the presence of 0.1 and 1 M chloride ions and increasing pH (5.5-9) decreased the binding of both enantiomers. The same result was obtained with addition of octanoic acid. Conversely, the addition of oleic, palmitic, or stearic acid to the protein solution increased the binding of (R)-etodolac, but decreased that of its antipode. All these findings suggest that (R)- and (S)-etodolac interact mainly with site II of HSA, and that the (R)-isomer is also bound to site I under physiological conditions. © 1996 Wiley-Liss, Inc.     

Cantharidin inhibits competitively heme‐Fe(III) binding to the FA1 site of human serum albumin

Cantharidin, a monoterpene isolated from the insect blister beetle, has long been used as a medicinal agent in the traditional Chinese medicine. Cantharidin inhibits a subgroup of serine/threonine phosphatases, thus inducing cell growth inhibition and cytotoxicity. Cantharidin has anticancer activity in vitro, since it is able of inducing p53‐dependent apoptosis and double‐strand breakage of DNA in cancer cells. Although the toxicity of cantharidin to the gastrointestinal and urinary tracts prevents its medical use, it is a promising lead compound for chemical modification to develop new anticancer therapeutics. In fact, cantharidin does not cause myelosuppression and displays anticancer activity against cells with a multidrug resistance phenotype. Here, the competitive inhibitory effect of cantharidin on heme‐Fe(III) binding to the fatty acid site 1 (FA1) of human serum albumin (HSA) is reported. Docking and molecular dynamics simulations support functional data indicating the preferential binding of cantharidin to the FA1 site of HSA. Present results may be relevant in vivo as HSA could transport cantharidin, which in turn could affect heme‐Fe(III) scavenging by HSA.     

Binding of fatty acids facilitates oxidation of cysteine-34 and converts copper-albumin complexes from antioxidants to prooxidants

As a transition metal capable of undergoing one-electron oxidation-reduction conversions, copper (Cu) is essential for life and fulfills important catalytic functions. Paradoxically, the same redox properties of copper can make it extremely dangerous because it can catalyze production of free radical intermediates from molecular oxygen. Factors involved in regulation of redox activity of albumin-bound copper have not been well characterized. In the present study, effects of modification of the albumin cysteine-34 (Cys-34) and binding of nonesterified fatty acids on the redox-cycling activity of the complex of copper with human serum albumin (Cu/HSA) were studied. Because ascorbate is the most abundant natural reductant/scavenger of free radicals in blood plasma, the electron paramagnetic resonance assay of ascorbate radical formation was used as a method to monitor Cu/HSA redox-cycling activity. At Cu/HSA ratios below 1:1, the bound Cu was virtually redox inactive, as long as Cys-34 was in reduced state (Cu/HSA-SH). Alkylation, nitrosylation, or oxidation of Cu/HSA resulted in the appearance of redox-cycling activity. Experiments with ultrafiltration of Cu/HSA alkylated with N-ethylmaleimide (Cu/HSA-NEM) showed that at Cu/HSA-NEM ratios below 1:1, the ascorbate radicals were produced by Cu tightly bound to HSA rather than by Cu released in solution. The rate of ascorbate radical production in HSA-NEM and S-nitrosylated HSA (HSA-NO) was, however, more than one order of magnitude lower than that in a solution containing equivalent concentration of free copper ions. While Cu/HSA-SH was redox inactive, binding of oleic or linoleic acids induced Cu-dependent redox-cycling with maximal activity reached at a fatty acid to protein molar ratio of 3:1 for oleic acid and 2:1 for linoleic acid. Binding of fatty acids caused profound conformational changes and facilitated oxidation of the Cys-34 SH-group at essentially the same ratios as those that caused redox-cycling activity of Cu/HSA. We conclude that fatty acids regulate anti-/prooxidant properties of Cu-albumin via controlling redox status of Cys-34.     

Probing the structure of the warfarin-binding site on human serum albumin using site-directed mutagenesis

The binding of warfarin to the following human serum albumin (HSA) mutants was examined: K195M, K199M, F211V, W214L, R218M, R222M, H242V, and R257M. Warfarin bound to human serum albumin (HSA) exhibits an intrinsic fluorescence that is approximately 10-fold greater than the corresponding signal for warfarin in aqueous solution. This property of the warfarin/HSA complex has been widely used to determine the dissociation constant for the interaction. In the present study, such a technique was used to show that specific substitutions in subdomain 2A altered the affinity of HSA for warfarin. The fluorescence of warfarin/mutant HSA complexes varied widely from the fluorescence of the warfarin/wild-type HSA complex at pH = 7.4, suggesting changes in the structure of the complex resulting from specific substitutions. The fluorescence of the warfarin/wild-type HSA complex increases about twofold as the pH is increased from 6.0 to 9.0 due to the neutral-to-base (N-B) transition, a conformational change that occurs in HSA as a function of pH. Changes in the fluorescence of warfarin/mutant HSA complexes as a function of pH suggests novel behavior for most HSA species examined. For the HSA mutants F211V and H242V, the midpoint of the N-B transition shifts from a wild-type pH of 7.8 to a pH value of 7.1-7.2.     

重组人血白蛋白临床研究进展

人血白蛋白是人血浆中最丰富的蛋白质,具有许多重要的生理特性,用途广泛。目前主要以毕赤酵母作为宿主表达的重组人血白蛋白,开发了重组人血白蛋白的纯化技术,同时对重组人血白蛋白结构进行了分析,结果表明与人血浆白蛋白基本一致。临床研究结果表明重组人血白蛋白与人血浆白蛋白有着几乎相同的疗效和安全性。综述了重组人血白蛋白的性质结构分析及酵母表达系统;重点介绍了重组人血白蛋白在临床方面研究进展。     

A fluorescent fatty acid probe, DAUDA, selectively displaces two myristates bound in human serum albumin

11-(Dansylamino) undecanoic acid (DAUDA) is a dansyl-type fluorophore and has widely used as a probe to determine the binding site for human serum albumin (HSA). Here, we reported that structure of HSA-Myristate-DAUDA ternary complex and identified clearly the presence of two DAUDA molecules at fatty acid (FA) binding site 6 and 7 of HSA, thus showing these two sites are weak FA binding sites. This result also show that DAUDA is an appropriate probe for FA site 6 and 7 on HSA as previous studied, but not a good probe of FA binding site 1 that is likely bilirubin binding site on HSA.     

Differential effects of albumin on microglia and macrophages; implications for neurodegeneration following blood–brain barrier damage

Microglial activation by blood-borne factors following blood–brain barrier damage may play a significant role in subsequent neuropathogenesis of several neurodegenerative diseases. Exposure of primary cultured rat brain microglia to pure, fatty acid- and lipid-deficient rat serum albumin or fraction V, (fatty acid and lipid-containing rat serum albumin), caused inducible nitric oxide synthase (iNOS) expression, glutamate release, tumour necrosis factor alpha (TNFα) and transforming growth factor-beta1 release. iNOS expression was attenuated by the MAPK/extracellular signal-regulated kinase pathway inhibitor U0126 and the phosphorylated forms of extracellular signal-regulated kinase 1 and 2 were detectable in microglia treated with albumin or fraction V. Glutamate release was prevented by l -α-aminoadipate and glutathione levels in microglia rose on exposure to albumin. Conditioned medium from microglia exposed to albumin or fraction V was neurotoxic. Peripheral macrophages were resistant to the effects of albumin but both microglia and macrophages responded to lipopolysaccharide, which induced interleukin-1 beta and tumour necrosis factor alpha release, cyclooxygenase-2 and iNOS expression in both cell types, indicating a discrete desensitised pathway in macrophages for albumin which was not desensitised in microglia. Thus, exposure of microglia in the brain to albumin may contribute to neuronal damage following blood–brain barrier breakdown and point to resident microglia rather than infiltrating macrophages as therapeutic targets.     

Effects of ferrous iron and transferrin on cell proliferation of human diploid fibroblasts in serum-free culture

Summary Iron-free RITC 80-7 defined medium was used to examine effects of ferrous iron and transferrin on cell proliferation of human diploid fibroblasts. Both ferrous iron and holotransferrin stimulated cell proliferation in the medium, but apotransferrin did not. When 5 g/l human serum albumin (HSA) was added to the defined medium, excellent growth was obtained under hypoxic conditions, whereas a reduction of cellular growth during the culture periods was observed under aerobic conditions. When ferrous iron was added to the HSA medium alone, the reduction in growth increased in proportion to the concentrations, whereas the addition of transferrin prevented this reduction in a concentration-dependent manner. This suggests that the ferrous iron concentration in media causes a reduction in growth under aerobic conditions and transferrin prevents this reduction because it decreases the ferrous iron concentration. Further, serum albumin seems to be a source of iron in media.     

Kinetics of fatty acid binding ability of glycated human serum albumin

Kinetics of fatty acid binding ability of glycated human serum albumin (HSA) were investigated by fluorescent displacement technique with 1-anilino-8-naphtharene sulphonic acid (ANS method), and photometric detection of nonesterified-fatty-acid (NEFA method). Changing of binding affinities of glycated HSA toward oleic acid, linoleic acid, lauric acid, and caproic acid, were not observed by the ANS method. However, decreases of binding capacities after 55 days glycation were confirmed by the NEFA method in comparison to control HSA. The decrease in binding affinities was: oleic acid (84%), linoleic acid (84%), lauric acid (87%), and caproic acid (90%), respectively. The decreases were consistent with decrease of the intact lysine residues in glycated HSA. The present observation indicates that HSA promptly loses its binding ability to fatty acid as soon as the lysine residues at fatty acid binding sites are glycated.     

Artificial lipid-protein complexes accelerate cholesterol crystallisation in model bile

Cholesterol gallstone disease is one of the major health problems in the world. Substances which can affect the crystallisation of cholesterol from human bile have been given considerable attention. Various substances (among them natural lipid-protein complexes) have been tested for cholesterol crystallisation-promoting activity. Various artificial lipid-albumin complexes have been prepared of which taurodeoxycholate-human serum albumin-calcium ions (TDC-HSA-Ca(2+)) had the highest cholesterol crystallisation-promoting activity. This cholesterol crystallisation-promoting activity is similar to that for the lipid-protein complex isolated from native human bile [concanavalin A nonbinding fraction (con A(-) fraction)]. Addition of cholesterol to the TDC-HSA-Ca(2+) complex further increased the cholesterol crystallisation-promoting activity whereas the addition of lecithin had an opposite effect. The interaction of individual components of the TDC-HSA-Ca(2+) complex was followed using several methods. A new effect of Ca(2+) ions (increase in the number of binding sites for bile salts) on the interaction of TDC with HSA was found by equilibrium dialysis. Interaction of TDC with albumin and Ca(2+) did not induce any modification of the secondary structure of albumin. The results of fluorescence spectroscopy may indicate that TDC is at least partially bound to not essentially fatty acid free HSA somehow via admixtures, probably fatty acids. Difference absorption spectrum of the TDC-HSA-Ca(2+)-cholesterol complex was very similar to that of the "natural" lipid-protein complex (con A(-) fraction). From the three drugs with different albumin binding characteristics, only sulphadimethoxin had an observable effect on the cholesterol crystallisation-promoting activity. The action of the TDC-HSA-Ca(2+) complex decreased significantly after the addition of sulphadimethoxin. The addition of TDC modified the absorption spectrum of the sulphadimethoxin-HSA-Ca(2+) complex. It can be suggested that the complex of HSA with bile salts (TDC mainly) and Ca(2+) forms a nucleation centre for cholesterol crystallisation in bile.     

生长激素释放激素和人血清白蛋白融合蛋白的克隆表达

目的:通过与人血清白蛋白(HSA)融合,延长生长激素释放激素(GHRH)在体内的半衰期。方法:根据毕赤酵母偏爱密码子重新设计GHRH的核酸序列,并通过化学合成和重叠PCR法将GHRH的N端与HSA的C端通过一个11肽的接头连接,获得GHRH和HSA融合的全长基因序列。构建pPIC9-HSA-GHRH表达载体,电击转化毕赤酵母GS115感受态细胞,通过表型筛选和诱导表达实验得到蛋白表达工程菌,对表达产物进行分离纯化和生物学活性分析。结果:克隆了HSA-GHRH融合基因,构建了pPIC9-HSA-GHRH融合表达载体;电击转化后通过表型筛选和诱导表达实验得到蛋白表达工程菌;经分离纯化后,对表达产物的生物学活性分析显示其在体内有促进生长的作用。结论:与人血清白蛋白的融合有效地提高了GHRH的表达水平,并延长了GHRH的半衰期。     

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     

Unfolding properties of recombinant human serum albumin products are due to bioprocessing steps

We have used differential scanning calorimetry (DSC) to determine the unfolding properties of commercial products of human serum albumin (HSA) prepared from pooled human blood, transgenic yeast, and transgenic rice. The initial melting temperatures (T_m1) for the unfolding transitions of the HSA products varied from 62°C to 75°C. We characterized the samples for purity, fatty acid content, and molecular weight. The effects of adding fatty acids, heat pasteurization, and a low pH defatting technique on the transition temperatures were measured. Defatted HSA has a structure with the lowest stability (T_m of ～62°C). When fatty acids are bound to HSA, the structure is stabilized (T_m of ～64–72°C), and prolonged heating (pasteurization at 60°C) results in a heat‐stabilized structural form containing fatty acids (T_m of ～75–80°C). This process was shown to be reversible by a low pH defatting step. This study shows that the fatty acid composition and bioprocessing history of the HSA commercial products results in the large differences in the thermal stability. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 31:62–69, 2015     

Isolation of esterified fatty acids bound to serum albumin purified from human plasma and characterised by MALDI mass spectrometry.

Human serum albumin (HSA) is the most abundant protein in plasma. It is known to transport drugs as well as endogenous ligands, like free fatty acids (FFA). A mass spectrometry based method was applied to analyze the albumin bound lipid ligands. HSA was isolated from a human plasma pool by cold ethanol fractionation and ion exchange chromatography. HSA was defatted using a solvent extraction method to release the copurified lipids bound to the protein. The extracts were then analyzed by matrix-assisted laser desorption ionisation (MALDI) mass spectrometry (MS). Using this method, phospholipids and acylglycerols were detected. The phospholipids were identified to be lyso-phosphatidylcholine (lyso-PC) with distribution of different fatty acids (palmitic, stearic, oleic, and linoleic acids). An abundant species in the HSA lipid extract was found to be a diacylglycerol, composed of two linoleic and/or oleic acid chains. The identified motifs reflect structures that are known to be present in plasma. The binding of lysophospholipids has already been described but it is the first ever-reported evidence of native diacylglycerol ligands bound to HSA. Besides the native ligands from plasma a triacylglycerol was detected that has been added during the albumin preparation steps.     

Production of human serum albumin by sugar starvation induced promoter and rice cell culture

Human serum albumin (HSA) is the most widely used clinical serum protein. Currently, commercial HSA can only be obtained from human plasma, due to lack of commercially feasible recombinant protein expression systems. In this study, inducible expression and secretion of HSA by transformed rice suspension cell culture was established. Mature form of HSA was expressed under the control of the sucrose starvation-inducible rice α Amy3 promoter, and secretion of HSA into the culture medium was achieved by using the α Amy3 signal sequence. High concentrations of HSA were secreted into culture medium in a short time (2–4 days) by sucrose depletion after cell concentrations had reached a peak density in culture medium containing sucrose. The recombinant HSA had the same electrophoretic mobility as commercial HSA and was stable and free from apparent proteolysis in the culture medium. In a flask scale culture with repeated sucrose provision-depletion cycles, HSA was stably produced with yields up to 11.5% of total medium proteins or 15 mg/L per cycle after each sucrose provision-depletion cycle. A bubble column type bioreactor was designed for production of HSA. In the bioreactor scale culture, HSA was produced with yields up to 76.4 mg/L 4 days after sucrose depletion. HSA was purified from the culture medium to high purity by a simple purification scheme. Enrichment of HSA in culture medium simplifies downstream purification, minimizes protease degradation, and may reduce production cost. The combination of a DNA construct containing the α Amy3 promoter and signal sequence, and the use of a rice suspension cell culture can provide an effective system for the production of recombinant pharmaceutical proteins.     

Copper inhibits activated protein C: protective effect of human albumin and an analogue of its high-affinity copper-binding site, d-DAHK

Activated protein C (APC) is useful in the treatment of sepsis. Ischemia and acidosis, which often accompany sepsis, cause the release of copper from loosely bound sites. We investigated (i) whether physiological concentrations of copper inhibit APC anticoagulant activity and (ii) if any copper-induced APC inhibition is reversible by human serum albumin (HSA) or a high-affinity copper-binding analogue of the human albumin N-terminus, d-Asp-d-Ala-d-His-d-Lys (d-DAHK). APC activity after 30 min of incubation with CuCl2 (10 microM) was decreased 26% below baseline. HSA, both alone and when combined with various ratios of CuCl2, increased APC activity significantly above baseline. d-DAHK alone and 2:1 and 4:1 ratios of d-DAHK:CuCl2 also increased APC activity. APC contained 1.4 microM copper, which helps explain the increased APC activity with HSA and d-DAHK alone. These in vitro results indicate that copper inhibits APC activity and that albumin and d-DAHK reverse the copper-induced APC deactivation.     

Analysis of tryptophan fluorescence lifetimes in a series of human serum albumin mutants with substitutions in subdomain 2 A

Tryptophan 214, the only tryptophan residue in human serum albumin, is located in the physiologically important subdomain 2A ligand binding site. In the present study the fluorescence lifetime of tryptophan 214 in the following human serum albumin (HSA) mutants with substitutions in subdomain 2A were determined: K195M, K199M, F211V, R218M, R218H, R218A, R222M, H242V, and R257M. An HSA mutant in which tryptophan was moved from subdomain 2A to subdomain 3A (W214L/Y411W) was also examined. Additionally, the fluorescence lifetime of tryptophan 214 in an HSA fragment consisting of subdomains 1A, 1B, and 2A (1A-1B-2A HSA) was determined. For those species expected to have the most dramatic changes in tryptophan microenvironment, W214L/Y411W and 1A-1B-2A HSA, clear changes in tryptophan lifetimes were observed. Significant changes were also seen for those species with mutations at position 218, which is next to tryptophan in the X-ray structure of HSA. However, significant changes were also observed for H242V and R257M, which contain substitutions at positions not immediately adjacent to tryptophan 214, highlighting the conformational flexibility of subdomain 2A.