Electron spin resonance study of the copper(II) complexes of human and dog serum albumins and some peptide analogs

Electron spin resonance spectra of the first Cu(II) complexes of human serum albumin, dog serum albumin, l-aspartyl-l-histidine N-methylamide and glycyl-glycyl-l-histidine N-methylamide have been studied using isotopically pure ⁶⁵Cu in its chloride form. At 77° K, the esr spectra of Cu(II) complex of human serum albumin exhibited only one form of esr signal between pH 6.5 and 11. No intermediate forms were detected. The presence of an equally spaced nine-line superhyperfine structure with spacing ～15 G indicated considerable covalent bonding between Cu(II) and four nitrogen atoms derived from the protein. The esr spectrum form of Cu(II) bound to human serum albumin detected at neutral pH would be consistent with the participation of four nitrogens from the α-NH₂ group, two peptide groups, and the imidazole group of a histidine residue. In contrast, the esr spectra of Cu(II)-dog serum albumin complex showed a transition from a low pH form to a high pH form as the pH was increased to 9.5. These spectral changes were found to be reversible upon lowering the pH. Ligand superhyperfine splittings in the low pH form of the esr signal of Cu(II)-dog albumin were not resolved. The distinct pH dependence of the esr signals observed in human and dog serum albumin complexes could be correlated to their respective optical spectra changes as a function of pH. At room temperature and in the pH range between 6 and 11, the esr spectra of Cu(II) complexes of l-aspartyl-l-alanyl-l-histidine N-methylamide and glycyl-glycyl-l-histidine N-methylamide exhibited a well-resolved nine-line superhyperfine structure indicating metal coordination with four equivalent nitrogen atoms of peptide.     

Studies of copper(II) binding to glycylglycyl-L-tyrosine-N-methyl amide, a peptide mimicking the NH2-terminal copper(II)-binding site of dog serum albumin by analytical potentiometry, spectrophotometry, CD, and NMR spectroscopy

Unlike human serum albumin (HSA), dog serum albumin (DSA) does not possess the characteristics of the specific first binding site for Cu(II). In DSA, the important histidine residue in the third position, responsible for the Cu(II)-binding specificity in HSA, is replaced by a tyrosine residue. In order to study the influence of the tyrosine residue in the third position of DSA, a simple model of the NH2-terminal native sequence tripeptide of DSA, glycylglycyl-L-tyrosine-N-methylamide (GGTNMA) was synthesized and its Cu(II)-binding properties studied by analytical potentiometry, spectrophotometry, CD, and NMR spectroscopy. The species analysis indicated the existence of five mono-complexes at different protonation states: MHA, MA, MH-1A, MH-2A, MH-3A, and only one bis-complex MH-2A-2. The complexing ability of GGTNMA to Cu(II) was found to be weaker than that of the Cu(II) binding peptide models of HSA. The visible absorption spectra of Cu(II)-GGTNMA complexes are similar to those observed in the case of DSA-Cu(II) complexes. The weaker binding and the spectral properties of Cu(II)-GGTNMA complexes are consistent with less specific Cu(II)-binding properties of the peptide of this sequence similar to what was noted with DSA. CD results are in excellent agreement with species analysis and visible spectra where it is clearly evident that Cu(II) binds to GGTNMA starting from the alpha-NH2 group and step by step to deprotonated amide nitrogens as the pH is raised. The absence of any charge transfer band around 400 nm strongly indicates that Cu(II) does not bind to the phenolate group. Furthermore, NMR results are consistent with the noninvolvement of the tyrosine residue of GGTNMA in Cu(II) complexation. Thus, it is clear that the low Cu(II)-binding affinity of DSA is due to the genetic substitution of tyrosine for histidine at the NH2-terminal region of the protein.     

4,4-Dimethyl-4-silapentane-1-ammonium trifluoroacetate (DSA), a promising universal internal standard for NMR-based metabolic profiling studies of biofluids, including blood plasma and serum

Nuclear magnetic resonance (NMR)-based metabolic profiling of biofluids and tissues are of key interest to enhance biomarker discovery for disease, drug efficacy and toxicity studies. Urine and blood plasma/serum are the biofluids of most interest as they are the most accessible in both clinical and preclinical studies. However, proteinaceous fluids, such as blood serum or plasma, represent the greatest technical challenge since the chemical shift (δ) and line-width (ν_1/2) of internal standards currently used for aqueous NMR samples are greatly affected by protein binding. We have therefore investigated the suitability of 4,4-dimethyl-4-silapentane-1-ammonium trifluoroacetate (DSA) as a universal internal standard for biofluids. Proton (¹H) NMR spectroscopy was used to determine the effect of serum pH (3, 7.4 and 10) and DSA concentration on the overall lineshape and position of the trimethylsilyl resonance of DSA. The results were compared to that of 3-(trimethylsilyl)propionic acid sodium salt (TSP). Both the chemical shift and line-width of the DSA peak were not significantly affected by pH or DSA concentration, whereas these parameters for TSP showed large variations due to protein binding. Furthermore, the peak area of DSA correlated linearly with its concentration under all pH conditions, whilst no linear correlation was observed with TSP. Overall, in contrast to TSP, these results support the use of DSA as an accurate universal internal chemical shift reference and concentration/normalisation standard for biofluids. In the case of proteinaceous biofluids such as serum, where no current standard is available, this offers a considerable saving in both operator and spectrometer time.     

人血清白蛋白纯化技术研究进展

本文综述了国内外关于血浆人血清白蛋白（pHSA）和基因重组人血清白蛋白（rHSA）分离纯化的进展和发展趋势。以冷乙醇沉淀为主的pHSA分离方法仍是目前多数工业采用的工艺,但近年来发展的离子交换色谱、凝胶过滤色谱、亲和色谱等多种色谱分离技术具有自动化程度高、生产周期短、更符合GMP等特点,正在逐步取代传统的冷乙醇沉淀法。当前用基因工程技术表达的人血清白蛋白对分离纯化提出了新的挑战。为获得高纯度、安全、稳定的产品,各种色谱分离技术得到更多的应用,其中扩张床离子交换色谱可以省去离心、过滤等传统固液分离操作。尽管rHSA的纯化技术已有一定的发展,但纯化过程仍需进一步优化以提高产品纯度及收率。     

Mechanism of the binding of 2-(4'-hydroxyphenylazo)benzoic acid to bovine serum albumin

The mechanism of the binding of 2-(4'-hydroxyphenylazo)benzoic acid (HABA) to bovine serum albumin was studied by relaxation methods as well as the binding isotherm using gel chromatography. A single relaxation was observed over a wide range of HABA concentration except at the extremes of high concentration where another slow process was observed. The concentration dependence of the reciprocal relaxation time of the fast process decreased monotonically with increase in concentration of HABA at constant polymer concentration. The data were analyzed on the basis of Brown's domain structure model and were found to be consistent with a sequential binding mechanism. The azohydrazon tautomerism of HABA was identified with the intramolecular step of the complex. The activation parameters of the step, determined from the temperature dependence of the relaxation time of the fast process, showed that this step is rate limited by an enthalpy barrier in both forward and backward directions. Comparison of the activation parameters with those of other serum albumin-ligand systems suggests that there is an enthalpy-entropy compensation in the activation process of the intramolecular step with the compensation temperature at about 270 K; the enthalpy-entropy compensation is thought to be related to the hydrophobic nature of the ligand.     

Understanding the effects of two bound glucose in Sudlow site I on structure and function of human serum albumin: theoretical studies

Human serum albumin (HSA) is the most abundant protein found in blood serum. It carries essential metabolites and many drugs. The glycation of HSA causes abnormal biological effects. Importantly, glycated HSA (GHSA) is of interest as a biomarker for diabetes. Recently, the first HSA structure with bound pyranose (GLC) and open-chain (GLO) glucose at Sudlow site I has been crystallised. We therefore employed molecular dynamics (MD) simulations and ONIOM calculations to study the dynamic nature of two bound glucose in a pre-glycated HSA (pGHSA) and observe how those sugars alter a protein structure comparing to wild type (Apo) and fatty acid-bound HSA (FA). Our analyses show that the overall structural stability of pGHSA is similar to Apo and FA, except Sudlow site II. Having glucose induces large protein flexibility at Sudlow site II. Besides, the presence of glucose causes W214 to reorient resulting in a change in W214 microenvironment. Considering sugars, both sugars are exposed to water, but GLO is more solvent-accessible. ONIOM results show that glucose binding is favoured for HSA (?115.04 kcal/mol) and GLO (?85.10 kcal/mol) is more preferable for Sudlow site I over GLC (?29.94 kcal/mol). GLO can strongly react with K195 and K199, whereas K195 and K199 provide slightly repulsive forces for GLC. This can confirm that an open-chain GLO is more favourable inside a pocket.     

Effect of prototypic drugs ibuprofen and warfarin on global chaotropic unfolding of human serum heme-albumin: a fast-field-cycling 1H-NMR relaxometric study

Human serum albumin (HSA) is the most prominent protein in plasma, but it is also found in tissues and secretions throughout the body. The three-domain design of HSA provides a variety of binding sites for many ligands, including heme and drugs. HSA has been used as a model multidomain protein to investigate how interdomain interactions affect the global folding/unfolding process. Here, we report on the reversible chemical denaturation of heme-HSA involving three different conformational states (F, N, and B, occurring at pH 4.0, 7.0, and 9.0, respectively) and on the effect of prototypic drugs ibuprofen and warfarin on thermodynamics of the reversible unfolding process. Chaotropic unfolding of heme-HSA in the F, N, and B conformations is governed by different thermodynamic regimes, with the B form showing an entropic stabilization of the structure that compensates an enthalpic destabilization, and the F form easily unfolding under entropic control. Warfarin and ibuprofen binding stabilizes heme-HSA in both N and B states.     

Microcalorimetric method to determine competitive binding action of a psychotropic drug (dipotassium chlorazepate) on L-tryptophan · human serum albumin complex

A mathematical treatment and an original microcalorimetric method are developed to verify an eventual competitive binding between any two substances for the same macromolecule. To apply this method, a competitive binding of L-tryptophan and one benzodiazepin (dipotassium chlorazepate) for human serum albumin is perfectly demonstrated.The association constants and the enthalpy variations are equal to 14 000 ± 2000 M^?1 and ?6.6 ± 0.2 kcal/mol for human serum albumin · tryptophan complex and 13 000 ± 1000 M^?1 and ?10.0 ± 0.2 kcal/mol for human serum albumin · chlorazepate complex. In all cases the stoichiometry is equal to one.The binding of tryptophan to human serum albumin is partially stereospecific; the association constant and the enthalpy variation for D-tryptophan complex are equal, respectively, to 1000 ± 200 M^?1 and ?2.6 ± 0.3 kcal/mol.     

Crystallographic analysis of human serum albumin complexed with 4Z,15E-bilirubin-IXalpha

Bilirubin, an insoluble yellow-orange pigment derived from heme catabolism, accumulates to toxic levels in individuals with impaired or immature liver function. The resulting jaundice may be managed with phototherapy to isomerize the biosynthetic 4Z,15Z-bilirubin-IXα to more soluble and excretable isomers, such as 4Z,15E-bilirubin. Bilirubin and its configurational isomers are transported to the liver by human serum albumin (HSA) but their precise binding location(s) on the protein have yet to be determined. To investigate the molecular details of their interaction, we co-crystallised bilirubin with HSA. Strikingly, the crystal structure—determined to 2.42 Å resolution—revealed the 4Z,15E-bilirubin-IXα isomer bound to an L-shaped pocket in sub-domain IB. We also determined the co-crystal structure of HSA complexed with fusidic acid, an antibiotic that competitively displaces bilirubin from the protein, and showed that it binds to the same pocket. These results provide the first crystal structure of a natural bilirubin pigment bound to serum albumin, challenge some of the present conceptions about HSA-bilirubin interactions, and provide a sound structural framework for finally resolving the long-standing question of where 4Z,15Z-bilirubin-IXα binds to the protein.     

Affinity chromatographic studies on antigen-antibody dissociation

An analytical affinity chromatography assay has been developed for the investigation of the dissociation of antigen-antibody complexes. Albumin-coupled Sepharose 4B and anti-albumin has been used as a model system. At extremely low or high pH, in the presence of highly concentrated chaotropic ions at pH 7 or by elution with 100% ethylene glycol after pretreating with high pH buffer, most of the bondings could be ruptured. The latter two-step desorption procedure provides recovery of intact antibody with high yield. The technique was also utilized for the preparation of antibody against human growth hormone.     

Expression of human serum albumin in the milk of transgenic mice

We have tested the feasibility of producing large quantities of human serum albumin (HSA) in the milk of transgenic livestock by generating transgenic mice as a model system. The sheep β-lactoglobulin (BLG) 5′-regulatory promoter sequences were used to support expression of BLG or HSA in transgenic mice. Transgenic animals generated from the entire BLG gene including 3, 5.5 or 10.8 kb of 5′-sequences demonstrated that 3 kb of 5′-sequences were sufficient to support high levels of expression of BLG, and that the longer 5′-sequences did not improve upon the levels of expression. As such, the 3 kb 5′-sequences were used to drive expression of HSA in BLG-HSA constructs. HSA was not detectably expressed in eight transgenic lines generated from a BLG-HSA construct containing the HSA cDNA. Two transgenic lines of 26 generated, using five different constructs, with an HSA minigene possessing the first intron expressed HSA in their milk. One of these expressed HSA at high levels (2.5 mg ml⁻¹) and has stably transmitted this ability to its progeny. A high percentage of transgenic mouse lines (four of six) generated from a vector containing an HSA minigene possessing introns 1 and 2 expressed HSA in their milk at levels which ranged from 1 to 35 μg ml⁻¹. In a similar trend, levels of expression of HSA by transfected tissue culture cells from BLG-HSA vectors containing an introduced SV40 enhancer were low with the HSA cDNA, increased with the HSA minigene with intron 1 and increased further with the minigene containing introns 1 and 2. This study demonstrates that high levels of HSA can be expressed in the milk of transgenic animals, that introns of the HSA gene play a role in its expression and that transfected cell lines may be used to quickly evaluate the relative expression efficiencies of various vector constructs intended for future transgenic evaluation.     

Characterization of Sulfhydryl Heterogeneity in Human Serum Albumin and Recombinant Human Serum Albumin for Clinical Use

Since human serum albumin has one sulfhydryl group and 17 disulfides, reactive sulfhydryl groups give rise to heterogeneity. The present paper presents a comparison of sulfhydryl heterogeneity in human serum albumin and recombinant human serum albumin for clinical use. Low molecular weight sulfhydryl compounds were identified from both sources. The recombinant albumin had a much higher sulfhydryl content than plasma serum albumin.     

Serum albumins—Unusual allergens

Background

Albumins are multifunctional proteins present in the blood serum of animals. They can bind and transport a wide variety of ligands which they accommodate due to their conformational flexibility. Serum albumins are highly conserved both in amino acid sequence and three-dimensional structure. Several mammalian and avian serum albumins (SAs) are also allergens. Sensitization to one of the SAs coupled with the high degree of conservation between SAs may result in cross-reactive antibodies in allergic individuals. Sensitivity to SA generally begins with exposure to an aeroallergen, which can then lead to cross-sensitization to serum albumins present in food.

Scope of review

This review focuses on the allergenicity of SAs presented in a structural context.

Major conclusions

SA allergenicity is unusual taking into account the high sequence identity and similarity between SA from different species and human serum albumin. Cross-reactivity of human antibodies towards different SAs is one of the most important characteristics of these allergens.

General significance

Establishing a relationship between sequence and structure of different SAs and their interactions with antibodies is crucial for understanding the mechanisms of cross-sensitization of atopic individuals. Structural information can also lead to better design and production of recombinant SAs to replace natural proteins in allergy testing and desensitization. Therefore, structural analyses are important for diagnostic and treatment purposes. This article is part of a Special Issue entitled Serum Albumin.     

血清蛋白与4,5-二溴荧光素相互作用及其分析应用的研究

在 0 .10mol/mL的醋酸溶液中 ,4,5 二溴荧光素能与血清蛋白形成稳定的复合物 ,最大吸收波长 482nm ,与试剂比较 ,红移了 12nm。据此建立了测定血清蛋白的方法 ,用于BSA和HSA的测定 ,分别在 2～ 14mg·L-1有线性关系 ,表观摩尔吸光系数分别为 3.12× 10 5L·mol-1·cm-1和 3.2 7× 10 5L·mol-1·cm-1。应用该法测定了人血清样品总蛋白含量 ,结果令人满意。