Conformational transitions of the three recombinant domains of human serum albumin depending on pH

Human serum albumin (HSA) is a protein of 66.5 kDa that is composed of three homologous domains, each of which displays specific structural and functional characteristics. HSA is known to undergo different pH-dependent structural transitions, the N-F and F-E transitions in the acid pH region and the N-B transition at slightly alkaline pH. In order to elucidate the structural behavior of the recombinant HSA domains as stand-alone proteins and to investigate the molecular and structural origins of the pH-induced conformational changes of the intact molecule, we have employed fluorescence and circular dichroic methods. Here we provide evidence that the loosening of the HSA structure in the N-F transition takes place primarily in HSA-DOM III and that HSA-DOM I undergoes a structural rearrangement with only minor changes in secondary structure, whereas HSA-DOM II transforms to a molten globule-like state as the pH is reduced. In the pH region of the N-B transition of HSA, HSA-DOM I and HSA-DOM II experience a tertiary structural isomerization, whereas with HSA-DOM III no alterations in tertiary structure are observed, as judged from near-UV CD and fluorescence measurements.     

Conformational transitions of human alpha-1 fetoprotein and serum albumin at acid and alkaline pH

Conformational transitions of HAFP in the pH-range 2-12 were studied by fluorescence spectroscopy, fluorescence polarization measurements, circular dichroism and hydrophobic chromatography in order to compare molecular architecture of HAFP and that of human serum albumin. It was found that HAFP has a remarkably hydrophilic exposed molecular surface at neutral pH and possesses extensive hydrophobic binding sites located in crevices. Conformational changes occur in HAFP in the acid and alkaline pH regions; extensive hydrophobic areas in HAFP are exposed by both acid and alkaline transitions. The alpha-helix contents of HAFP were determined as 67% at pH 7.6, 47% at pH 2.11.     

Conformational changes in human serum albumin around the neutral pH from circular dichroic measurements.

The molar ellipticity of the warfarin-albumin complex at 310 nm increases with pH from 6 to 9. This pH dependence runs parallel with that of the molar ellipticity of the albumin alone at 292 nm. The change in molar ellipicity with pH occurs in a smaller pH interval after addition of the physiological concentration of calcium ions. These findings give support to the assumption that the binding site for warfarin on the albumin molecule is affected by the neutral-to-base transition in the protein.     

Conformational changes in the bilirubin-human serum albumin complex at extreme alkaline pH.

Light-absorption, c.d. and fluorescence of the bilirubin-albumin complex were investigated at extreme alkaline pH. Above pH 11.1 albumin binds the bilirubin molecule, twisted oppositely to the configuration at more neutral pH. On the basis of light-absorption it is shown that two alkaline transitions occur. The first alkaline transition takes place at pH between 11.3 and 11.8, co-operatively dissociating at least six protons. The second alkaline transition takes place at pH between 11.8 and 12.0. It probably implies a reversible unfolding of the albumin molecule, increasing the distance between tryptophan-214 and bilirubin, and partly exposing the liganded bilirubin to the solvent.     

Conformational analysis of human serum albumin and its non-enzymatic glycation products using monoclonal antibodies

Monoclonal antibodies (mAbs) were prepared to analyse the conformation of human serum albumin (HSA) and its non-enzymatic glycation (NEG) products. We first determined the epitopes of the mAbs using HSA subdomains expressed on the surface of yeast. Each mAb was classified as belonging to one of two groups; Type I mAbs which recognized a single subdomain structure and Type II mAbs which bound to plural subdomains. We analysed the pH-dependent conformational change in HSA. We found that one Type II mAb, HAy2, detected the normal to base form (N-B) transition while the other did not, suggesting that N-B transition occurred around Domain I accompanied by topological isomerization of subdomains without changing the subdomain structure itself. Next, we analysed the conformations of the NEG products. Since all mAbs reacted with the early NEG products, no structural change was thought to have occurred in the early NEG products. On the other hand, only a Type I mAb, HAy1, had full binding activity with the advanced glycation end products (AGE) while the other mAbs had lost or had diminished activity, suggesting that the over-all tertiary structure of HSA was altered except for a subdomain, sDOM Ia, in AGE.     

Study of the structure of human serum albumin, liberated from fatty acids, by a tritium marker method]

To elucidate the natural fatty acids effect on the human serum albumin (HSA) structure a new method of tritium labelling was used. The main peculiarity of the method consists in the possibility to get information on the qualitative and quantitative amino acid composition of the surface layer of the protein globule at different conformational states of the globule. Defatted HSA was shown to be characterized a higher accessibility of Asx, Glx, Thr, Ser, Gly, Pro, Ile, Tyr residues while the other residues remain unchanged. Asx residues are characterized by the largest changes (about 8 folds). Full accessible protein surface during defatting increases from 39,000 to 48,000 A2. Fatty acids connected with albumin in the relation 1-3 moles/mol of protein are noted to be the factor increasing the globule compactness and stipulating for the conformational protein stability to warmth, urine and guanidine salts effect.     

Molten globule-like state of human serum albumin at low pH.

Human serum albumin (HSA), under conditions of low pH, is known to exist in two isomeric forms, the F form at around pH 4.0 and the E form below 3.0. We studied its conformation in the acid-denatured E form using far-UV and near-UV CD, binding of a hydrophobic probe, 1-anilinonaphthalene-8-sulfonic acid (ANS), thermal transition by far-UV and near-UV CD, tryptophan fluorescence, quenching of tryptophan fluorescence using a neutral quencher, acrylamide and viscosity measurements. The results show that HSA at pH 2.0 is characterized by a significant amount of secondary structure, as evident from far-UV CD spectra. The near-UV CD spectra showed a profound loss of tertiary structure. A marked increase in ANS fluorescence signified extensive solvent exposure of non-polar clusters. The temperature-dependence of both near-UV and far-UV CD signals did not exhibit a co-operative thermal transition. The intrinsic fluorescence and acrylamide quenching of the lone tryptophan residue, Trp214, showed that, in the acid-denatured state, it is buried in the interior in a non-polar environment. Intrinsic viscosity measurements showed that the acid-denatured state is relatively compact compared with that of the denatured state in 7 M guanidine hydrochloride. These results suggest that HSA at pH 2.0 represents the molten globule state, which has been shown previously for a number of proteins under mild denaturing conditions.     

Thermal aggregation of bovine serum albumin at different pH: comparison with human serum albumin

We report here a study on thermal aggregation of BSA at two different pH values selected to be close to the isoelectric point (pI) of this protein. Our aim is to better understand the several steps and mechanisms accompanying the aggregation process. For this purpose we have performed kinetics of integrated intensity emission of intrinsic and extrinsic dyes, tryptophans and ANS respectively, kinetics of Rayleigh scattering and of turbidity. The results confirm the important role played by conformational changes in the tertiary structure, especially in the exposure of internal hydrophobic regions that promote intermolecular interactions. We also confirm that the absence of electrostatic repulsion favours the disordered non-specific interactions between molecules and consequently affects the aggregation rate. Finally, the comparison between BSA and another relative protein, HSA, allows us to clarify the role of different domains involved in the aggregation process. Proceedings of the XVIII Congress of the Italian Society of Pure and Applied Biophysics (SIBPA), Palermo, Sicily, September 2006.     

Conformational changes in human serum albumin studied by fluorescence and absorption spectroscopy. Distance measurements as a function of pH and fatty acids.

pH- and fatty acid-induced conformational changes in human serum albumin were investigated by fluorescence-energy transfer, determining the distance between Trp-214 and bound bilirubin at 25 degrees C. This distance changes significantly with the pH, being 2.52 +/- 0.01 nm at pH 6, 2.31 +/- 0.04 nm at pH 9, 2.13 +/- 0.07 nm at pH 11.0 and 2.77 nm at pH 11.9. The influence of different fatty acids on the distance was also determined. At pH 7.4 medium-chain fatty acids seem to increase this distance, whereas long-chain fatty acids, at low concentrations, decrease the distance between the two chromophores. The contraction of the protein carrying long-chain saturated fatty acids is even more pronounced at pH 9.     

Anion-induced refolding of human serum albumin under low pH conditions

We studied the effect of various anions (of acids and salts) on the acid denatured state of HSA by near-UV circular dichroism (CD), far-UV CD, 1-anilinonaphthalene-8-sulfonate (ANS) binding, tryptophan fluorescence and thermal transition. Addition of different acids and salts caused an induction of alpha-helical structure as evident from the increase in the mean residue ellipticity (MRE) value at 222 nm and loss of ANS binding sites exhibited by the decrease in the ANS fluorescence intensity at 480 nm. However, the concentration range of acids/salts required to bring about the transition varied greatly among different acids and salts. Among various acids/salts tested, K(3)Fe(CN)(6) was found to be most effective whereas HCl and KCl were least effective in inducing the properties close to native structure. Further, they followed the electroselectivity series. The near-UV CD spectra showed an increase in MRE towards the native state, whereas the tryptophan fluorescence emission spectra produced a red shift of about 6 nm on addition of KClO(4). The temperature-induced transition in the presence of 40 mM KClO(4) monitored by ellipticity measurements at 222 nm was characterized by the presence of an intermediate state in the temperature range 30-50 degrees C having abundant secondary structure. These results suggest that human serum albumin at low pH and in the presence of acids or salts exists in a partially folded state characterized by native-like secondary structure and tertiary folds.     

Interaction of human serum albumin with luminol]

Chemoluminescent and fluorescent studies of the interaction between serum albumine and luminole have been carried out. An increase of chemoluminescent intensity and quenching of luminole fluorescence dependent on protein concentration has been observed. Possibility of complex formation and the mechanism of fluorescence quenching with luminole are discussed.     

Relations between high-affinity binding sites of markers for binding regions on human serum albumin.

Binding of warfarin, digitoxin, diazepam, salicylate and Phenol Red, individually or in different pair combinations, to defatted human serum albumin at ligand/protein molar ratios less than 1:1 was studied at pH 7.0. The binding was determined by ultrafiltration. Some of the experiments were repeated with the use of equilibrium dialysis in order to strengthen the results. Irrespective of the method used, all ligands bind to one high-affinity binding site with an association constant in the range 10(4)-10(6) M-1. High-affinity binding of the following pair of ligands took place independently: warfarin-Phenol Red, warfarin-diazepam, warfarin-digitoxin and digitoxin-diazepam. Simultaneous binding of warfarin and salicylate led to a mutual decrease in binding of one another, as did simultaneous binding of digitoxin and Phenol Red. Both effects could be accounted for by a coupling constant. The coupling constant is the factor by which the primary association constants are affected; in these examples of anti-co-operativity the factor has a value between 0 and 1. In the first example it was calculated to be 0.8 and in the latter 0.5. Finally, digitoxin and salicylate were found to compete for a common high-affinity binding site. The present findings support the proposal of four separate primary binding sites for warfarin, digitoxin (and salicylate), diazepam and Phenol Red. An attempt to correlate this partial binding model for serum albumin with other models in the literature is made.     

Conformational transitions in phosvitin with pH variation. Vibrational circular dichroism study

The vibrational circular dichroism (VCD) spectra of metal-free phosvitin are presented as a function of pH and analyzed both qualitatively and by using a factor analysis approach referenced to a protein data set. The qualitative pattern of both the IR and VCD changes is consistent with a coil-to-sheet transition occurring as pH is progressively decreased to values lower than 3. A similar transition was seen in commercial preparation of phosvitin which still contained metal ions, but there the transition was more gradual and occurred at somewhat different pH values. Such a gradual change is also evident in the solution phase absorption band profile but is made clearer using Fourier deconvolution. Based on VCD results, the low pH transition appears to occur with two distinct manifestations of the beta-sheet form. However, at the lowest pH values the sample may precipitate. These two forms are not distinguishable with Fourier transform infrared alone and may be due to a twist of the beta-sheet form or to aggregation.     

共振瑞利散射测试血清蛋白酸碱度影响的探讨

提出了一种基于共振瑞利散射（RRS）原理测量人体血清蛋白的新方法。在缓冲溶液的作用下,把配制好的人体血清蛋白稀释液按比例与四羧基酞菁锌混合,经过化学作用后在波长为400 nm左右蓝色波段强光照射下,散射出480 nm左右的共振瑞利散射光强信号。考察在不同pH对共振瑞利散射光强信号与混合物中的血清蛋白反应线性关系的影响。结果表明,pH在6.0～8.0范围内混合溶液共振瑞利散射光强信号与血清蛋白的线性关系良好。     

Conformational changes and allosteric communications in human serum albumin due to ligand binding

It is well recognized that knowledge of structure alone is not sufficient to understand the fundamental mechanism of biomolecular recognition. Information of dynamics is necessary to describe motions involving relevant conformational states of functional importance. We carried out principal component analysis (PCA) of structural ensemble, derived from 84 crystal structures of human serum albumin (HSA) with different ligands and/or different conditions, to identify the functionally important collective motions, and compared with the motions along the low-frequency modes obtained from normal mode analysis of the elastic network model (ENM) of unliganded HSA. Significant overlap is observed in the collective motions derived from PCA and ENM. PCA and ENM analysis revealed that ligand selects the most favored conformation from accessible equilibrium structures of unliganded HSA. Further, we analyzed dynamic network obtained from molecular dynamics simulations of unliganded HSA and fatty acids- bound HSA. Our results show that fatty acids-bound HSA has more robust community network with several routes to communicate among different parts of the protein. Critical nodes (residues) identified from dynamic network analysis are in good agreement with allosteric residues obtained from sequence-based statistical coupling analysis method. This work underscores the importance of intrinsic structural dynamics of proteins in ligand recognition and can be utilized for the development of novel drugs with optimum activity.