Independent denaturation of albumin and globulins in human serum

The independent melting of albumin, gamma-globulin, transferrin, and protease inhibitors in the composition of donor blood serum was studied by differential scanning microcalorimetry. It was found that the number of domains in gamma-globulin in donor blood serum and in diluted solutions is the same, whereas the number of domains of albumin in solution and in the composition of blood serum is three and two, respectively. In blood serum, the N-terminal domain melts by the "all-or-none" mechanism. Therefore, the decomposition of peaks of the denaturation curve was made under the assumption that the denaturation of blood serum proteins occurs by the "all-or-none" principle. It is assumed that comparing the calculated melting parameters (Td, delta Td, delta Hd, delta Cd) of domains of blood serum proteins of donor with the corresponding parameters of patients with oncological and nononcological diseases can be used as a basis for a more precise diagnostics of these diseases.     

Urea-induced denaturation of human serum albumin labeled with acrylodan

We induced the denaturation of unlabeled human serum albumin (HSA) and of similar albumin labeled with acrylodan (6-acryloyl-2-dimethylamino naphthalene) with urea and studied the transition profiles using circular dichroism and fluorescence spectroscopy. The circular dichroism spectra for both albumin preparations resulted in the same curves, thus indicating that labeling with acrylodan does not perturb the conformation of HSA. Our results indicate that the denaturation of both albumin preparations takes place at a single, two-state transition with midpoint at about 6 M urea, due to the unfolding of its domain II. It is important to point out that even at 8 M urea, some residual structure remains in the HSA. Great changes in the fluorescence of the dye bound to the protein were observed by addition of solid guanidine hydrochloride to the protein labeled with acrylodan dissolved in 8 M urea, indicating that domain I of this protein was not denatured by urea.     

The effect of solvated ions on the thermal denaturation of human serum albumin in water-dimethylsulfoxide solutions

The effects of solvated ions on the thermal denaturation of human serum albumin (HAS) in water-dimethylsulfoxide (DMSO) solutions were studied by the method of electron absorption spectroscopy. It was shown that depending on the DMSO concentration, electrolytes (LiCl, LiNO₃, LiClO₄, NaCl, and NaNO₃) contained in these solutions were characterized by different anion and cation solvation degrees: unlike cations, anions were only negligibly solvated, which affected HAS thermal denaturation. Electrostatic interactions between anions and positively charged amino acid residues supporting protein denaturation subsided in the line Cl⁻ > NO₃⁻ > ClO₄⁻.     

Study of heat denaturation of human serum albumin in water-alcohol and water-salt solutions in the presence of organic ligands

The method of differential scanning microcalorimetry was used to show a decrease in heat stability of serum albumin in the presence of aliphatic alcohols. In aqueous-alcohol media, the melting temperature, denaturation transition enthalpy were decreased, and the protein intermolecular aggregation enhanced. When the alcohol concentration in aqueous solution was elevated, the number of epsilon-amino groups of lysine residues in human serum albumin exposed to the solvent rose from 6-7 in aqueous solution to maximum 20 groups in the aqueous-alcohol solution, respectively. The elevation of ionic strength also induced an increase in the number of exposed lysine residues and was accompanied by an enhancement of protein aggregation. The modification of six amino groups by pyridoxal phosphate or three by glucose in the initial albumin stabilized the protein incubated at 65 degrees-70 degrees C both in the aqueous-alcohol media. At the given concentration and temperature the native protein was denatured and fully aggregated. Aliphatic alcohols displaced fatty acids from the binding sites on the molecule of serum albumin, which resulted in a change in the number of peaks of the melting curve.     

Heat denaturation of human serum albumin. Migration of bound fatty acids.

Prolonged heat treatment of solutions of human serum albumin at 60 degrees C resulted in formation of one aggregate fraction and one fraction that was stable against further heat treatment. Fatty acid analyses of these fractions indicate that the heat stable fraction is formed by migration of fatty acids from the aggregating molecules to the remaining monomer thereby stabilizing the latter against heat denaturation. Disulphide and SH groups are involved in the aggregation process.     

Anion-induced stabilization of human serum albumin prevents the formation of intermediate during urea denaturation

The unfolding of human serum albumin (HSA), a multidomain protein, by urea was followed by far-UV circular dichroism (CD), intrinsic fluorescence, and ANS fluorescence measurements. The urea-induced transition, which otherwise was a two-step process with a stable intermediate at around 4.8 M urea concentration as monitored by far-UV CD and intrinsic fluorescence, underwent a single-step cooperative transition in the presence of 1.0 M KCl. The free energy of stabilization (DeltaDelta G(H2O)D) in the presence of 1 M KCl was found to be 1,090 and 1,200 cal/mol as determined by CD and fluorescence, respectively.The salt stabilization occurred in the first transition (0-5.0 M urea), which corresponded to the formation of intermediate (I) state from the native (N) state, whereas the second transition, corresponding to the unfolding of I state to denatured (D) state, remained unaffected. Urea denaturation of HSA as monitored by tryptophan fluorescence of the lone tryptophan residue (Trp(214)) residing in domain II of the protein, followed a single-step transition suggesting that domain(s) I and/or III is (are) involved in the intermediate formation. This was also confirmed by the acrylamide quenching of tryptophan fluorescence at 5 M urea, which exhibited little change in the value of Stern-Volmer constant. ANS fluorescence data also showed single-step transition reflecting the absence of accumulation of hydrophobic patches. The stabilizing potential of various salts studied by far-UV CD and intrinsic fluorescence was found to follow the order: NaClO(4) > NaSCN >Na(2)SO(4) >KBr >KCl >KF. A comparison of the effects of various potassium salts revealed that anions were chiefly responsible in stabilizing HSA. The above series was found similar to the electroselectivity series of anions towards the anion-exchange resins and reverse of the Hofmeister series, suggesting that preferential binding of anions to HSA rather than hydration, was primarily responsible for stabilization. Further, single-step transition observed with GdnHCl can be ascribed to its ionic character as the free energy change associated with urea denaturation in the presence of 1.0 M KCl (5,980 cal/mol) was similar to that obtained with GdnHCl (5,870 cal/mol).     

The effect of microwave thermal denaturation on release properties of bovine serum albumin and gluten matrices

The purpose of this study was to compare the effects of denaturation by microwave irradiation on release properties of 2 physically different proteins. Matrices were prepared from water-soluble bovine serum albumin loaded with metoclopramide and sorbed with adequate amount of moisture were thermally denatured in a microwave oven. The release profile of the rather insoluble denatured albumin matrices followed the classical Fickian diffusion profile. The release rate was dependent on the degree of denaturation, which was highly dependent on the level of moisture originally absorbed by the albuminoidal matrices and the period of exposure to microwave energy. Consersely, attempts to reduce the rate of drug release through microwave irradiation of metoclopramide-loaded matrices prepared from water-insoluble gluten were futile. The denaturation process was shown to be limited to the relatively water-soluble protein core fraction, while aggregation between neighboring gluten proteins in the matrix was not achieved even in the presence of considerable amounts of sorbed water. Published: February 10, 2006     

Urea denaturation of bovine serum albumin at pH 9

The action of 5 m urea on bovine serum albumin has been studied at pH 9.0 and 25°C. Analysis by the acrylamide gel electrophoresis revealed the presence of a few components 1, 1′, 2, 3, 4 and 5. The components 1 and 1′ are monomers, component 2 is a dimer, and components 3, 4 and 5 are aggregates. In presence of SH blocking reagent, bovine serum albumin gave only the zone 1, indicating that the components 1′-5 were formed by the SH to S-S exchange reactions. Component 1′ was formed by the intramolecular SH to S-S exchange reaction, and components 2–5 were formed by the intermolecular exchange reaction. Addition of cysteine either to bovine serum albumin or to the SH-blocked bovine serum albumin increased the percent of zone 1′, indicating that a complex bovine serum albumin-cysteine was formed or that the SH-catalyzed structural alteration occurred in bovine serum albumin. Components 1, 1′, 2 and 3 were isolated separately by the preparative disc gel electrophoresis. The sedimentation coefficients 1 and 1′ differed slightly indicating that they were different monomers, and values were slightly smaller than the normal value of bovine serum albumin, indicating that these components were in slightly expanded state. Isolated component 1 was exposed to 5 m urea again, but no further change occurred. This supports the concept of microheterogeneity of bovine serum albumin.     

A dilatometric study of the denaturation of bovine serum albumin by guanidine hydrochloride

The denaturation of bovine serum albumin by guanidine hydrochloride was studied using the dilatometric method. From dilatometric measurements the differences between the partial specific volume of the protein in denaturant solutions and water, respectively, were determined. The differences reflect the extent of unfolding as well as the binding of the denaturant. From the differences and the known partial specific volume of the native protein, the partial specific volumes at individual denaturant concentrations were obtained.     

Guanidine hydrochloride denaturation of human serum albumin originates by local unfolding of some stable loops in domain III

Thiamine triphosphate (ThTP) is found in most organisms and may be an intracellular signal molecule produced in response to stress. We have recently cloned the cDNA coding for a highly specific mammalian 25-kDa thiamine triphosphatase. The enzyme was active in all mammalian species studied except pig, although the corresponding mRNA was present. In order to determine whether the very low ThTPase activity in pig tissues is due to the absence of the protein or to a lack of catalytic efficiency, we expressed human and pig ThTPase in E. coli as GST fusion proteins. The purified recombinant pig GST-ThTPase was found to be 2-3 orders of magnitude less active than human GST-ThTPase. Using site-directed mutagenesis, we show that, in particular, the change of Glu85 to lysine is responsible for decreased solubility and catalytic activity of the pig enzyme. Immunohistochemical studies revealed a distribution of the protein in pig brain very similar to the one reported in rodent brain. Thus, our results suggest that a 25-kDa protein homologous to hThTPase but practically devoid of enzyme activity is expressed in pig tissues. This raises the possibility that this protein may play a physiological role other than ThTP hydrolysis.     

Conformational change of bovine serum albumin by heat treatment

The thermal denaturation of bovine serum albumin (BSA) was studied at pH 2.8 and 7.0 in the range of 2–65°C. The relative proportions of -helix, -structure, and disordered structure in the protein conformation were determined as a function of temperature, by the curve-fitting method of circular dichroism spectra. With the rise of temperature at pH 7.0, the proportion of -helix decreased above 30°C and those of -structure and disordered structure increased in the same temperature range. The structural change was reversible in the temperature range below 45°C. However, the structural change was partially reversible upon cooling to room temperature subsequent to heating at 65°C. On the other hand, the structural change of BSA at pH 2.3 was completely reversible in the temperature range of 2–65°C, probably because the interactions between domains and between subdomains might disappear due to the acid expansion. The secondary structure of disulfide bridges-cleaved BSA remained unchanged during the heat treatment up to 65°C at pH 2.8 and 7.0.     

Interaction of ergosterol with bovine serum albumin and human serum albumin by spectroscopic analysis

This study was designed to examine the interactions of ergosterol with bovine serum albumin (BSA) and human serum albumin (HSA) under physiological conditions with the drug concentrations in the range of 2.99-105.88?μM and the concentration of proteins was fixed at 5.0?μM. The analysis of emission spectra quenching at different temperatures revealed that the quenching mechanism of HSA/BSA by ergosterol was the static quenching. The number of binding sites n and the binding constants K were obtained at various temperatures. The distance r between ergosterol and HSA/BSA was evaluated according to F?ster non-radioactive energy transfer theory. The results of synchronous fluorescence, 3D fluorescence, FT-IR, CD and UV-Vis absorption spectra showed that the conformations of HSA/BSA altered in the presence of ergosterol. The thermodynamic parameters, free energy change (ΔG), enthalpy change (ΔH) and entropy change (ΔS) for BSA-ergosterol and HSA-ergosterol systems were calculated by the van't Hoff equation and discussed. Besides, with the aid of three site markers (for example, phenylbutazone, ibuprofen and digitoxin), we have reported that ergosterol primarily binds to the tryptophan residues of BSA/HSA within site I (subdomain II A).