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.     

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.     

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.     

Copper(II)-DNA denaturation. II. The model of DNA denaturation

In a continuing study of the denaturation of DNA as brought abought about by Cu(II) ions, results are presented for the dependence of T_m and τ (the terminal relaxation time) on ionic strength, pH, reactant concentrations, and temperature. Maximum stability of the double helix, as reflected by the longest relaxation times and highest T_m values, was observed between pH 5.3 and 6.2. Outside this range both T_m and τ decreased sharply. A second, faster relaxation time was deduced from the kinetic cureves. The apparent activation energies of the rapid and slow (“terminal”) relaxations were found to be 12 and 55 kcal/mole, respectively. Several lines of evidence led to the conclusions that (1) the rate-determining step in DNA denaturation, when occurring in the transition region, is determined by chemical events and (2) the interactions which are disrupted kinetically in the rate-determining step are those which account for the major portion of the thermal (T_m) stability of helical DNA.     

Cold denaturation and heat denaturation of Streptomyces subtilisin inhibitor. 1. CD and DSC studies

Cold denaturation and heat denaturation of the protein Streptomyces subtilisin inhibitor (SSI) were studied in the pH range 1.84-3.21 and in the temperature range -3-70 degrees C by circular dichroism and scanning microcalorimetry. The native structure of the protein was apparently most stabilized at about 20 degrees C and was denatured upon heating and cooling from this temperature. Each denaturation was reversible and cooperative, proceeding in two-state transitions, that is, from the native state to the cold-denatured state or from the native state to the heat-denatured state. The two denatured states, however, were not perfect random-coiled structures, and they differed from each other, indicating that there exist three states in this temperature range, i.e., cold denatured, native, and heat denatured. The difference between the cold and heat denaturations was indicated first by circular dichroism. The isodichroic point for the transition from the native state to the cold-denatured state was different from that from the native state to the heat-denatured state in the pH range between 3.21 and 2.45. Moreover, molar ellipticity for the cold-denatured state was different from that of the heat-denatured state, and the transition from the former to the latter was observed at pH values below 2. Values of van't Hoff enthalpies from the native state to the heat-denatured state at pH values between 3.21 and 2.45 were obtained by curve fitting of the CD data, and delta Cp = 1.82 (+/- 0.11) [kcal/(mol.K)] was obtained from the linear plot of the enthalpies against temperature. The parameters obtained from the heat denaturation studies gave curves for delta G zero which were not in agreement with the experimental data in the cold denaturation region when extrapolated to the low temperature. Moreover, the value of the apparent delta Cp for the cold denaturation in the pH range 3.03-2.45 was estimated to be different from that for the heat denaturation, indicating that the mechanism of the cold denaturation of SSI is different from a simple cold denaturation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cold denaturation and heat denaturation of Streptomyces subtilisin inhibitor. 2. 1H NMR studies

Structural transitions of the protein Streptomyces subtilisin inhibitor (SSI) from the native state to the cold-denatured and heat-denatured states were studied by 1H NMR spectroscopy in the temperature range from -10 to 60 degrees C in the acidic pH range. Assignments of some of the 1H NMR signals of SSI in the cold-denatured and heat-denatured states were performed by a combined use of selective deuteration and site-directed mutagenesis. Throughout the pH range from 2.1 to 3.1, both transitions were cooperative and basically only three distinct spectra corresponding to structures in the cold-denatured, native, and heat-denatured states were detected. In the cold-denatured state, the side-chain signals of Met73, His106, at least one Val, and two Leu were observed at distinctly shifted positions from those for a random-coiled structure, suggesting the formation of a tertiary structure, while those of Met70, His43, and Ala2 were observed at positions for a random-coiled structure. This tertiary structure in the cold-denatured state is entirely different from that in the native state, as some amino acid residues exposed to the solvent in the native state (e.g., Met73, His106) are buried while those sequestered in the native state (e.g., His43) are exposed. In the heat-denatured state, however, most 1H NMR signals were observed at random-coiled positions, indicating that there is much less tertiary structure in the heat-denatured state than in the cold-denatured state. At pH values below 2.09, a structural transition was observed from the cold-denatured state to the heat-denatured state without passing through the native state.(ABSTRACT TRUNCATED AT 250 WORDS)     

Urea-induced denaturation of apolipoprotein serum amyloid A reveals marginal stability of hexamer

Serum Amyloid A (SAA) is an acute phase reactant protein that is predominantly found bound to high-density lipoprotein in plasma. Upon inflammation, the plasma concentration of SAA can increase dramatically, occasionally leading to the development of amyloid A (AA) amyloidosis, which involves the deposition of SAA amyloid fibrils in major organs. We previously found that the murine isoform SAA2.2 exists in aqueous solution as a hexamer containing a central channel. Here we show using various biophysical and biochemical techniques that the SAA2.2 hexamer can be totally dissociated into monomer by approximately 2 M urea, with the concerted loss of its alpha-helical structure. However, limited trypsin proteolysis experiments in urea showed a conserved digestion profile, suggesting the preservation of major backbone topological features in the urea-denatured state of SAA2.2. The marginal stability of hexameric SAA2.2 and the presence of residual structure in the denatured monomeric protein suggest that both forms may interconvert in vivo to exert different functions to meet the various needs during normal physiological conditions and in response to inflammatory stimuli.