Koichiro Aoki

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 α-helix, β-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 α-helix decreased above 30°C and those of β-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.

Secondary structure changes of disulfide bridge-cleaved bovine serum albumin in solutions of urea, guanidine hydrochloride, and sodium dodecyl sulfate

Abstract Secondary structure of bovine serum albumin, disulfide bridges of which were reduced and blocked by iodoacetamide, was examined in solutions of urea, guanidine hydrochloride, and sodium dodecyl sulfate. The profile of structural changes in the reduced albumin was compared with that in the unreduced protein with the intact disulfide bridges. The relative proportions of α-helix, β-structure, and random coil were estimated by simulating a mixed circular dichroism (CD) spectrum of reference spectra of the corresponding structures to the experimentally obtained spectrum of the protein. The helical proportion was 25% for the reduced albumina dn the proportion increased up to 50% in a dodecyl sulfate solution. On the other hand, the helical proportion is 66% for the unreduced protein and it decreases down to 50% by the addition of the identical surfactant [K. Takeda et al., J. Colloid Interface Sci. 117, 120 (1987)]. The helical proportions of both the reduced and the unreduced proteins decreased in the presence of urea and guanidine. the unreduced albumin resisted the unfolding of the helix below 4 M urea and a 1.5 M guanidine. In contrast, the helical proportion in the reduced protein began to decrease even in lower concentrations of the two denaturants.

The effect of pressure on solubilities of ionic surfactants in water

Abstract The solubilities of ionic surfactants, sodium dodecylsulfate and dodecylamine hydrobromide, have been determined measuring the electric conductivity at pressures up to 3000 atm and at several temperatures in the range 7 to 35°C. Influences of pressure and temperature on the solubility were similar on these ionic surfactants. With increase in pressure, the solubility decreases abruptly until the “critical solution pressure” Pc, but decreases gradually above this pressure. The critical micelle concentration (CMC) has also been determined under pressure. The relation between the solubility, the CMC, and the pressure is given by a phase diagram. The CMC vs pressure curve and the solubility vs pressure curve cross at a point Q. (The pressure at which the point Q exists is designated Pc.) The point Q is found to be the Krafft point at the pressure Pc. Micelles above this pressure are in a state of superpressing and metastable. The pressure dependences of the solubility and of the CMC are considered thermodynamically. Comparisons are made among the partial molal volumes of surfactant in micellar state ( V m ), in singly dispersed state ( V s ) and in hydrated solid state ( V c ).

Bovine serum albumin exposed to high pressure

Abstract Pressures of 1000–6000 kg/cm 2 were applied to a solution of bovine serum albumin for 15–60 min at 15–40°. The bovine serum albumin solution at pH 8.9 was compressed, for instance, at 3000 kg/cm 2 for 60 min at 35°. The product was resolved into five zones by acrylamide gel electrophoresis, and into two main and a trace boundaries by the ultracentrifuge. When results by the ultracentrifuge and those by gel electrophoresis at various gel concentrations were combined, it was found that a modified monomer, a dimer and probably higher aggregates were formed. A pressure-temperature curve was constructed; the pressure denaturation occurred in the upper region of the curve. The heat-denatured bovine serum albumin and pressure-denatured bovine serum albumin gave similar patterns in the ultracentrifuge, and gave zones with the same mobilities in gel electrophoresis. The mechanism of heat denaturation is interpreted to be identical with that of pressure denaturation, at least in the initial steps. High pressure lowered the temperature at which heat denaturation began to occur. The dimerization was irreversible. Direct measurement of the electric conductivity under high pressure revealed that another kind of aggregation, which is reversible, occurred at higher pressures than 4000 kg/cm 2 at 25–30°.

Isolation of domain-sized fragments of bovine serum albumin by limited peptic digestion and their secondary structural changes in solutions of urea, guanidine hydrochloride, and sodium dodecyl sulfate

Abstract Three fragments of bovine serum albumin (BSA) by limited peptic hydrolysis were isolated and their positions in the parent molecule were identified on the basis of the analyses of plural amino acid residues at N- and C-terminals of each fragment. The sequences of the three fragments correspond to residue Nos. 1–306, 193–368, and 383–511. The molecular weights of the three fragments were determined to be 34,900, 20,100, and 14,700 from their sequences. As is well known, BSA consists of three domains, each comprising three subdomains (disulfide double loops) and containing about 190 amino acid residues with a molecular weight of approximately 22,000. The present three fragments approximately corresponded to regions representing 1.5, 1, and 0.5 domains of BSA based on their positions in the parent molecule and their molecular weights. The relative proportions of a-helix, β-structure, and disordered structure in conformations of the fragments were determined by the curve-fitting method of circular dichroism (CD) spectra. The a-helical portions occupied 50–60% in these fragments. Most of the helices in the intact BSA molecule appeared to remain after digestion into the fragments. The helical proportions of the fragments decreased stepwise in SDS solutions in the same way as intact BSA. This suggests that the decrease in helical content of BSA might represent the sum of the reductions in helical content of the three domains by SDS binding. The helices in the fragments resisted structural changes by dilute urea and guanidine in a manner similar to that of BSA.

Effect of metallic cations on human serum: Study by starch-gel electrophoresis: II. Effect of Hg++, Cr+++, Ag+, Ni++, Cd++, Zn++, Ba++, Mg++, Al+++, and Fe+++

Abstract The effect of Hg ++ and of Cr +++ on human serum was pronounced. Almost all the protein components were precipitated when serum was made 8 m M In Hg ++ or in Cr +++ . Only acid α 1 -glycoprotein was unusually stable toward Hg ++ . A serum kept in the cold for A month In The presence Of 55 m M Ag + gave a pattern with A series of aggregates of albumin. The effect of Ni ++ on human serum was less pronounced than the effect of Pb ++ , Cu ++ , and Hg ++ . The amount of γ-globulin was minimal at 10 m M of Ni ++ with The ph of the serum at 7.9. the effect of Cd ++ and Zn ++ was also mild. the serum produced larger amounts of precipitate with Cd ++ or Zn ++ at room temperature than in the cold. This behavior is occasioned by albumin which precipitates more extensively at room temperature. the effect of Ba ++ and Mg ++ was less pronounced than that of Cd ++ and Zn ++ . Only pre-albumins were precipitated at 0.1 M Ba ++ . The effect of Mgso 4 resembled that of (NH 4 ) 2 SO 4 . γ-Globulin and acid α 1 -glycoprotein were stable in a wider range of Ph and Al +++ Concentration.