SummaryThe thermal stability of free and immobilized a-chymotrypsin was investigated experimentally and theoretically. The inactivation process of free a-chymotrypsin was analyzed with a kinetic model which included a first-order reaction process and autolysis. The effects of ionic strength, Ca2+ concentration, and temperature are discussed here in terms of the estimated kinetic parameters included in this model. The inactivation process of a-chymotrypsin immobilized onto various supports by several methods was investigated. The contribution of thermal denaturation and autolysis to the inactivation depended upon the method of immobilization. To interpret quantitatively the non-first-order thermal denaturation process of the immobilized enzyme, a model in which the heterogeneity of the immobilized enzyme was taken into account is proposed.

The reconstitution reaction of deoxy hemoglobin (Hb) tetramer from isolated alpha and beta chains was kinetically studied by measuring circular dichroism (CD) changes in the Soret and the ultraviolet regions and optical absorbance change in the Soret region with a stopped-flow apparatus. The CD change in the Soret region was a fast reaction, followed by that in the ultraviolet region and the absorbance change in the Soret region. This fast reaction followed a simple second-order rate law with a rate constant of 6.4 x 10(5) M-1 . s-1. These results indicated that the combination of alpha and beta monomers into an alpha beta dimer accompanied the CD change in the Soret region and was the rate-limiting step of the overall reconstitution reaction of deoxyHb tetramer. On the other hand, the CD change in the ultraviolet region was ascribed to the combination of two alpha beta dimers into an Hb tetramer. The absorbance change in the Soret region was related to both the combination of alpha and beta monomers and that of two alpha beta dimers. From the analyses of these reactions the rate constant of the combination of two alpha beta dimers was determined to be 1.0 x 10(6) M-1 . s-1.

The kinetics of assembly of oxyhemoglobin from isolated alpha and beta chains was investigated by the use of a circular dichroism (CD) stopped-flow apparatus. The CD change in the Soret region was observed after mixing equivalent concentrations of the isolated chains. The intensity of the CD change was proportional to the protein concentration. The dilution of the isolated chains did not produce any detectable CD change. These results indicate that the CD change could be ascribed to the combination of alpha and beta monomers into alpha beta dimer. The time courses of the CD change showed a rapid phase and a slow phase. The slow phase was a first-order reaction with a rate constant of 2.8 X 10(-3) s-1 (independent of the protein concentration), which suggested that the slow phase reflected the dissociation of self-associated beta chain. The rapid phase depended on the protein concentration: (1) the ratio of the rapid phase to the total CD change decreased with increase in the protein concentration, and (2) the half-life of the rapid phase decreased with increasing protein concentration. The ratio of the rapid phase coincided with the fraction of beta monomer which was calculated from the self-association constant of beta chain. The constant was estimated to be 2.4 X 10(16) M-3 by frontal gel chromatography on the assumption that the isolated beta chain was in a monomer-tetramer equilibrium. This result indicated that the rapid phase could be ascribed to the combining of alpha and beta monomers initially present. Therefore, the half-life of the rapid phase was analyzed on the basis of a scheme which included the monomer-tetramer equilibrium of the beta chain and a second-order combination reaction of alpha and beta monomers. The analysis yielded a second-order rate constant of 7.5 X 10(5) M-1 X S-1. These results suggest that alpha and beta monomers rapidly combine to form alpha beta dimer followed by assembly into Hb, though at high protein concentration the rate of the assembly is limited by the dissociation of self-associated beta chain.

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