Assembly of Oxyhemoglobin from Isolated α and β Chains
1983 年 93 巻 4 号 p. 1159-1166
詳細
1983 年 93 巻 4 号 p. 1159-1166
The kinetics of assembly of oxyhemoglobin from isolated α and β 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 a and β monomers into αβ 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 2.8×10-3 s-1 (independent of the protein concentration), which suggested that the slow phase reflected the dissociation of self-associated β 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 β monomer which was calculated from the self-association constant of β chain. The constant was estimated to be 2.4×1016 M-3 by frontal gel chromatography on the assumption that the isolated β chain was in a monomer-tetramer equilibrium. This result indicated that the rapid phase could be ascribed to the combining of a and β 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 β chain and a second-order combination reaction of α and β monomers. The analysis yielded a second-order rate constant of 7.5×105M-1•S-1.
These results suggest that α and β monomers rapidly combine to form αβ dimer followed by assembly into Hb, though at high protein concentration the rate of the assembly is limited by the dissociation of self-associated β chain.
