| Abstract: | The kinetics of assembly of oxygenated hemoglobin from isolated alpha and beta chains was investigated under various buffer conditions by use of a circular dichroism (CD) stopped-flow apparatus. The difference CD spectra of hemoglobin against its constituent chains were independent of the buffer conditions, while the time courses of the Soret CD after mixing equimolar amounts of the alpha and beta chains changed with the buffer conditions. The time courses were analyzed on the basis of a scheme which included a monomer-tetramer equilibrium of the beta chain (beta 4 in equilibrium 4 beta), dissociation of the beta 4 (beta 4 leads to 4 beta), and a second-order combination of alpha and beta monomers (alpha + beta leads to alpha beta). The analysis showed that buffer conditions affected the dissociation of the beta 4 rather than the monomer combination: The rate of the dissociation of the beta 4 accelerated with decreasing phosphate concentration, while the rate of the monomer combination was less sensitive to the phosphate concentration. This result indicates that the stability of the beta 4 depends on the phosphate concentration. It was furthermore suggested that the inorganic phosphate was bound to the beta 4 with an association constant of 133 M-1 and a Hill coefficient of 1.2. |
