Abstract
Dissociation kinetics of Limulus polyphemus hemocyanin, induced by calcium removal was studied by the stopped-flow X-ray scattering (SFXS) method and fluorescence stopped-flow method at various pH. Between pH 6.3 and 8.4, the time course of the kinetics showed a lag followed by a single exponential decay, while it obeyed a single exponential decay without a lag at pH 8.8. To analyze the process, equations to calculate zero angle intensity, I 0, and radius of gyration, R g, were presented. Simulated curves from the equations fitted the data well. Concerning either of I 0 and R g, the lag-time decreased with increasing EDTA concentration, while the apparent rate constant of the dissociation, k app, increased. Based on the value of I 0 and R g at infinite time, it is most likely that the observed kinetics between pH 6.3 and 7.9 reflects the dissociation of the protein (48-mer) into two equal halves (24-mer), for which a sequential two-step reaction was suggested. On the other hand the kinetics above pH 8.0 indicated the dissociation into constituent subunits.
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