An oxygenation-linked dye binding to Limulus polyphemus hemocyanin

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Abstract
The reaction of L. polyphemus hemocyanin with a dye, bromthymol blue, was examined by equilibrium dialysis, spectrophotometric titration and stopped-flow methods. Oxy-hemocyanin contained 1 binding site per hexamer unit. The dye binding was linked to oxygenation, and the affinity of the dye for the oxy form was .apprx. 10 times as high as that for the deoxy form. The dye increased the O2 affinity of hemocyanin. Hemocyanin showed a simple hyperbolic binding curve in the bromthymol blue titration; the time course of the reaction was generally biphasic. It was inferred from the kinetic analyses that the reaction proceeds in 2 steps. The 1st bimolecular step is characterized by an increase in the apparent pKa of the bound dye; the 2nd unimolecular step by a red shift of the absorption band of the unionized dye. The dye binding to partially oxygenated hemocyanin was examined spectrophotometrically; the fractional change in the binding was ahead of the increase in the average degree of O2 saturation. The structural changes in the hemocyanin which lead to the increased dye affinity take place at an early stage of the ligand binding sequence.