Allosteric Modulation of Haemocyanin Oxygen-affinity by L-Lactate And Urate in the Lobster Homarus Vulgaris: II. Characterization of Specific Effector Binding Sites

The haemocyanin of Homarus vulgaris possesses specific binding sites for L-lactate and urate, two allosteric modulators of haemocyanin oxygen-affinity. The affinities for both ligands have been determined. The dissociation constants, KD, are 0.87±0.26mmoll−1 for L-lactate and 0.03±0.01mmoll−1 for urate at 15°C and pH 8.0. The affinity of the haemocyanin is about 40 times larger for urate than for L-lactate. The stoichiometry of the binding is two ligands per dodecamer in both cases. l-Lactate does not compete with urate for its binding site and vice versa, indicating that the ligand binding sites are independent of each other. The specificity of urate binding to haemocyanin was investigated in competition experiments with allantoin, caffeine and hypoxanthine. The purine derivatives caffeine and hypoxanthine reduce the binding of urate to haemocyanin, whereas allantoin has no effect. Thus, the purine ring system seems to be essential for the binding of urate to haemocyanin. Note: To whom reprint requests should be addressed.