Binding of urate and caffeine to hemocyanin of the lobster Homarus vulgaris (E.) as studied by isothermal titration calorimetry

Hemocyanin serves as an oxygen carrier in the hemolymph of the European lob ster Homarus vulgaris. The oxygen binding behavior of the pigment is modula ted by metabolic effecters such as lactate and urate. Urate and caffeine bi nding to 12-meric hemocyanin (H. vulgaris) was studied using isothermal tit ration calorimetry (ITC). Binding isotherms were determined for fully oxyge nated hemocyanin between PPI 7.55 and 8.15. No pH dependence of the binding parameters could be found for either effector. Since the magnitude of the Bohr effect depends on the urate concentration, the absence of any pH depen dence of urate and caffeine binding to oxygenated hemocyanin suggests two c onformations of the Figment under deoxygenated conditions. Urate binds to t wo identical binding sites (n = 2) each with a microscopic binding constant K of 8500 M-1 and an enthalpy change Delta H degrees of -32.3 kcal mol(-1) . Caffeine binds cooperatively to hemocyanin with two microscopic binding c onstants: K-1 = 14 100 M-1 and K-2 = 40 400 M-1. The corresponding enthalpy changes in binding are as follows: Delta H(1)degrees = -23.3 kcal mol(-1) and Delta H(2)degrees = -27.1 kcal mol(-1). The comparison of urate and caf feine binding to the oxygenated pigment indicates the existence of two prot ein conformations for oxygen-saturated hemocyanin. Since effector binding i s not influenced by protons, four different conformations are required to c reate a convincing explanation for caffeine and urate binding curves. This was predicted earlier on the basis of the analysis of oxygen binding to lob ster hemocyanin, employing the nesting model.