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 lobster Homarus vulgaris. The oxygen binding behavior of the pigment is modulated by metabolic effectors such as lactate and urate. Urate and caffeine binding to 12-meric hemocyanin (H. vulgaris) was studied using isothermal titration calorimetry (ITC). Binding isotherms were determined for fully oxygenated hemocyanin between pH 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 dependence of urate and caffeine binding to oxygenated hemocyanin suggests two conformations of the pigment under deoxygenated conditions. Urate binds to two identical binding sites (n = 2) each with a microscopic binding constant K of 8500 M(-1) and an enthalpy change DeltaH degrees of -32.3 kcal mol(-1). Caffeine binds cooperatively to hemocyanin with two microscopic binding constants: K(1) = 14 100 M(-1) and K(2) = 40 400 M(-1). The corresponding enthalpy changes in binding are as follows: DeltaH degrees (1) = -23.3 kcal mol(-1) and DeltaH degrees (2) = -27.1 kcal mol(-1). The comparison of urate and caffeine binding to the oxygenated pigment indicates the existence of two protein conformations for oxygen-saturated hemocyanin. Since effector binding is not influenced by protons, four different conformations are required to create a convincing explanation for caffeine and urate binding curves. This was predicted earlier on the basis of the analysis of oxygen binding to lobster hemocyanin, employing the nesting model.
Menze
M A
MA
Institut für Zoophysiologie, Heinrich-Heine Universität, Universitätsstrasse 1, 40225 Düsseldorf, Germany. mmenze@hotmail.com
Hellmann
N
N
Decker
H
H
Grieshaber
M K
MK
D000818:Animals; D001665:Binding Sites; D002021:Buffers; D002110:Caffeine / Q000737:chemistry / Q000378:metabolism*; D002151:Calorimetry / Q000379:methods; D006433:Hemocyanins / Q000737:chemistry / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008297:Male; D008121:Nephropidae / Q000737:chemistry / Q000378:metabolism*; D013816:Thermodynamics; D017186:Titrimetry; D014325:Tromethamine; D014527:Uric Acid / Q000737:chemistry / Q000378:metabolism*
D002021:Buffers; D014325:Tromethamine; D014527:Uric Acid; D002110:Caffeine; D006433:Hemocyanins