Menze M A, Hellmann N, Decker H, Grieshaber M K
Institut für Zoophysiologie, Heinrich-Heine Universität, Universitätsstrasse 1, 40225 Düsseldorf, Germany.
Biochemistry. 2000 Sep 5;39(35):10806-11. doi: 10.1021/bi000008l.
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.
血蓝蛋白是欧洲龙虾（Homarus vulgaris）血淋巴中的一种氧载体。该色素的氧结合行为受乳酸和尿酸盐等代谢效应物的调节。使用等温滴定量热法（ITC）研究了尿酸盐和咖啡因与12聚体血蓝蛋白（欧洲龙虾）的结合。测定了pH值在7.55至8.15之间的完全氧合血蓝蛋白的结合等温线。两种效应物的结合参数均未发现pH依赖性。由于波尔效应的大小取决于尿酸盐浓度，尿酸盐和咖啡因与氧合血蓝蛋白的结合不存在任何pH依赖性，这表明在脱氧条件下该色素有两种构象。尿酸盐结合到两个相同的结合位点（n = 2），每个位点的微观结合常数K为8500 M⁻¹，焓变ΔH°为 -32.3 kcal mol⁻¹。咖啡因与血蓝蛋白协同结合，有两个微观结合常数：K(1) = 14100 M⁻¹和K(2) = 40400 M⁻¹。相应的结合焓变如下：ΔH°(1) = -23.3 kcal mol⁻¹和ΔH°(2) = -27.1 kcal mol⁻¹。尿酸盐和咖啡因与氧合色素结合的比较表明，氧饱和血蓝蛋白存在两种蛋白质构象。由于效应物结合不受质子影响，需要四种不同的构象才能对咖啡因和尿酸盐的结合曲线做出令人信服的解释。这是早期基于嵌套模型对龙虾血蓝蛋白氧结合分析所预测的。
