Van Kempen L H, Breepoel P M, Kreuzer F
Respir Physiol. 1975 Mar;23(2):223-41. doi: 10.1016/0034-5687(75)90062-6.
emoglobin-bound CO2 was estimated by a procedure first described by Rossi-Bernardi et al. (1969) in which the carbamate compound is stabilized by rapid mixing with alkali and then separated from other CO2 constituents in solution by gel filtration and ion-exchange chromatography. Carbamate equilibrium of bovine hemoglobin was studied at constant PCO2 (44 mm Hg) and varying pH as well as at constant pH (7.4) and varying PCO2 (ionic strength 0.18, temperature 37.0 degrees C). The difference in Z (deltaZ) between hemoglobin and oxyhemoglobin appeared to be 0.11 plus or minus 0.04 (pH=7.40; PCO2=44 mm Hg) i.e. about half the value observed in human hemoglobin. DeltaZ was shown to account completely for the difference in CO2 content (CCO2) between hemoglobin and oxyhemoglobin when in total equilibrium with CO2. Carbamate determinations on bovine hemoglobin specifically modified at all terminal amino groups (double-blocked carbamylated derivative) did not show any CO2 binding at all, thus giving a final proof for the exclusive role of the terminal amino groups in CO2 binding under physiological conditions. Attempts to calculate the ionization constant (Kz) and the carbamate equilibrium constant (Kc) of the terminal amino groups failed, suggesting that both terminal groups are not equivalent in their CO2 binding properties. This was confirmed by the fact that carbamate data obtained at constant PCO2 and varying pH fitted binding curves derived from two sets of independent but non-equivalent binding sites. Association constants for both kinds of binding sites appeared to differ by a factor of at least 3 in hemoglobin and of about 10 in oxyhemoglobin. From determinations of hemoglobin-bound CO2 and CO2 content of hemoglobin and oxyhemoglobin solutions in total equilibrium with CO2, the apparent first dissociation constant of carbonic acid was calculated as 5.71 plus or minus 0.0061 pH and found to be independent of the oxygenation state of hemoglobin. In contrast with hemoglobin of other species bovine hemoglobin appeared to be not influenced by the presence of 2.3-diphosphoglycerate as far as its CO2-binding properties are concerned.
与血红蛋白结合的二氧化碳是通过Rossi - Bernardi等人（1969年）首次描述的方法估算的。在该方法中，氨基甲酸盐化合物通过与碱快速混合来稳定，然后通过凝胶过滤和离子交换色谱法与溶液中的其他二氧化碳成分分离。在恒定的PCO2（44毫米汞柱）和不同的pH值下，以及在恒定的pH值（7.4）和不同的PCO2（离子强度0.18，温度37.0摄氏度）下，研究了牛血红蛋白的氨基甲酸盐平衡。血红蛋白和氧合血红蛋白之间的Z值差异（ΔZ）似乎为0.11±0.04（pH = 7.40；PCO2 = 44毫米汞柱），即约为在人血红蛋白中观察到的值的一半。当与二氧化碳完全平衡时，ΔZ被证明完全解释了血红蛋白和氧合血红蛋白之间二氧化碳含量（CCO2）的差异。对所有末端氨基均经过特异性修饰的牛血红蛋白（双封闭氨基甲酰化衍生物）进行的氨基甲酸盐测定根本未显示任何二氧化碳结合，从而最终证明了在生理条件下末端氨基在二氧化碳结合中的独特作用。计算末端氨基的电离常数（Kz）和氨基甲酸盐平衡常数（Kc）的尝试失败了，这表明两个末端基团在其二氧化碳结合特性方面并不等同。在恒定PCO2和不同pH值下获得的氨基甲酸盐数据符合源自两组独立但不等同的结合位点的结合曲线，这一事实证实了这一点。两种结合位点的缔合常数在血红蛋白中似乎相差至少3倍，在氧合血红蛋白中相差约10倍。通过测定与二氧化碳完全平衡的血红蛋白溶液和氧合血红蛋白溶液中与血红蛋白结合的二氧化碳和二氧化碳含量，计算出碳酸的表观一级解离常数为5.71±0.0061pH，并且发现其与血红蛋白的氧合状态无关。就其二氧化碳结合特性而言，与其他物种的血红蛋白不同，牛血红蛋白似乎不受2,3 - 二磷酸甘油酸存在的影响。
