Gros G, Rollema H S, Forster R E
J Biol Chem. 1981 Jun 10;256(11):5471-80.
We have investigated the carbamate equilibrium of human adult hemoglobin, human cord blood hemoglobin, methemoglobin, and carbamylated hemoglobin using a stopped flow, rapid reaction pH apparatus described previously. The carbamate formation of human adult hemoglobin at 37 degrees C and ionic strength 0.15 was measured at pH values ranging from 6.2 to 8.8 and at CO2 partial pressures between 15 and 140 Torr. From experiments with unmodified hemoglobin as well as with hemoglobin specifically carbamylated at the four NH2 termini, it was found that already at pH 8, the epsilon-amino groups contribute significantly to carbamate formation in addition to the alpha-amino groups. At pH 8.5, about 70% of the total carbamate is due to epsilon-amino groups. The carbamate formation of alpha- and epsilon-amino groups can be suppressed by complete carbamylation of the hemoglobin. The results obtained from human adult deoxy- and oxyhemoglobin were used to calculate the equilibrium constants governing carbamate formation of these hemoglobins: Kc, the carbamate equilibrium constant, Kz, the R-NH2 ionization constant, and n, the number of binding sites per hemoglobin tetramer. Accordingly, two types of alpha-amino groups, each comprising two groups per tetramer, participate in carbamate formation of deoxyhemoglobin, one of low CO2 affinity (pKc = 5.2; pKz = 7.1; n = 2) and one of high CO2 affinity (pKc = 4.4; pKz = 6.1; n = 2). The pKz values derived from carbamate measurements agree within experimental error with figures obtained by difference titration of unmodified and specifically carbamylated hemoglobin. In addition to the alpha-amino groups, 15 epsilon-amino groups with pKc = 5.0 and pKz = 9.8 form carbamate in deoxyhemoglobin. In oxyhemoglobin, the carbamate data could be fitted with only two similar alpha-amino groups per tetramer in addition to 15 epsilon-amino groups, the latter with pKc = 4.7 and pKz = 10.2. The difference titration of the alpha-amino groups of oxyhemoglobin showed abnormal titration behavior of the beta-chain alpha-NH2. The pKc and pKz values obtained for the epsilon-amino groups of unmodified hemoglobin also provide a good description of the carbamate formed by hemoglobin specifically carbamylated at the four alpha-amino groups. The oxylabile carbamate, according to these results and in agreement with earlier reports, is only formed by alpha-amino groups; it amounts to 0.18 mol/mol of hemoglobin monomer at physiological conditions of pH 7.2 and pCO2 = 40 Torr. The amount of CO2 bound by methemoglobin equals that of oxyhemoglobin. Experiments carried out in the presence of 2,3-diphosphoglycerate provided evidence for competitive binding of CO2 and diphosphoglycerate to adult and cord blood oxy- as well as deoxyhemoglobin, the sites of competition probably being alpha-amino groups.
我们使用先前描述的停流快速反应pH装置，研究了成人血红蛋白、人脐血血红蛋白、高铁血红蛋白和氨甲酰化血红蛋白的氨基甲酸酯平衡。在37℃和离子强度0.15条件下，于pH值6.2至8.8以及二氧化碳分压15至140托范围内，测定了成人血红蛋白的氨基甲酸酯形成情况。通过对未修饰血红蛋白以及在四个NH2末端特异性氨甲酰化的血红蛋白进行实验发现，在pH 8时，除α-氨基外，ε-氨基对氨基甲酸酯的形成也有显著贡献。在pH 8.5时，约70%的总氨基甲酸酯归因于ε-氨基。血红蛋白的完全氨甲酰化可抑制α-和ε-氨基的氨基甲酸酯形成。从成人脱氧血红蛋白和氧合血红蛋白获得的结果用于计算这些血红蛋白氨基甲酸酯形成的平衡常数：Kc，氨基甲酸酯平衡常数；Kz，R-NH2电离常数；n，每个血红蛋白四聚体的结合位点数。因此，两种类型的α-氨基（每个四聚体各含两个基团）参与脱氧血红蛋白的氨基甲酸酯形成，一种对二氧化碳亲和力低（pKc = 5.2；pKz = 7.1；n = 2），另一种对二氧化碳亲和力高（pKc = 4.4；pKz = 6.1；n = 2）。由氨基甲酸酯测量得出的pKz值在实验误差范围内与通过未修饰和特异性氨甲酰化血红蛋白的差值滴定获得的数据一致。除α-氨基外，15个ε-氨基（pKc = 5.0，pKz = 9.8）在脱氧血红蛋白中形成氨基甲酸酯。在氧合血红蛋白中，除15个ε-氨基（pKc = 4.7，pKz = 10.2）外，氨基甲酸酯数据仅能用每个四聚体两个类似的α-氨基拟合。氧合血红蛋白α-氨基的差值滴定显示β链α-NH2的滴定行为异常。未修饰血红蛋白ε-氨基的pKc和pKz值也很好地描述了在四个α-氨基处特异性氨甲酰化的血红蛋白形成的氨基甲酸酯。根据这些结果并与早期报告一致，对氧不稳定的氨基甲酸酯仅由α-氨基形成；在pH 7.2和pCO2 = 40托的生理条件下，其含量为0.18摩尔/摩尔血红蛋白单体。高铁血红蛋白结合的二氧化碳量与氧合血红蛋白相等。在2,3-二磷酸甘油酸存在下进行的实验证明，二氧化碳和二磷酸甘油酸与成人和脐血氧合血红蛋白以及脱氧血红蛋白存在竞争性结合，竞争位点可能是α-氨基。
