Rieder R F
J Clin Invest. 1970 Dec;49(12):2369-76. doi: 10.1172/JCI106456.
Several unstable mutant hemoglobins have alterations which affect areas of the molecule involved in the attachment of heme to globin. Loss of heme from globin has been demonstrated during the denaturation of some of these unstable mutants. The importance of heme ligands for the stability of hemoglobin was illustrated in the present experiments on the denaturation of several hemoglobins and hemoglobin derivatives by heat, oxidative dyes, and alkali. Heating of normal hemolysates diluted to 4 g of hemoglobin per 100 ml at 50 degrees C for 20 hr in 0.05 M sodium phosphate, pH 7.4, caused precipitation of 23-54% of the hemoglobin. Dialysis against water or dilution of the sample decreased denaturation to 12-20%. Precipitation was decreased to less than 3.5% by the presence of 0.015 M potassium cyanide. Increasing the ionic strength of the medium increased precipitation. Cyanide prevented the formation of inclusion bodies when red cells containing unstable hemoglobin Philly, beta35 tyr --> phe, were incubated with the redox dye new methylene blue. Conversion to methemoglobin increased the rate of alkali denaturation of hemoglobin but the presence of potassium cyanide returned the denaturation rate to that of ferrohemoglobin. The ability of cyanide to decrease heat precipitation of hemoglobin may depend on a dimeric or tetrameric state of the hemoglobin molecule. Purified beta-chains, which exist as tetramers, were stabilized but purified monomeric alpha-chains were not rendered more heat resistant by the ligand. Stabilization of hemoglobin by cyanide required binding of the ligand to only one heme of an alphabeta-dimer. Hemoglobin Gun Hill, an unstable molecule with heme groups present only on the alpha-chains was quite heat stable in the presence of cyanide. The binding of cyanide to the iron atom in methemoglobin is thought to be associated with increased planarity of the heme group and increased stability of the heme-globin complex. The stabilizing effect of cyanide in the above experiments suggests that Heinz body formation, heat precipitation of hemoglobin, and the increased alkali denaturation of methemoglobin depend on changes of heme-globin binding.
几种不稳定的突变血红蛋白存在一些改变，这些改变影响了血红素与珠蛋白结合部位的分子区域。在某些不稳定突变体变性过程中，已证实珠蛋白中的血红素会丢失。在当前关于几种血红蛋白和血红蛋白衍生物经热、氧化染料及碱变性的实验中，说明了血红素配体对血红蛋白稳定性的重要性。将正常溶血产物稀释至每100毫升含4克血红蛋白，于50℃在pH 7.4的0.05M磷酸钠中加热20小时，会导致23% - 54%的血红蛋白沉淀。对水透析或稀释样品可使变性降至12% - 20%。0.015M氰化钾的存在可使沉淀降至低于3.5%。增加介质的离子强度会增加沉淀。当含不稳定血红蛋白Philly（β35 tyr→phe）的红细胞与氧化还原染料新亚甲蓝孵育时，氰化物可防止包涵体形成。转化为高铁血红蛋白会增加血红蛋白的碱变性速率，但氰化钾的存在可使变性速率恢复到亚铁血红蛋白的水平。氰化物降低血红蛋白热沉淀的能力可能取决于血红蛋白分子的二聚体或四聚体状态。以四聚体形式存在的纯化β链得到了稳定，但纯化的单体α链并未因该配体而变得更耐热。氰化物对血红蛋白的稳定作用需要该配体仅与αβ二聚体的一个血红素结合。血红蛋白Gun Hill是一种不稳定分子，其血红素基团仅存在于α链上，在氰化物存在下相当耐热。氰化物与高铁血红蛋白中铁原子的结合被认为与血红素基团平面度增加以及血红素 - 珠蛋白复合物稳定性增加有关。上述实验中氰化物的稳定作用表明，亨氏小体形成、血红蛋白热沉淀以及高铁血红蛋白碱变性增加取决于血红素 - 珠蛋白结合的变化。
