Wind M, Stern A, Simon S, Law L
Biochemistry. 1976 Nov 16;15(23):5161-7. doi: 10.1021/bi00668a033.
The pH dependence of several functional properties of human fetal and adult hemoglobins have been studied to determine the relative stabilities of the high and low affinity (R and T) quaternary conformations of the two proteins under different conditions. Fetal aqumethemoglobin undergoes changes in sulfhydryl reactivity, absorption spectrum, and circular dichroism in the presence of insitol hexaphospahte which are consistent with a transition from the R to T quaternary state, but only at pH values below 6.8. In adult hemoglobin this transition can be induced pH values below 7.2. Even in the absence of phosphates, the ultraviolet (uv) circular dichroism spectrum of fetal aquomethemoglobin at low pH indicates the presence of some T conformation. The initial value for the second-order rate constant for combination of fetal deoxyhemoglobin with carbon monoxide is comparable to that for adult hemoglobin in the absence of organic phosphates and is not reduced by organic phosphates as much as that for the adult protein. The apparent first-order rate constant for dissociation of CO from fully liganded fetal hemoglobin, measured by replacement with NO, increases threefold in the absence of organic phosphates, and fourfold in the presence of organic phosphates, with decreasing pH; the midpoint of the pH dependent transition occurs around 6.8. A similar increase in the apparent first-order rate constant for O2 dissociation as measured by replacement with CO, can also be seen with decreasing pH. NO-hemoglobin F can be converted to the T state even when fully liganded simply by lowering the pH, as judged by uv circular dichroism, visible difference spectrum in the region of the alpha and beta bands, and a dramatic increase in the rate of NO dissociation, measured by replacement with CO in the presence of dithionite. These results are all consistent with a model for fetal hemoglobin in which the organic phosphate site may be functionally weakened by replacement of a residue involved in ionic interactions with the negatively charged phosphate groups, but in which the low affinity T conformation is intrinsically more stable than that of adllt hemoglobin. According to this model,the differences between fetal and adult hemoglobin can be accounted for primarily in terms of the relative stabilities of R and T conformations in each of the proteins with differences in the intrinsic properties of the individual conformations contributing effects of only secondary importance.
研究了人胎儿血红蛋白和成人血红蛋白几种功能特性的pH依赖性，以确定这两种蛋白质在不同条件下高亲和力和低亲和力（R态和T态）四级构象的相对稳定性。胎儿高铁血红蛋白在肌醇六磷酸存在下，其巯基反应性、吸收光谱和圆二色性会发生变化，这与从R态到T态的四级状态转变一致，但仅在pH值低于6.8时发生。在成人血红蛋白中，这种转变在pH值低于7.2时即可诱导。即使在没有磷酸盐的情况下，低pH值下胎儿高铁血红蛋白的紫外圆二色光谱也表明存在一些T构象。胎儿脱氧血红蛋白与一氧化碳结合的二级速率常数的初始值与没有有机磷酸盐时成人血红蛋白的相当，并且不像成人蛋白质那样被有机磷酸盐显著降低。通过用NO替代来测量，从完全配位的胎儿血红蛋白中解离CO的表观一级速率常数，在没有有机磷酸盐时增加三倍，在有有机磷酸盐时随着pH降低增加四倍；pH依赖性转变的中点出现在6.8左右。通过用CO替代来测量，随着pH降低，O2解离的表观一级速率常数也有类似增加。通过紫外圆二色性、α和β带区域的可见差光谱以及在连二亚硫酸盐存在下用CO替代测量的NO解离速率的显著增加来判断，即使完全配位，仅通过降低pH值，NO-血红蛋白F也可转变为T态。这些结果都与胎儿血红蛋白的模型一致，在该模型中，有机磷酸盐位点可能因参与与带负电荷的磷酸基团离子相互作用的残基被取代而在功能上减弱，但低亲和力的T构象本质上比成人血红蛋白的更稳定。根据该模型，胎儿血红蛋白和成人血红蛋白之间的差异主要可以用每种蛋白质中R态和T态构象的相对稳定性来解释，而各个构象内在性质的差异仅起次要作用。
