Pennathur-Das R, Heath R, Mentzer W, Lubin B
Biochim Biophys Acta. 1984 Dec 7;791(2):259-64. doi: 10.1016/0167-4838(84)90017-7.
Dimethyl adipimidate (DMA), an effective antisickling agent in vitro, reacts with free amino groups producing chemically modified and cross-linked molecules. In this report, we have investigated the effect of cross-linked hemoglobin tetramers on sickle hemoglobin polymerization. Since the extent of cross-linking is pH-dependent, we first compared the solubilities of deoxygenated hemolysates prepared from sickle cells previously treated with dimethyl adipimidate at either pH 7.4 or 8.4. The solubility of the hemolysate increased from 18.6 +/- 0.8 g/dl in the untreated sample to 20.9 +/- 1.5 g/dl (pH 7.4) and to 25.4 +/- 3.0 g/dl (pH 8.4) after dimethyl adipimidate treatment. Removal of cross-linked hemoglobin tetramers from hemolysate obtained from dimethyl adipimidate-treated cells abolished part of this effect; at pH 7.4, the solubility decreased from 20.9 +/- 1.5 to 19.4 +/- 0.2 and at pH 8.4 from 25.4 +/- 3.0 to 21.0 +/- 1.5. However, the ratio of [14C]DMA-labelled hemoglobin in the sol phase to that in the gel phase in the unfractionated hemolysate was 1.17 at pH 7.4 and 1.25 at pH 8.4, suggesting that part of the cross-linked hemoglobin tetramers was incorporated into the gel. In order to further investigate the effect of cross-linked hemoglobin tetramers on sickle hemoglobin polymerization, we separated cross-linked hemoglobin tetramers on a gel-filtration column, prepared mixtures of untreated sickle hemoglobin and cross-linked hemoglobin tetramers and studied the polymerization of these mixtures. The Csat of the untreated hemolysate increased progressively from 18.6 +/- 0.8 to 22.5 +/- 0.8 g/dl with 33% cross-linked hemoglobin tetramers. The hemoglobin concentration in the gel decreased from 43 +/- 1.0 to 33.8 +/- 1.0 g/dl with 33% cross-linked hemoglobin tetramers, while the pellet volume fraction, phi p, increased with and almost approached 1 at 50% cross-linked hemoglobin tetramers. In addition, the sol phase contained a higher molecular weight distribution of cross-linked hemoglobin tetramers than the gel phase. These observations suggest that a loose polymer was formed in the gel phase with a hemoglobin concentration much lower than that of the control. Thus, polymerization of sickle hemoglobin is inhibited by: (1) exclusion of higher molecular weight cross-linked hemoglobin tetramers from the gel, and (2) loose incorporation of cross-linked hemoglobin tetramers into the gel, perhaps preventing lateral packing and formation of tightly ordered fibers.
己二酸二甲酯（DMA）是一种在体外有效的抗镰变剂，它与游离氨基反应生成化学修饰和交联的分子。在本报告中，我们研究了交联血红蛋白四聚体对镰状血红蛋白聚合的影响。由于交联程度依赖于pH值，我们首先比较了在pH 7.4或8.4下用己二酸二甲酯预处理的镰状细胞制备的脱氧溶血产物的溶解度。溶血产物的溶解度从未处理样品中的18.6±0.8 g/dl增加到己二酸二甲酯处理后的20.9±1.5 g/dl（pH 7.4）和25.4±3.0 g/dl（pH 8.4）。从己二酸二甲酯处理的细胞获得的溶血产物中去除交联血红蛋白四聚体消除了部分这种影响；在pH 7.4时，溶解度从20.9±1.5降至19.4±0.2，在pH 8.4时从25.4±3.0降至21.0±1.5。然而，未分级溶血产物中溶胶相[14C]DMA标记的血红蛋白与凝胶相中[14C]DMA标记的血红蛋白的比率在pH 7.4时为1.17，在pH 8.4时为1.25，这表明部分交联血红蛋白四聚体被纳入凝胶中。为了进一步研究交联血红蛋白四聚体对镰状血红蛋白聚合的影响，我们在凝胶过滤柱上分离交联血红蛋白四聚体，制备未处理的镰状血红蛋白和交联血红蛋白四聚体的混合物，并研究这些混合物的聚合。未处理溶血产物的Csat随着33%的交联血红蛋白四聚体逐渐从18.6±0.8增加到22.5±0.8 g/dl。随着33%的交联血红蛋白四聚体，凝胶中的血红蛋白浓度从43±1.0降至33.8±1.0 g/dl，而沉淀体积分数φp增加，在50%的交联血红蛋白四聚体时几乎接近1。此外，溶胶相比凝胶相含有更高分子量分布的交联血红蛋白四聚体。这些观察结果表明，在凝胶相中形成了一种松散的聚合物，其血红蛋白浓度远低于对照。因此，镰状血红蛋白的聚合受到以下抑制：（1）凝胶中排除较高分子量的交联血红蛋白四聚体，以及（2）交联血红蛋白四聚体松散地纳入凝胶中，可能阻止侧向堆积和紧密排列纤维的形成。
