Eisinger J, Flores J, Salhany J M
Proc Natl Acad Sci U S A. 1982 Jan;79(2):408-12. doi: 10.1073/pnas.79.2.408.
The problem of demonstrating hemoglobin binding to the erythrocyte membrane in intact cells was approached by observing the quenching of fluorescent membrane probes by hemoglobin as a function of pH. This quenching was studied by measuring fluorescence intensities and decay rates of membrane-bound donors by both right-angle and front-face fluorometry. The donors included 4,4'-bis(isothiocyano)-2,2'-stilbene disulfonate (DIDS) bound to the band 3 protein and 2-, 6-, and 12-(9-anthroyloxy(stearic acid (2-AS, 6-AS, and 12-AS) in the lipid portion of the membrane. The probe fluorescence is quenched progressively as the intracellular pH is decreased from 7.2 to 5.9 and does not depend on the oxygenation state. Since the fluorescence characteristic of the DIDS and x-AS probes in ghosts are independent of pH over this range, this quenching is due to the greater proximity of the donors and hemoglobin. Because hemoglobin binding to the band 3 protein in ghosts has previously been shown under conditions of low ionic strength and hemoglobin concentration, the present results were analyzed by a model in which a hemoglobin molecule is bound or in close proximity to a band 3 protein at pH 6. By using resonance energy transfer theory, we found the distance from DIDS to the bound hemoglobin to be approximately 4 A, which is within the range of distances measured between the DIDS binding site and the band 3 protein cytoplasmic sulfhydryl groups. Furthermore, the pH-dependent fluorescence quenching of 12-AS was stronger than that of 2-AS, showing that a cytoplasmic acceptor is involved and that the average distance between hemoglobin and these probes was greater than that for DIDS.
通过观察血红蛋白对荧光膜探针的猝灭作用随pH的变化，来研究完整细胞中血红蛋白与红细胞膜结合的问题。通过直角荧光法和前表面荧光法测量膜结合供体的荧光强度和衰减率，对这种猝灭现象进行了研究。供体包括与带3蛋白结合的4,4'-双（异硫氰酸）-2,2'-二苯乙烯二磺酸（DIDS）以及膜脂质部分中的2-、6-和12-（9-蒽氧基）硬脂酸（2-AS、6-AS和12-AS）。随着细胞内pH从7.2降至5.9，探针荧光逐渐猝灭，且不依赖于氧合状态。由于在此pH范围内，空泡中DIDS和x-AS探针的荧光特性与pH无关，所以这种猝灭是由于供体与血红蛋白的距离更近所致。因为先前已在低离子强度和血红蛋白浓度条件下，证明了空泡中血红蛋白与带3蛋白的结合，所以通过一个模型对目前的结果进行了分析，该模型认为在pH 6时，一个血红蛋白分子与一个带3蛋白结合或紧密相邻。通过共振能量转移理论，我们发现从DIDS到结合的血红蛋白的距离约为4 Å，这在DIDS结合位点与带3蛋白细胞质巯基之间测量的距离范围内。此外，12-AS的pH依赖性荧光猝灭比2-AS更强，表明涉及一个细胞质受体，且血红蛋白与这些探针之间的平均距离大于DIDS的平均距离。
