Chong S, Dill K, McGown E
Division of Biophysical Research, Letterman Army Institute of Research, Presidio of San Francisco, California 94129-6800.
J Biochem Toxicol. 1989 Spring;4(1):39-45. doi: 10.1002/jbt.2570040108.
The purpose of the study was to identify binding sites of organic arsenic in the erythrocyte and to explain species differences in binding. Washed erythrocytes were exposed to graded concentrations of [U-14C]phenyldichloroarsine (PDA) in phosphate-buffered saline containing 0.1% glucose and 0.1% bovine serum albumin. At low PDA concentrations, all cells bound the arsenical rapidly (within 10 min) and quantitatively. Human, pig, hamster, guinea pig, and mouse erythrocytes approached saturation at 0.02-0.3 mumol PDA/10(9) cells, depending on the species. Saturation points correlated well with each respective species' erythrocyte glutathione content. In contrast, rat erythrocytes showed no sign of saturation at PDA loads as high as 3.0 mumol/10(9) cells. Hemolysates of PDA-treated erythrocytes were subjected to Sephadex G-75 gel filtration chromatography. 14C from rat hemolysate was distributed between the hemoglobin and small molecular weight (glutathione-containing) fractions. In all other species, the 14C eluted almost exclusively with the glutathione-containing fractions. In equilibrium dialysis experiments, human hemoglobin did not bind PDA, whereas rat hemoglobin bound 2 PDA/mol with Kd approximately 5 microM. In conclusion, glutathione is the principal binding site of phenyldichloroarsine in erythrocytes. In most species, the arsenical does not bind to hemoglobin, even though it has free (titratable) sulfhydryls considerably in excess of the glutathione concentration. In rat erythrocytes, phenlydichloroarsine binds both to glutathione and to hemoglobin. Arsenical binding by rat hemoglobin is presumably due to the unique location of the extra titratable cysteine in that protein.
本研究的目的是确定有机砷在红细胞中的结合位点，并解释结合的种属差异。将洗涤后的红细胞暴露于含0.1%葡萄糖和0.1%牛血清白蛋白的磷酸盐缓冲盐水中，使其接触不同浓度梯度的[U-14C]苯基二氯胂（PDA）。在低PDA浓度下，所有细胞均能迅速（10分钟内）且定量地结合砷化物。人、猪、仓鼠、豚鼠和小鼠的红细胞在0.02 - 0.3 μmol PDA/10⁹个细胞时接近饱和，具体取决于物种。饱和点与各物种红细胞的谷胱甘肽含量密切相关。相比之下，大鼠红细胞在PDA负载高达3.0 μmol/10⁹个细胞时仍无饱和迹象。对经PDA处理的红细胞溶血产物进行Sephadex G - 75凝胶过滤色谱分析。大鼠溶血产物中的¹⁴C分布于血红蛋白和小分子质量（含谷胱甘肽）组分之间。在所有其他物种中，¹⁴C几乎仅随含谷胱甘肽的组分洗脱。在平衡透析实验中，人血红蛋白不结合PDA，而大鼠血红蛋白以Kd约5 μM的亲和力结合2个PDA/摩尔。总之，谷胱甘肽是苯基二氯胂在红细胞中的主要结合位点。在大多数物种中，砷化物不与血红蛋白结合，尽管其游离（可滴定）巯基大大超过谷胱甘肽浓度。在大鼠红细胞中，苯基二氯胂既与谷胱甘肽结合，也与血红蛋白结合。大鼠血红蛋白对砷化物的结合可能归因于该蛋白质中额外可滴定半胱氨酸的独特位置。
