三氧化二砷中间代谢产物二甲基亚砷酸（DMA^Ⅲ）与大鼠血红蛋白相互作用的分子机制研究

The molecular mechanism of interaction of trivalent dimethylarsinous acid （DMA^Ⅲ） binding to rat hemoglobin

为了研究三氧化二砷的中间代谢产物三价二甲基亚砷酸DMAIII与大鼠血红蛋白的作用位点及其机制,本研究将SD大鼠随机分成对照组和给药组,对照组单次经口给蒸馏水,给药组单次经口给予三价亚砷酸(iAsIII),分离大鼠血红细胞→溶血→获得血红蛋白。利用高效液相质谱(HPLC)联用结合电感耦合等离子质谱仪(ICP-MS)分析砷结合血红蛋白的化学形态。利用时间飞行质谱(LC/ESI-TOF-MS)分析DMAIII与血红蛋白结合位置,进一步采用分子对接(molecular docking)方法预测了DMAIII与血红蛋白的结合模式,从分子水平上分析了DMAIII与血红蛋白的作用机制。实验结果表明,砷以DMAIII与大鼠血红蛋白结合,主要结合位点为血红蛋白的alpha链螺旋的Cys13周围的氨基酸口袋,该位置使DMAIII更容易结合半胱氨酸,并阻止了DMAIII的游离。指出DMAIII与血红蛋白的高亲和力与Cys13周围的空间结构有直接关系。

In our previous work, we found that trivalent dimethylarsinous acid （DMA^Ⅲ） have high affinity binding to cysteine residue 13 of rat hemoglobin. However, it is still unknown why arsenic intermediate metabolite DMA^Ⅲ has high binding affinity for Cys13 but not for other cysteine residues 93, 140, 111 and 125. In order to better understand the molecular mechanism of DMA^Ⅲ with rat hemoglobin, we have done current study. So, SD rats were divided into control and arsenic-treated groups randomly. Arsenic species in lysate of red blood cells were analyzed by HPLC-ICP-MS, and then determined by a hybrid quadrupole TOF MS. In addition, trivalent DMA^Ⅲ binds to different cysteine residues in rat hemoglobin alpha and beta chains were also simulated by Molecular Docking. Only Cys13 in alpha chain is able to bind to DMA^Ⅲ from the experiment results. Cys 13 of alpha chain in rat hemoglobin is a specific binding site for DMA^Ⅲ, and we found that amino acids compose pockets structure and surround Cysl3 （but not other cysteine residues）, makeDMA^Ⅲ much easy to bind cysteine 13. Taken together, the DMA^Ⅲ specific binding to Cys13 is related to spatial structure of Cys13.