APPLICATION OF RANEY-NICKEL TO MEASURE ADDUCTS OF STYRENE OXIDE WITH HEMOGLOBIN AND ALBUMIN
APPLICATION OF RANEY-NICKEL TO MEASURE ADDUCTS OF STYRENE OXIDE WITH HEMOGLOBIN AND ALBUMIN
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DOI：
10.1021/tx00032a014
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发表时间：
1993-03-01
影响因子：
4.1
通讯作者：
MCDONALD, T
中科院分区：
文献类型：
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作者：
RAPPAPORT, SM;TING, D;MCDONALD, T
用雷尼镍 (Ra-Ni) 处理蛋白质以裂解 2-苯基乙醇后，测量苯乙烯 7,8-氧化物 (SO) 与血液蛋白质、Hb 和白蛋白 (Alb) 的加合物，随后对其进行测定。通过用 4-甲基-SO 修饰 Hb 和 Alb 来制备内标，以产生类似的加合物。 在体外用 [C-14]SO 修饰的人血的初步实验中，确定 Ra-Ni 释放了与珠蛋白总结合的 6% 和与 Alb 总结合的 76%。由于 Ra-Ni 主要裂解共价结合的硫物质，这表明游离半胱氨酸残基与人血清 Alb 的结合比与人 Hb 的结合要多得多。 SO在37℃体外的总反应速率在人血液中的半衰期为0.70小时，在Sprague-Dawley大鼠血液中的半衰期为0.36小时。在 37 摄氏度下对人类和大鼠的血液进行 SO 修饰后，测量 SO 与 Hb 和 Alb 的加合物。加合物水平和 SO 浓度之间线性关系的斜率允许估计以下二阶速率常数 [(L/mol 蛋白质)/h]：大鼠 Hb，72；人血红蛋白，2.4；大鼠白蛋白，63；人 Alb，32。这些速率常数表明，SO 对人 Hb 半胱氨酸残基的固有反应性远低于大鼠 Hb 或来自任一物种的 Alb。给大鼠注射 SO 和苯乙烯后，还测量了 Hb 和 Alb 加合物。通过比较加合物水平与给药剂量之间线性关系的斜率，估计只有 2% 的苯乙烯剂量以 SO 形式从肝脏释放。由于无论是否测量 Hb 或 Alb 的 SO 加合物，都会发现该结果，因此在 SO 释放到血液中之前，肝细胞中可能形成相对较少的 SO-Alb 加合物。
Adducts of styrene 7,8-oxide (SO) with the blood proteins, Hb and albumin (Alb), were measured following treatment of the proteins with Raney nickel (Ra-Ni) to cleave 2-phenylethanol, which was subsequently assayed. Internal standards were prepared by modifying Hb and Alb with 4-methyl-SO to produce similar adducts. In a preliminary experiment with human blood, which had been modified with [C-14]SO in vitro, it was determined that Ra-Ni released 6% of the total binding to globin and 76% of the total binding to Alb. Since Ra-Ni primarily cleaves covalently-bound sulfur species, this suggests that much more binding to human serum Alb occurred at a free cysteine residue than to human Hb. The overall rate of reaction of SO at 37-degrees-C in vitro had a half-time of 0.70 h in human blood and of 0.36 h in blood from Sprague-Dawley rats. Adducts of SO with Hb and Alb were measured following SO modification of blood from hu ans and rats in vitro at 37-degrees-C. Slopes of the linear relationships between adduct level and SO concentration allowed the following second-order rate constants [(L/mol of protein)/h] to be estimated: rat Hb, 72; human Hb, 2.4; rat Alb, 63; human Alb, 32. These rate constants show that intrinsic reactivity of SO toward the cysteine residue of human Hb is much lower than that of rat Hb or of Alb from either species. Adducts of Hb and Alb were also measured following administration of both SO and styrene to rats. By comparing the slopes of the linear relationships between adduct levels and administered doses, it was estimated that only 2% of the dose of styrene was released from the liver as SO. Since this result was found regardless of whether the SO adduct of Hb or Alb was measured, relatively little of the SO-Alb adduct could have been formed in the hepatocyte prior to release of SO into the blood.