Site-directed mutagenesis studies of human serum albumin define tryptophan at amino acid position 214 as the principal site for nitrosation
Site-directed mutagenesis studies of human serum albumin define tryptophan at amino acid position 214 as the principal site for nitrosation
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DOI：
10.1159/000048199
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发表时间：
2002-01-01
影响因子：
11
通讯作者：
Bhagavan, NV
中科院分区：
文献类型：
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作者：
Harohalli, K;Petersen, CE;Bhagavan, NV
比较了亚硝化牛血清白蛋白 (BSA)、人血清白蛋白 (HSA) 和一些重组 HSA 突变体的一氧化氮 (NO) 释放模式。所有白蛋白种类均通过与酸化NO2-一起温育而被亚硝化。用酸化 NO2- 亚硝化的 BSA 释放 NO 的模式与之前的报道一致，表明 Cys-34 是 BSA 亚硝化的主要目标。相反，用酸化NO2-亚硝化的HSA释放NO的模式表明主要亚硝化目标是除Cys-34之外的氨基酸残基。根据我们的初步发现和之前的报告（色氨酸是酸化 NO2- 亚硝化的潜在靶标），在酵母种毕赤酵母中合成了几种重组 HSA 突变体。产生了以下重组 HSA 物种：野生型、C34S、W214L、W214E 和 W214L/Y411W HSA。使用酸化的 NO2- 对这些突变体进行亚硝化表明 Trp-214 是 HSA 中的主要亚硝化靶标。 Trp-214 的突变导致 Cys-34 亚硝化作用增加，表明这两个残基之间可能存在与 N2O3 反应的竞争，N2O3 是在酸化 NO2 水溶液中形成的反应性亚硝化物质。版权所有 (C) 2002 国家科学委员会、ROC 和巴塞尔 S. Karger AG。
The patterns of nitric oxide (NO) release from nitrosated bovine serum albumin (BSA), human serum albumin (HSA) and a number of recombinant HSA mutants were compared. All albumin species were nitrosated by, incubation with acidified NO2-. The pattern of NO release from BSA nitrosated with acidified NO2- was in agreement with previous reports which indicated that Cys-34 is the primary target for nitrosation in BSA. In contrast, the pattern of NO release from HSA nitrosated with acidified NO2- indicated that the primary nitrosation target was an amino acid residue other than Cys-34. Based on our initial findings and a previous report that tryptophan is a potential target for nitrosation by acidified NO2-, several recombinant HSA mutants were synthesized in the yeast species Pichia pastoris. The following recombinant HSA species were produced: wild-type, C34S, W214L, W214E and W214L/Y411W HSA. Nitrosation of these mutants using acidified NO2- showed that Trp-214 is the primary nitrosation target in HSA. Mutation of Trp-214 led to an increase in Cys-34 nitrosation, indicating possible competition between these two residues for reaction with N2O3, the reactive nitrosating species formed in aqueous acidified NO2- solutions. Copyright (C) 2002 National Science Council, ROC and S. Karger AG, Basel.