Kinetics and Mechanisms of the Reaction of Hypothiocyanous Acid with 5-Thio-2-nitrobenzoic Acid and Reduced Glutathione
Kinetics and Mechanisms of the Reaction of Hypothiocyanous Acid with 5-Thio-2-nitrobenzoic Acid and Reduced Glutathione
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DOI：
10.1021/tx900249d
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发表时间：
2009-11-01
影响因子：
4.1
通讯作者：
Winterbourn, Christine C.
中科院分区：
文献类型：
--
作者：
Nagy, Peter;Jameson, Guy N. L.;Winterbourn, Christine C.
次硫氰酸盐是哺乳动物过氧化物酶产生的主要氧化剂。尽管有报道称其与生物分子中的硫醇基团发生特异性反应，但尚未对该反应进行详细的机理研究。我们通过停流光谱研究了次硫氰酸/亚硫氰酸盐与5-硫代-2-硝基苯甲酸和还原型谷胱甘肽的反应。观察到的钟形 pH 曲线表明，在 2.5 < pH < 8 范围内，与 5-硫代-2-硝基苯甲酸的反应通过硫醇盐和次硫氰酸进行。得到的反应二级速率常数为(1.26+/-0.02)×10(8)M(-1)s(-1)，pH 7.4下的有效速率常数为(4.37+/-0.03)×10(5)M(-1)s(-1)，动力学数据分析，pK(a)值为4.38+/-0.01 5-硫代-2-硝基苯甲酸硫醇（通过光谱分析独立测量），在生理盐浓度（I = 120 mM；氯化钠和磷酸盐缓冲液）和 25 摄氏度下，次硫氰酸的 pK(a) 为 4.85 +/- 0.01。 二阶速率常数为 (8.0 +/- 0.5) x 10(4) M(-1) s(-1)测定了次硫氰酸/次硫氰酸盐与还原型谷胱甘肽在pH 7.4下的反应。谷胱甘肽数据也与通过硫醇盐和次硫氰酸进行的反应一致。我们的结果表明，次硫氰酸/次硫氰酸盐与硫醇具有非常高的反应性，并且在生理浓度的谷胱甘肽和硫醇蛋白存在下，其寿命是短暂的。由于反应严格与硫醇盐发生，因此该氧化剂应选择性地靶向含有低 pK(a) 硫醇的蛋白质。
Hypothiocyanite is a major oxidant generated by mammalian peroxidases. Although reported to react specifically with thiol groups in biological molecules, a detailed mechanistic study of this reaction has not been conducted. We have investigated the reaction of hypothiocyanous acid/ypothiocyanite with 5-thio-2-nitrobenzoic acid and with reduced glutathione by stopped-flow spectroscopy. The observed bell-shaped pH profile established that the reaction with 5-thio-2-nitrobenzoic acid proceeds via the thiolate and hypothiocyanous acid in the 2.5 < pH < 8 region. The obtained second-order rate constant of the reaction is (1.26 +/- 0.02) x 10(8) M(-1) s(-1), and the effective rate constant at pH 7.4 is (4.37 +/- 0.03) x 10(5) M(-1) s(-1), Analysis of the kinetic data, using a value of 4.38 +/- 0.01 for the pK(a) of 5-thio-2-nitrobenzoic acid thiol (measured independently by spectroscopic analysis), gave a pK(a) of 4.85 +/- 0.01 for hypothiocyanous acid at physiological salt concentration (I = 120 mM; NaCl and phosphate buffer) and 25 degrees C. A second-order rate constant of (8.0 +/- 0.5) x 10(4) M(-1) s(-1) for the reaction of hypothiocyanous acid/hypothiocyanite with reduced glutathione at pH 7.4 was determined. The glutathione data are also consistent with the reaction proceeding via the thiolate and hypothiocyanous acid. Our results demonstrate that hypothiocyanous acid/hypothiocyanite has very high reactivity with thiols and will be short-lived in the presence of physiological concentrations of glutathione and thiol proteins. As the reaction occurs strictly with the thiolate, this oxidant should selectively target proteins containing low pK(a) thiols.