Department of Pathology, University of Otago Christchurch, P.O. Box 4345, Christchurch, New Zealand.
Chem Res Toxicol. 2009 Nov;22(11):1833-40. doi: 10.1021/tx900249d.
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/hypothiocyanite 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 + or - 0.02) x 10(8) M(-1) s(-1), and the effective rate constant at pH 7.4 is (4.37 + or - 0.03) x 10(5) M(-1) s(-1). Analysis of the kinetic data, using a value of 4.38 + or - 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 + or - 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 + or - 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.
次碘酸盐是哺乳动物过氧化物酶产生的主要氧化剂。尽管有报道称它专门与生物分子中的巯基反应，但对该反应的详细机制研究尚未进行。我们通过停流光谱法研究了次碘酸/次碘酸盐与 5-硫-2-硝基苯甲酸和还原型谷胱甘肽的反应。观察到的钟形 pH 曲线确定，与 5-硫-2-硝基苯甲酸的反应在 2.5 < pH < 8 区域内通过硫醇盐和次碘酸进行。反应的获得的二级速率常数为（1.26 ± 0.02）x 10（8）M（-1）s（-1），在 pH 7.4 时的有效速率常数为（4.37 ± 0.03）x 10（5）M（-1）s（-1）。使用通过光谱分析独立测量的 5-硫-2-硝基苯甲酸硫醇的 pK（a）值（4.38 ± 0.01）对动力学数据进行分析，在生理盐浓度（I = 120 mM；NaCl 和磷酸盐缓冲液）和 25°C 下，次碘酸的 pK（a）值为 4.85 ± 0.01。在 pH 7.4 时，次碘酸/次碘酸盐与还原型谷胱甘肽的反应的二级速率常数为（8.0 ± 0.5）x 10（4）M（-1）s（-1）。谷胱甘肽数据也与通过硫醇盐和次碘酸进行的反应一致。我们的结果表明，次碘酸/次碘酸盐与巯基具有很高的反应性，并且在生理浓度的谷胱甘肽和巯基蛋白存在下将很快失活。由于反应严格地与硫醇盐发生，因此这种氧化剂应选择性地针对含有低 pK（a）巯基的蛋白质。
