Peskin Alexander V, Midwinter Robyn G, Harwood David T, Winterbourn Christine C
Department of Pathology, Christchurch School of Medicine and Health Sciences, P.O. Box 4345, Christchurch, New Zealand.
Free Radic Biol Med. 2005 Feb 1;38(3):397-405. doi: 10.1016/j.freeradbiomed.2004.11.006.
Hypochlorous acid formed by activated neutrophils reacts with amines to produce chloramines. Chloramines vary in stability, reactivity, and cell permeability. We have examined whether chloramine exchange occurs between physiologically important amines or amino acids and if this affects interactions of chloramines with cells. We have demonstrated transchlorination reactions between histamine, glycine, and taurine chloramines by measuring chloramine decay rates with mixtures as well as by mass spectrometry. Kinetic analysis suggested the formation of an intermediate complex with a high Km. Apparent second-order rate constants, determined for concentrations <Km, were 19.4, 23.8, 6.0, and 7.5 M(-1) min(-1) for glycine chloramine (Gly-Cl) and taurine, Gly-Cl and histamine, histamine chloramine and glycine, and taurine chloramine (Tau-Cl) and glycine, respectively. Thus with 10 mM amine concentrations, half-lives for chloramine exchange are of the order of a few minutes. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity in cells was measured as an indicator of permeability of the chloramines. When endothelial or Jurkat cells were treated in Hanks' buffer, Gly-Cl inhibited GAPDH, whereas Tau-Cl, which does not penetrate the cells, did not. Adding glycine to Tau-Cl brought about inhibition, whereas taurine mitigated the effect of Gly-Cl. For cells in full medium, high chloramine concentrations were needed to inhibit GAPDH because of scavenging by methionine and other constituents. In methionine-free medium, chlorine exchange resulted in GAPDH inhibition by Tau-Cl, whereas Gly-Cl was less effective than in Hanks' buffer. Thus interchange between chloramines occurs readily and modulates their cellular effects.
活化的中性粒细胞产生的次氯酸与胺反应生成氯胺。氯胺在稳定性、反应活性和细胞通透性方面存在差异。我们研究了生理上重要的胺或氨基酸之间是否发生氯胺交换，以及这是否会影响氯胺与细胞的相互作用。我们通过测量混合物中氯胺的衰减率以及通过质谱法证明了组胺、甘氨酸和牛磺酸氯胺之间的转氯反应。动力学分析表明形成了具有高Km的中间复合物。对于浓度 <Km测定的表观二级速率常数，甘氨酸氯胺（Gly-Cl）与牛磺酸、Gly-Cl与组胺、组胺氯胺与甘氨酸、牛磺酸氯胺（Tau-Cl）与甘氨酸的分别为19.4、23.8、6.0和7.5 M(-1) min(-1)。因此，在胺浓度为10 mM时，氯胺交换的半衰期约为几分钟。测量细胞中的甘油醛-3-磷酸脱氢酶（GAPDH）活性作为氯胺通透性的指标。当内皮细胞或Jurkat细胞在汉克斯缓冲液中处理时，Gly-Cl抑制GAPDH，而不穿透细胞的Tau-Cl则不会。向Tau-Cl中添加甘氨酸会导致抑制作用，而牛磺酸会减轻Gly-Cl的作用。对于完全培养基中的细胞，由于蛋氨酸和其他成分的清除作用，需要高氯胺浓度才能抑制GAPDH。在无蛋氨酸的培养基中，氯交换导致Tau-Cl抑制GAPDH，而Gly-Cl的效果不如在汉克斯缓冲液中。因此，氯胺之间的交换很容易发生，并调节它们的细胞效应。
