Oxidation and reduction of thymosin beta(4) and its influence on the interaction with G-actin studied by reverse-phase HPLC and post-column derivatization with fluorescamine
Oxidation and reduction of thymosin beta(4) and its influence on the interaction with G-actin studied by reverse-phase HPLC and post-column derivatization with fluorescamine
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DOI：
10.1016/s0003-2670(97)00132-3
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发表时间：
1997-10-10
影响因子：
6.2
通讯作者：
Hannappel, E
中科院分区：
文献类型：
--
作者：
Huff, T;Hannappel, E
已显示甲硫氨酸残基的氧化提供了体内蛋白质功能的潜在调节机制。与G-肌动蛋白形成一对一复合物的胸腺素β（4）是哺乳动物组织中最丰富的β-胸腺素成员，在6位具有甲硫氨酸残基。在哺乳动物组织的制备中，胸腺素β（4）在大多数情况下伴随有少量的亚砜。使用反相HPLC，我们发现，胸腺素β（4）的甲硫氨酸残基的氧化可以在体外通过毫摩尔浓度的H2 O2实现，并且伴随着其与G-肌动蛋白复合物的表观解离常数增加18倍。使用RP-18柱，采用20 mM吡啶-0.11 M甲酸-0.05 M高氯酸锂中的正丙醇线性梯度，通过反相HPLC分离肽，并在用荧光胺柱后衍生后通过荧光检测。分别使用3 mM或1 mM H2 O2在3.5或6小时后氧化50%的胸腺素P4。在0.5 mM H2 O2的情况下，约45%的蛋氨酸残基在18小时后被氧化。用亚硫酸氢钠水溶液还原所得亚砜。该减少伴随着对G-肌动蛋白的初始亲和力的恢复。用90%饱和Na 2S 2 O 5溶液，我们发现亚砜在约5小时内还原50%，12小时后还原80%，而用二硫苏糖醇（0.81 M）在25小时后仅还原30%。还原所需的大量亚硫酸氢钠可以通过固相萃取从肽中分离出来。(C)1997年Elsevier Science B.V.
Oxidation of methionine residues has been shown to provide a potential regulatory mechanism in protein function in vivo. Thymosin beta(4) which forms a one-to-one complex with G-actin is the most abundant member of beta-thymosins in mammalian tissues and possesses a methionine residue at position 6. In preparations of mammalian tissues thymosin beta(4) is, in most cases, accompanied by a small amount of its sulfoxide. Using reverse-phase HPLC we showed that the oxidation of the methionine residue of thymosin beta(4) can be achieved by millimolar concentrations of H2O2 in vitro and is accompanied by an 18-fold increase in the apparent dissociation constant of its complex with G-actin. Peptides were separated by reverse-phase HPLC using a RP-18 column applying a linear gradient of n-propanol in 20 mM pyridine - 0.11 M formic acid - 0.05 M lithiumperchlorate and were detected by fluorescence after postcolumn derivatization with fluorescamine. 50% of thymosin P4 is oxidized after 3.5 or 6 hours using 3 mM or 1 mM H2O2, respectively. In the case of 0.5 mM H2O2, about 45% of the methionine residues are oxidized after 18 hours. The resulting sulfoxide is reduced with aqueous solutions of sodiumbisulfite. The reduction is accompanied by the recovery of the initial affinity to G-actin. With a solution of 90% saturated Na2S2O5 we find 50% reduction of the sulfoxide in about 5 hours and 80% after 12 hours while only 30% is reduced with dithiothreitol (0.81 M) after 25 hours. The large amount of sodiumbisulfite necessary for reduction can be separated from the peptide by solid phase extraction. (C) 1997 Elsevier Science B.V.