双监测高效液相色谱法测定色氨酸酶中臭氧敏感色氨酸残留。

Journal of applied biochemistry Pub Date : 1985-02-01
N Ida, M Tokushige
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引用次数: 0

摘要

从大肠杆菌B/1t7-A中纯化的色氨酸酶被轻度臭氧化灭活,遵循准一级动力学。作者先前的数据表明,在臭氧失活的同时,酶亚基中的两个色氨酸残基中有一个优先被氧化,并与辅酶吡哆醛磷酸(PLP)直接相互作用[M. Tokushige, Y. Fukuda, and Y. Watanabe, 1979, Biochem]。Biophys。《共同法典》,86,976-981)。为了确定哪一种色氨酸更容易被臭氧化并与PLP相互作用,用胰蛋白酶裂解天然和臭氧化的酶蛋白,用反相高效液相色谱分析两种含色氨酸的肽,并配备由紫外和荧光监视器串联组成的双监测系统,以选择性检测含色氨酸肽。该装置有助于快速检测和定量臭氧化后减少的含色氨酸肽。结果表明,Trp在臭氧化过程中优先被氧化,参与与PLP相互作用的是肽T-15中的Trp,而不是Kagamiyama等人最初认定的T-23中的Trp (H. Kagamiyama, H. Wada, H. Matsubara, and E. E. Snell, 1972, J. Biol)。化学,247,1576-1585)。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Assignment of ozone-sensitive tryptophan residue in tryptophanase by a dual-monitoring high-performance liquid chromatography system.

Tryptophanase purified from Escherichia coli B/1t7-A is inactivated by mild ozonization following pseudo-first-order kinetics. Previous data from the authors suggest that one out of two tryptophan residues (Trp's) in the enzyme subunit is preferentially oxidized concomitant with the ozone inactivation and has a direct interaction with the coenzyme, pyridoxal phosphate [PLP (M. Tokushige, Y. Fukuda, and Y. Watanabe, 1979, Biochem. Biophys. Res. Commun. 86, 976-981)]. To determine which Trp is more susceptible to ozonization and interacts with PLP, the native and ozonized enzyme proteins were cleaved by trypsin and the two Trp-containing peptides were analyzed by reverse-phase HPLC equipped with a dual-monitoring system consisting of an uv and a fluorescence monitor connected in tandem for selective detection of Trp-containing peptides. This device facilitated rapid detection and quantitation of the Trp-containing peptides which decreased upon ozonization. The results showed that Trp preferentially oxidized upon ozonization and involved in the interaction with PLP was the one in peptide T-15 rather than that in T-23, which Kagamiyama et al. originally designated (H. Kagamiyama, H. Wada, H. Matsubara, and E. E. Snell, 1972, J. Biol. Chem. 247, 1576-1585).

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