豆壳过氧化物酶的盐酸胍变性与化学修饰研究

Chemical Modification and Guanidinium Chloride Denaturation of Soybean Hull Peroxidase

研究了盐酸胍对豆壳过氧化物酶(soybeanhullperoxidase,SHP,EC1.11.1.7)构象与活力的影响,发现去辅基SHP的盐酸胍变(复)性及荧光变化关系与SHP全酶分子的盐酸胍变(复)性及荧光变化关系明显不同。应用过碘酸氧化法去除SHP分子表面糖链,研究糖链去除对酶性质的影响,则证实了SHP分子表面的糖链去除导致酶热稳定性下降。应用不同的蛋白质侧链修饰剂对SHP进行化学修饰则表明,巯基、酪氨酸和色氨酸残基为酶活力非必需,而羧基、组氨酸和精氨酸残基为酶活力所必需。

The guanidinium chloride denaturation/renaturation of the holo and apo soybean hull peroxidase(SHP,EC 1.11.1.7) was studied by fluorescence spectroscopy.A distinct equilibrium intermediate of the apoprotein could be detected at low concentrations of guanidinium chloride.Renaturation of the apo SHP was reversible.The denaturation of the holo SHP was more complex.In addition,the stability of the enzyme following treatment with periodate was studied.This treatment resulted in a loss of specific activity which was shown not to be correlated with the removal of sugars.The thermal stability was found decreased by this chemical deglycosylation.Finally,the effects of some protein modification reagents on the activity of SHP were studied.The enzyme was not affected by DTNB,IAA,HNBB and NAI modification,suggesting amino groups,thiol groups,tyrosyl and tryptophanyl residues were non essential to enzyme activity.The enzyme activity was remarkably decreased after DEPC,PGH,WRK and EDC modification.The results indicated histidyl residues,arginyl residues and carboxyl groups seemed to be essential to the catalytic acticvity of SHP.