脉冲电场对木瓜蛋白酶影响的荧光光谱分析  

Fluorescence Spectra Analysis of Papain Treated by Pulsed Electric Field

作　　者：曾新安[1] 于淑娟[1] 徐娅莉[1] 

机构地区：[1]华南理工大学轻工与食品学院,广东广州510640

出　　处：《光谱学与光谱分析》2007年第12期2558-2561,共4页Spectroscopy and Spectral Analysis

基　　金："十一五"国家科技支撑计划课题项目(2006BAD05A02);广东省自然科学基金项目(04020063)资助

摘　　要：木瓜蛋白酶溶液在电场强度为50 kV.cm-1,频率1 500 Hz,脉冲宽度40μs的脉冲电场下接受19 800个脉冲处理后,其活性降低了56.5%。文章采用荧光偏光光谱对处理前后的样品进行了分析。处理后酶样的荧光强度明显大于处理前,在峰值位置其荧光强度增加超过50%,峰位从342 nm移到了346 nm左右,其荧光偏振幅度明显减小。由此推断出木瓜蛋白酶经脉冲电场处理后酶蛋白的α-螺旋结构松散拉伸,分子内部的氨基酸残基暴露于分子表面,并有部分发生离解游离于溶液中,导致活性部位的结构发生变化,最终导致酶失活。Tryptophane, tyrosine and phenylalanine are there kinds of fluorescent amino acids and exist in papain, so the structure change of papain can be measured by fluorescence spectra analysis without exterior fluorescence probe. Fluorescence excitation spectrum, emission spectrum and polarization spectrum were used to analyze the possible mechanisms of papain＇ s activity change after being treated by pulsed electric field （PEF）. Results demonstrated that the relative activity of papain was decreased by 56.50/00 after PEF treatment under the condition of electric field strength 50 kV·cm^-1 , frequency 1 500 Hz, pulse width 40 lus and pulse number 19 800. The spectra of fluorescence excitation showed that the relative fluorescence strength of the treated sample was distinctly higher than the untreated one, even at peak position （280 urn） which was 80 and 120 for untreated and treated samples, respectively. The peak position in the fluorescence emission spectrum of treated sample was shifted from the o- riginal 342 nm to about 346 nm, and its fluorescence polarization degree was much smaller compared to the untreated sample. These phenomena indicated that the a-helix structure of papain was loosened or broken down after being treated by PEF. This treatment made the amino acid＇s residue exposed from inside to outside, and even some fluorescent amino acids such as tryptophane, tyrosine and phenylalanine were decomposed from the a-helix structure of protein and went into the solution which helped increase the fluorescent strength. This effect led to the active site change of the enzyme and finally inactivated it.

关 键 词：脉冲电场 木瓜蛋白酶 酶活 荧光光谱 

分 类 号：O657.3[理学—分析化学]