脲对果菠萝蛋白酶活力与构象的影响

Effect of Urea on the Activity and Conformation of Fruit Bromelain

本文用荧光光谱,紫外差示光谱和CD谱研究果菠萝蛋白酶在不同浓度的脲溶液中的构象及酶活力的变化情况。酶的荧光强度随脲浓度增大而明显增加,8mol/L脲使荧光强度增强65％,发射峰出现红移。差示谱表明在232nm和288nm出现二个正峰,它们均随脲浓度增大而加剧,前者与主链构象变化有关,而后者与生色基团(Trp、Tyr)的微环境变化相关。CD谱表明:天然酶在208nm和225nm处有二个负峰,脲变性后,225nm的负峰基本上不随脲浓度增大而变化,但208nm峰则明显发生变化并逐渐出现红移,6mol/L以上此峰则完全消失。

Changes of activity and conformation of fruit bromelain in different concentrations of urea solution have been studied by means of measuring the fluorescence spectra, differential UV-absorption spectra and circular dichroism spectra.The fluorescence intensity of the enzyme increased distinctly with increasing urea concentrations, and the emission peaks appeared red-shifted.The fluorescence intensity increased 65% when the concentration of urea was 8mol/L.The differential UV-absorption spectra showed that there were two positive peaks at 232nm and 288nm, both of them intensified with increasing urea concentrations, the 232nm peak is in concordance with the conformational change of the main chain of the enzyme molecule, while the 288nm peak is in concordance with the conformational change of the microenvironments of tyrosine and tryptophan.The circular dichroism spectra showed that the natural enzyme had two negative peaks at 208nm and 225nm.Denaturation by urea in a series of increasing concentrations indicated that the 225nm -peak did not change basically, whereas the 208nm-peak exhibited distinct change with gradual red-shifts in position, eventually the peak disappeared completely at urea concentrations above 6mol/L.