尿素对经动态高压微射流(DHPM)诱导去折叠胰蛋白酶构象变化的影响  

Effect of Urea on Conformation Changes of Dynamic High-pressure Microfluidization(DHPM)-induced Unfolded Trypsin

作　　者：刘军平[1] 刘伟[1] 邹立强[1] 刘成梅[1] 方丽淳[1] 张兆琴[1] 

机构地区：[1]南昌大学食品科学与技术国家重点实验室,江西南昌330047

出　　处：《食品科学》2011年第21期80-85,共6页Food Science

基　　金：国家自然科学基金项目(31060209);国家重点实验室重点青年骨干研究基金项目(SKLF-QN-201101)

摘　　要：以牛胰蛋白酶为原料,通过紫外吸收光谱、荧光发射光谱和圆二色谱研究尿素对天然胰蛋白酶和经动态高压微射流(DHPM)诱导去折叠胰蛋白酶的构象变化。结果显示:天然胰蛋白酶构象在尿素浓度4.0～6.0mol/L之间变化最显著,紫外吸光度从0.2746降至0.1824,吸收峰从波长279nm红移至283nm,相对荧光强度从105.9降至98.27,发射峰从波长352nm红移至355nm,β-折叠含量从46.9%降至30.6%;而去折叠胰蛋白酶构象在尿素浓度2.0～4.0mol/L之间变化最显著,紫外吸光度从0.3121降至0.2159,吸收峰从波长281nm红移至283nm,相对荧光强度从108.24降至99.07,发射峰从波长353nm红移至360nm,β-折叠含量从40.6%降至38.9%。表明尿素对经DHPM处理后处于部分去折叠胰蛋白酶构象的影响更为显著。不同浓度尿素导致胰蛋白酶产生不同构象的中间体,表现出"熔球态"模型特征。The conformational changes of native trypsin and DHPM-induced unfolded trypsin during the denaturation by urea have been investigated using the methods of ultraviolet（UV） spectroscopy,fluorescence spectroscopy and circular dichroism spectroscopy.Results indicated that native trypsin showed the most significant changes when the concentration of urea was from 4.0 to 6.0 mol/L.The absorbance at 280 nm decreased from 0.2746 to 0.1824,and the absorption peak showed red shift from 279 to 283 nm;the relative fluorescence intensity decreased from 105.9 to 98.27,and the emission peak showed red shift from 352 to 355 nm.The percentage of β-sheet content decreased from 46.9% to 30.6%.In contrast,the most significant changes were observed in unfolded trypsin when the concentration of urea was from 2.0 to 4.0 mol/L.The UV absorbance decreased from 0.3121 to 0.2159 and the peak showed red shift from 281 to 283 nm;the fluorescence intensity decreased from 108.24 to 99.07 and the emission peak showed red shift from 353 to 360 nm.The percentage of β-sheet content decreased from 40.6% to 38.9%.These findings show that urea has a more significant impact on DHPM-induced unfolded trypsin compared with native trypsin.In the presence of urea at different concentrations,trypsin has different conformations of intermediates that show characteristics of molten globule state.

关 键 词：尿素 胰蛋白酶 动态高压微射流 构象 

分 类 号：Q556.3[生物学—生物化学]