摘要
以多酚氧化酶(Polyhenol oxidase,PPO)和脂肪氧化酶(Lipoxygenase,LOX)为研究材料,利用圆二色谱(CD)和荧光光谱分析了脉冲电场(PEF)对此两种酶蛋白二级和三级构象的影响。PPO和LOX酶活在电场强度为20kV·cm-1的同轴处理室内处理320μs分别降低了60.3%和21.7%,并且,随着电场强度的增大,脉冲处理时间的延长,PPO与LOX酶的活性进一步降低。通过CD光谱图发现,PEF处理后,PPO和LOX的α-螺旋含量显著降低,而β-折叠含量则呈现增长,表明PPO与LOX酶蛋白中的二级构象在PEF处理后发生了极大的改变,酶活钝化和二级结构的破坏之间存在对应关系。PEF处理后LOX的荧光强度增加,随电场强度增强荧光强度增加幅度显著增大,表明PEF破坏了LOX三级构象,酶活钝化和三级结构的破坏之间存在对应关系。
Inactivation effect of pulsed electric field （PEF） on polyphenol oxidase （PPO） and lipoxygenase （LOX） was investiga- ted using a laboratory PEF system with a coaxial treatment chamber. Circular dichroism （CD） and fluorescence analysis were used to study the conformation change of the protein. The experimental results show that PPO and LOX can be effectively inactivated by the PEF treatment. Inactivation effect of PPO and LOX increases with the increase in the applied electric strength and the treatment time. Activity of PPO and LOX can be reduced by 60. 3% and 21.7% at 20 kV . cm^-1 after being treated for 320μs respectively. The decrease of the negative peaks （208 and 215 nm in PPO spectra, 208 nm and 218 nm in LOX spectra） in CD spectra of PPO and LOX shows that PEF treatment caused a loss of a-helix and increase in fl-sheet content, indicating that conformation changes occur in the secondary structure of PPO and LOX enzyme. This effect was strengthened as the applied electric field increased： a-helical content of PPO and LOX was 56% and 29% after being treated at 8 kV . cm ^-1, however, when the electric field was increased up to 20 kV . cm^-1, α-helical content of PPO and LOX decreased to 21% and 16% respectively. The decrease rate of α-helix and increase rate of β-sheet in PPO are higher than LOX, indicating that the second conformation of PPO is less resistant to PEF treatment than LOX. The fluorescence intensity of LOX increases after PEF treatment. At the same time, increasing the applied pulsed electric field increases the fluorescence intensity emitted. Fluorescence measurements confirm that tertiary conformation changes occur in the local structure of LOX. However the possible mechanism of the conformation change induced by the PEF treatment is beyond the scope of the present investigation.
出处
《光谱学与光谱分析》
SCIE
EI
CAS
CSCD
北大核心
2009年第8期2122-2125,共4页
Spectroscopy and Spectral Analysis
基金
国家"十一五"科技支撑计划课题项目(2006BAD05A02)
国家自然科学基金项目(30371003)资助
关键词
脉冲电场
脂肪氧化酶
多酚氧化酶
圆二色谱
荧光光谱
Pulsed electric field
Polyhenol oxidase
Lipoxygenase
Circular dichroism spectra
Fluorescence spectra
