圆二色谱表征芥蓝抗坏血酸过氧化物酶变性过程中的结构变化

摘要

Circular dichroism (CD) is a special absorption spectrum .The secondary structure of protein such asα-helix ,β-sheet andβ-turn in the far ultraviolet region (190~250 nm) has a characteristic CD spectrum .In order to understand the activity and structural changes of ascorbate peroxidase from Chinese kale (BaAPX) during denaturation ,specific activity and percentage of secondary structure of BaAPX under different time ,temperature and concentration were analyzed by CD dynamically .In addition ,the per-centage of four secondary structures in BaAPX was calculated by CD analysis software Dichroweb .The results show that BaAPX is a fullα-type enzyme whose specific activity is positively related to the percentage of α-helix .During denaturation of BaAPX , three kinds of structural changes were proposed:the one-step structural change from initial state (N state) to minimum state ofα-helix (R state) under low concentration and low temperature;the one-step structural change from N state to equilibrium state (T state) under high concentration and low temperature;the two-step structural changes from N state through R state to final T state under heat treatment and low temperature renaturation .%圆二色谱（CD ）是一种特殊的吸收光谱，蛋白质的二级结构如α-螺旋、β-折叠、β-转角在远紫外区（190～250 nm ）都具有特征性的CD图谱。为了了解芥蓝抗坏血酸过氧化物酶（BaA PX ）在变性过程中活性变化和结构变化的关系，动态监测了不同时间、温度及浓度条件下BaA PX的比酶活及远紫外CD图谱，并利用CD分析软件Dichroweb计算了BaAPX中四种二级结构的百分含量。结果表明：BaAPX属于全α型蛋白，α-螺旋的百分含量与比酶活之间存在比较明显的正相关关系。在低浓度低温的变性过程中，BaA PX发生了初始态（N态）→α-螺旋最少态（R态）一步结构变化；在高浓度低温的变性过程中，BaAPX发生了 N态→平衡态（ T态）一步结构变化；在热处理后低温复性的变性过程中，BaA PX发生了 N态→ R态→ T态两步结构变化。