有机溶剂来源污染物对中国鲎N-乙酰-β-D-氨基葡萄糖苷酶的影响

Effects of Organic Solvents on N-acetyl-β-D-glucosaminidase of Chinese Horseshoe Crab Tachypleus tridentatus

N-乙酰-β-D-氨基葡萄糖苷酶(EC3.2.1.52,NAGase)是几丁质酶系的主要组成分之一,节肢动物周期性脱皮生理和生长发育与NAGase密切相关。为了探讨有机溶剂对中国鲎(Tachypleus tridentatus)NAGase的影响,从中国鲎内脏分离提取了NAGase,研究甲醇、乙醇、正丙醇、正丁醇、乙二醇、丙三醇、甲醛和丙酮等8种有机溶剂对中国鲎NAGase的影响;利用酶抑制作用动力学方法,研究丙酮对NAGase的抑制机理和抑制作用类型。结果表明:甲醇对中国鲎NAGase有激活作用,乙醇、正丙醇、正丁醇、甲醛和丙酮对该酶均有不同程度的抑制作用;乙二醇和丙三醇对NAGase表现为低浓度下呈激活现象,随浓度增大则转为对酶的抑制作用;丙酮对中国鲎NAGase的抑制有剂量效应关系,0.1 mol/L丙酮可使酶活力丧失49.53%;丙酮对中国鲎NAGase的抑制作用是个可逆过程;动力学研究显示,该酶抑制作用类型属于混合型。丙酮对游离酶(E)的抑制常数KI和对酶-底物络合物(ES)的抑制常数KIS分别为0.117和0.608 mol/L,KI<KIS,说明底物对中国鲎NAGase在丙酮中的失活具有一定的保护作用。NAGase酶蛋白的紫外吸收光谱和内源荧光发射光谱显示:经丙酮作用后NAGase酶蛋白紫外吸收值增强,但其内源荧光发射强度减弱,荧光发射峰值并没有发生位移,0.60 mol/L丙酮作用下NAGase酶蛋白荧光几近淬灭,说明中国鲎NAGase在丙酮溶剂中酶蛋白空间构象发生了变化,从而导致了酶变性失活,证明了有机溶剂对中国鲎NAGase活力具有调控作用。

N-acetyl-β-D-glucosidase(EC3.2.1.52,NAGase)is one of the main components of chitinolytic enzymes.The molting and growth of arthropods is closely related to NAGase.In order to investigate the effects of organic solvents on NAGase from Chinese horseshoe crab Tachypleus tridentatus,the NAGase was extracted from the viscera of Chinese horseshoe crab.The effects of eight organic solvents,including methanol,alcohol,propanol,butanol,glycol,glycerol,formaldehyde and acetone,were studied on NAGase.Besides,the inhibition mechanism and inhibition type of acetone on NAGase were studied by using the kinetics method of enzymatic inhibition.The results indicated that the enzyme was activated in methanol.Alcohol,propanol,butanol,formaldehyde and acetone showed various degrees of inhibitory on the enzyme.The enzyme was activated in glycol and glycerol solutions at low concentration,but inhibited as increasing concentration.The effects of acetone on the enzyme were concentration dependent,the enzyme activity decreased by 49.53%when its concentration reached 0.1 mol/L.The inhibitory effect of acetone on NAGase was reversible.The kinetics showed that the inhibitory effect of the enzyme in acetone solution was mix typed.The inhibition constants of acetone on free enzyme(KI)and on enzyme-substrate complex(KIS)were determined to be 0.117 mol/L and 0.608 mol/L,respectively.The value of KI is less than that of KIS,indicating a marked protective effect of the substrate on the inactivation of the enzyme.Meanwhile,UV absorption spectra and fluorescence emission spectra of NAGase were measured.The UV absorption of NAGase treated by acetone was enhanced with increasing acetone concentration,but the fluorescence emission peak intensity of the enzyme gradually decreased,their fluorescence emission peak of NAGase did not shift.The fluorescence emission of enzyme was almost quenched when the concentration of acetone reached 0.60 mol/L.The results indicated that the inhibitory effect of acetone on NAGase was caused by the conformational changes,leading to inactivation of the enzyme.It suggested that organic solvents have regulatory effects on the activities of NAGase from Chinese horseshoe crab.