乙醇对文昌鱼酸性磷酸酶活力与构象的影响

Changes of conformation and activity of ACPase from Branchoistoma belcheri in ethanol solutions

文昌鱼酸性磷酸酶（ACPase，E．C．3．1．3．2）是一种含有铁离子的金属酶，酶的紫外-可见光谱（200～600nm）扫描，280nm，500nm有最大吸收峰，320nm有肩峰。荧光扫描：在281nm激发下，330nm有发射峰，在480nm激发下，515nm有一发射峰。可见光谱500nm；的吸收峰以及荧光515nm的发射峰为酶分子含铁的特征峰，与酶活力关系密切。不同浓度的乙醇对酶活力有明显的抑制作用，表现为反竞争抑制类型，其抑制常数为16％，测定酶随时间的变化的失活程度。可见光谱500nm吸收值随酶体系中乙醇含量增加而增加，当乙醇浓度达40％时，吸收值明显下降。在乙醇作用下，监测荧光330nm及515nm处的强度随时间变化的过程作为酶构象变化的指标，酶的330nm荧光发射峰值随乙醇浓度的增强而下降，515nm的荧光发射峰值随着乙醇浓度的增强而下降，峰位不变。在不同浓度乙醇作用下，酶的构象变化快于活力变化。

The previous studies by our research team have shown that Branchiostoma belcheri Acid Phosphatase (ACPase, E. C. 3. 1. 3. 2) was a kind of metalloenzyme which exhibits an absorption peak with the warelength at 500nm in its visible spectra and an emission peak with the warelength at 515nm in its fluorescence spectra because its containing iron. This two charactal peaks are sensitive to the enzyme activity (referance 7~9 for detail). Changes of conformation and activity at the active site of the ACPase in different eoncentration(V/V) of ethanol solutions has been investigated by means of fiuorescence emission and UV-visible spectra. Kinetic analysis of the inhibition shows that the inhibition of ethanol on the enzyme is found to be of uncompetitive type and the inhibition constants is found to be 16%. The rate constants of denaturation are faster than those of inactivation in the presence of ethanol.