脂肪酶活力测定中终止反应方法的比较分析

摘要

[Objective]Searching an accurate and effective termination reaction method in lipase activity assay for studying lipase enzymatic characters.[Methods]Comparative analyses were conducted in inhibiting effect and result stability of termination methods(absolute ethanol,ED-TA,trichloroacetic acid,sodium carbonate and boiling water bath),which predecessors used generally in lipase activity assay.[Results]Inhibition ratio of termination by absolute ethanol was 97.56%,and net absorbance reduced by 5% within 5min after termination;Inhibition ratio of terminaion by EDTA was 32.94%,and net absorbance increased by 5% within 10min after termination;Inhibition ratio of termination by trichloroacetic acid was 99.46%,and coloration net absorbance increased by 0.47% within 10min after termination;Inhibition ratio of termina-tion by sodium carbonate was 76.44%,and net absorbance increased by 0.53% within 10min after termination;Inhibition ratio of termination by boiling water was 99.51%,and net absor-bance reduced by 0.77% within 10min after termination, but it would result in the decomposition of most remaining substrates.[Conclusion]The termination method of trichlo-roacetic acid is an accurate and effective one in li-pase activity assay,and can be adopt in analyzing lipase enzymatic characters.%【目的】寻找一种准确有效的脂肪酶活力测定中的终止反应方法，以进行脂肪酶酶学性质研究。【方法】比较分析前人研究脂肪酶常用的终止方法（无水乙醇法、EDTA 法、三氯乙酸法、碳酸钠法、沸水浴法）对酶活力的抑制效果和吸光值的稳定性。【结果】无水乙醇法对酶活的抑制率为97．56％，终止反应后5min 内净吸光值减少5％；EDTA 法对酶活抑制率为32．94％，终止反应后10min 内净吸光值增加5％；三氯乙酸法对酶活的抑制率为99．46％，终止反应后10min 内净吸光值增加了0．47％；碳酸钠法对酶活抑制率为76．44％，终止反应后10min 内净吸光值增加了0．53％；沸水浴法对酶活抑制率为99．51％，终止反应后10min 内净吸光值减少0·77％，但沸水浴会导致剩余底物的大量分解。【结论】三氯乙酸法是一种准确有效的终止脂肪酶活力测定反应的方法，可用于脂肪酶酶学性质的研究。