酶标甲胺磷交联反应的pH及缓冲体系优化

Optimization of pH and Buffer System for Catalase-labeled Methamidophos Crosslinking Reaction

采用碳二亚胺法制备酶标农药(过氧化氢酶标记甲胺磷),以酶标农药对鸡肝酯酶的抑制率和抑制后酶标农药-酯酶复合物中标记酶酶活为指标,对制备酶标甲胺磷交联反应的pH和所用缓冲液类型进行优化,并对其偶联率进行了分析。结果发现,pH增加有效促进了甲胺磷和标记酶分子之间的交联,而缓冲体系类型对此交联反应的影响相对较小。然而随之持续上升的偶联率带来的空间位阻效应也逐渐明显。随着pH增加,标记物抑制率和抑制后的标记酶活均呈现先增加后略下降的趋势,且分别在pH为6.0和5.5时达到峰值,其峰值时偶联率分别为24和22。综合考量,采用pH 6.0的2-(N-吗啉)乙磺酸一水合物(MES)缓冲液制备的酶标农药,其抑制率和标记酶活均达到较优值,分别为45.29%和47.67U,此时其偶联率约为24。

The preparation of catalase-labeled methamidophos（CLM） was carried out with a carbodiimide（EDC and sulfo-NHS as crosslinking agents） method. Using the inhibition rate of CLM to chicken liver esterase（CLE）and the catalase enzyme activity of CLM-CLE compounds as criteria, the pH and kinds of buffer were optimized in the crosslinking reaction and the coupling rate（methamidophos to catalase） was analyzed. The results indicated that the crosslinking reaction between methamidophos and catalase was promoted by the increasing of pH, while different kinds of buffer had little influence on the reaction. However, the steric-hindrance effect appeared along with the increasing of coupling rate. As the increasing of pH, inhibition rate and catalase enzyme activity firstly increased and then slightly decreased. They reached their peak values separately at pH 6.0 and 5.5 when the coupling rates were 24 and 22 respectively. Upon comprehensive consideration, pH 6.0 MES buffer was chosen. The inhibition rate and enzyme activity of the prepared CLM reached the appropriate values of 45.29% and 47.67 U,when the coupling rate was 24.