铜Ⅱ化合物水解肌红蛋白活性位点的质谱研究  

Mass Spectrometric Study on Active Sites of Myoglobin Hydrolyzed by CuⅡ Compounds

作　　者：张琳[1] 温燕梅[1] 谢建英[1] 

机构地区：[1]广东海洋大学理学院,湛江524088

出　　处：《分析化学》2009年第9期1371-1374,共4页Chinese Journal of Analytical Chemistry

摘　　要：以铜Ⅱ化合物为金属模拟酶,通过聚丙烯酰胺凝胶电泳研究它们对肌红蛋白的水解作用,利用高效液相色谱-电喷雾质谱的测量方法替代传统的N端氨基酸序列分析结合基质辅助激光解吸电离飞行时间质谱或电喷雾质谱方法来检测得到的多肽片断分子量,据此确定水解反应的活性位点。结果得到分子量为10093.0,10388.0,6876.0和6581.0的4个片段,分别对应于Gly1-91,Gly1-Ala94,Ser92-Gly153和Thr95-Gly153,表明Cu2+通过与肌红蛋白中His93的侧链配位,水解断裂位于其前后沿的第2个肽键,即:Gln91-Ser92和Ala94-Thr95,水解反应具有很高的选择性。位于His93前沿的活性位点特征与过去的研究结果一致,而His93后沿的活性位点,即His93-Ala94-Thr95序列中Thr95N端是一个新位点。Hydrolyzation of myoglobin was studied by means of polyacrylamide gel electrophoresis using CuⅡ compounds as artificial metallopeptidase.High performance liquid chromatography-electrospray mass spectrometric method was used to replace the traditional N-terminal amino acid sequence combined with matrix-assisted laser desorption-ionization time of flight-mass spectrometry（MALDI-TOF-MS） and ESIMS to measure the molecular masses of fragments obtained from the hydrolyzation and determine the hydrolytic active sites.Four fragments with molecular mass of 10093.0,10388.0,6876.0 and 6581.0 were obtained,corresponding to fragment of Gly1-91,Gly1-Ala94,Ser92-Gly153 and Thr95-Gly153 respectively.The results indicated that Cu can degrade myoglobin through anchoring to the side chain of His93 which is detached from iron,and breaks the second peptide bonds of both upstream and downstream：Gln91-Ser92 and Ala94-Thr95.The active site of Gln91-Ser92 has been observed in other studies of protein hydrolysis by Cu compounds,however,the site of Ala94-Thr95 is a new active site located at the N-terminal of Thr in His93-Ala94-Thr95 sequence.This site has not been reported in the investigations of protein hydrolyzation with CuⅡ compounds.

关 键 词：铜 肌红蛋白 高效液相色谱-电喷雾质谱联用 水解作用 

分 类 号：R341[医药卫生—基础医学]