Jennings Lori L, Malecki Michael, Komives Elizabeth A, Taylor Palmer
Department of Pharmacology, University of California, San Diego, La Jolla, California 92093, USA.
Biochemistry. 2003 Sep 23;42(37):11083-91. doi: 10.1021/bi034756x.
A sensitive matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry procedure has been established for the detection and quantitation of acetylcholinesterase (AChE) inhibition by organophosphate (OP) compounds. Tryptic digests of purified recombinant mouse AChE (mAChE) were fractionally inhibited by paraoxon to form diethyl phosphoryl enzyme. The tryptic peptide of mAChE that contains the active center serine residue resolves to a molecular mass of 4331.0 Da. Phosphorylation of the enzyme by paraoxon results in covalent modification of the active center serine and a corresponding increase in molecular mass of the tryptic peptide by 136 Da. The relative abundance of AChE peptides containing a modified active center serine strongly correlates with the fractional inhibition of the enzyme, achieving a detection range of phosphorylated to nonphosphorylated enzyme of 5-95%. Modifications of AChE by OP compounds resulting in dimethyl, diethyl, and diisopropyl phosphoryl adducts have been monitored with subpicomole amounts of enzyme. The individual phosphorylated adducts of AChE that result from loss of one alkyl group from the inhibited enzyme (the aging reaction) and the reappearance of unmodified AChE (spontaneous reactivation) have been resolved by the kinetic profiles and relative abundance of species. Further, the tryptic peptide containing the active center serine of AChE, isolated from mouse brain by anion-exchange and affinity chromatography, has been monitored by mass spectrometry. Native brain AChE, purified from mice treated with sublethal doses of metrifonate, has demonstrated that enzyme modifications resulting from OP exposure can be detected in a single mouse brain. For dimethyl phosphorylated AChE, OP exposure has been monitored by the ratio of tryptic peptide peaks that correspond to unmodified (uninhibited and/or reactivated), inhibited, and aged enzyme.
已建立一种灵敏的基质辅助激光解吸/电离飞行时间（MALDI-TOF）质谱方法，用于检测和定量有机磷酸酯（OP）化合物对乙酰胆碱酯酶（AChE）的抑制作用。纯化的重组小鼠AChE（mAChE）的胰蛋白酶消化产物被对氧磷部分抑制，形成二乙基磷酰化酶。mAChE中含有活性中心丝氨酸残基的胰蛋白酶肽段的分子量为4331.0 Da。对氧磷对该酶的磷酸化作用导致活性中心丝氨酸的共价修饰，相应地，该胰蛋白酶肽段的分子量增加136 Da。含有修饰活性中心丝氨酸的AChE肽段的相对丰度与该酶的部分抑制作用密切相关，磷酸化酶与非磷酸化酶的检测范围为5%-95%。已用亚皮摩尔量的酶监测了OP化合物对AChE的修饰作用，这些修饰作用会生成二甲基、二乙基和二异丙基磷酰化加合物。通过动力学曲线和各物种的相对丰度，解析了抑制酶失去一个烷基（老化反应）导致的AChE的各个磷酸化加合物，以及未修饰AChE（自发再活化）的重新出现。此外，通过阴离子交换和亲和色谱从鼠脑中分离出的含有AChE活性中心丝氨酸的胰蛋白酶肽段，已用质谱法进行监测。从用亚致死剂量敌百虫处理的小鼠中纯化得到的天然脑AChE表明，在单个小鼠脑中可检测到OP暴露导致的酶修饰。对于二甲基磷酸化的AChE，通过对应于未修饰（未抑制和/或再活化）、抑制和老化酶的胰蛋白酶肽峰的比例来监测OP暴露情况。
