De Bisschop H C, Michiels K W, Vlaminck L B, Vansteenkiste S O, Schacht E H
Royal Military Academy, Brussels, Belgium.
Biochem Pharmacol. 1991;41(6-7):955-9. doi: 10.1016/0006-2952(91)90201-f.
Cholinesterases (EC 3.1.1.8, acylcholine acylhydrolase) from the sera of man, dog and pig were purified 400-600-fold using a combination of ion-exchange and affinity chromatography. In a first approach, phosphonylation by soman was studied by using the half-resolved epimers C(+)P(+/-)-soman and C(-)P(+/-)-soman. The degradation of soman at the nanomolar level was followed in time by determining the remaining soman by capillary gas chromatography with NP detection. In the three sera investigated the P-(-)-epimer phosphonylates at a higher rate than its corresponding P(+)-counterpart and the stereoselectivity is greater for the C(+)-epimers than for the C(-)-epimers. Individual soman isomers were isolated from C(+)- and C(-)-epimers and quantified by gas chromatography. Second-order rate constants were determined for the phosphonylation of purified cholinesterase by isolated soman isomers. The C(+)P(-)-isomer has the highest phosphonylation rate for the three species; the other toxic isomer, C(-)P(-), has a five to ten-fold lower rate. The overall stereoselectivity is more marked in human cholinesterase than in canine. Porcine serum cholinesterase is phosphonylated by the P(-)-isomers at a slightly higher rate than the human enzyme.
利用离子交换色谱法和亲和色谱法相结合的方法，将人、狗和猪血清中的胆碱酯酶（EC 3.1.1.8，酰基胆碱酰基水解酶）纯化了400 - 600倍。在第一种方法中，通过使用半拆分的差向异构体C(+)P(+/-)-梭曼和C(-)P(+/-)-梭曼研究了梭曼的膦酰化作用。通过使用氮磷检测的毛细管气相色谱法测定剩余的梭曼，及时跟踪纳摩尔水平下梭曼的降解情况。在所研究的三种血清中，P(-)-差向异构体的膦酰化速率高于其相应的P(+)-对应物，并且C(+)-差向异构体的立体选择性大于C(-)-差向异构体。从C(+)-和C(-)-差向异构体中分离出单个梭曼异构体，并通过气相色谱法定量。测定了分离出的梭曼异构体对纯化胆碱酯酶进行膦酰化作用的二级速率常数。对于这三种物种，C(+)P(-)-异构体具有最高的膦酰化速率；另一种有毒异构体C(-)P(-)的速率低五到十倍。人胆碱酯酶中的整体立体选择性比犬胆碱酯酶更明显。猪血清胆碱酯酶被P(-)-异构体膦酰化的速率略高于人酶。
