在偶联分光光度法中5,5'-二硫代双-(2-硝基苯甲酸)对胆碱酯酶活性的修饰。非催化性底物结合位点的证据。

The modification of cholinesterase activity by 5,5'-dithiobis-(2-nitrobenzoic acid) included in the coupled spectrophotometric assay. Evidence for a non-catalytic substrate-binding site.

Brownson C, Watts D C

Biochem J. 1973 Feb;131(2):369-74. doi: 10.1042/bj1310369.

Abstract

Compared with the acetylcholinesterase assay carried out in the absence of a dithiol, the presence of 5,5'-dithiobis-(2-nitrobenzoic acid) caused marked activation, 6,6'-dithiodinicotinic acid and 2,2'-dithiobis-(5-nitropyridine) less so and 2,2'-dithiodipyridine (aldrithiol-2) had no effect at all. Measurements are further complicated in that the 5-thio-2-nitrobenzoate ion also appears to interact with the enzyme, resulting in slightly lowered absorbance values. 2. Acetylthiocholine competes for the 5,5'-dithiobis-(2-nitrobenzoic acid)-binding site so that activation is essentially eliminated by saturating concentrations of substrate. The presence of the dithiol decreases the K(m) value of acetylthiocholine. 3. Similar results were obtained with pseudocholinesterase. However, with butyrylthiocholine clear activation was still observed under V(max.) conditions in addition to K(m) being lowered. 4. All the data yielded Hill coefficients of 1 and analysis of the results leads to the conclusion that activation results from the dithiol being bound to a site on the subunit that is actively catalysing ester hydrolysis. 5. The use of aldrithiol-2 is recommended for kinetic work where absolute quantitative measurements are required.

摘要

与在无二硫醇存在的情况下进行的乙酰胆碱酯酶测定相比，5,5'-二硫代双(2-硝基苯甲酸)的存在会引起显著激活，6,6'-二硫代烟酸和2,2'-二硫代双(5-硝基吡啶)的激活作用稍弱，而2,2'-二硫代二吡啶(双硫仑-2)则完全没有作用。测量进一步复杂化的原因是5-硫代-2-硝基苯甲酸离子似乎也与该酶相互作用，导致吸光度值略有降低。2. 乙酰硫代胆碱竞争5,5'-二硫代双(2-硝基苯甲酸)的结合位点，因此通过底物的饱和浓度可基本消除激活作用。二硫醇的存在降低了乙酰硫代胆碱的K(m)值。3. 假胆碱酯酶也得到了类似的结果。然而，对于丁酰硫代胆碱，除了K(m)降低外，在V(max.)条件下仍观察到明显的激活作用。4. 所有数据的希尔系数均为1，对结果的分析得出结论，激活作用是由于二硫醇与亚基上一个积极催化酯水解的位点结合所致。5. 对于需要进行绝对定量测量的动力学研究，建议使用双硫仑-2。