Effects of Oxygen on Determination of Thiocholine by 5,5′-Dithiobis (2-Nitrobenzoic Acid) . ∗

Summary

The DTNB (5,5′-dithiobis-2-nitrobenzoic acid) method has been recommended for measuring the hydrolysis of thiocholine esters. During studies on the alkaline hydrolysis of benzoylthiocholine, however, analytical errors have been encountered and traced to the presence of molecular oxygen. Although oxygen does not influence the rate of benzoylthiocholine hydrolysis, it does compete with DTNB for the hydrolysis product thiocholine. Furthermore it produces fading of the chromogen on which the assay is based by oxidizing the monothiol TNB back to the DTNB. Thus the amount of thiocholine released is significantly underestimated if hydrolysis periods of 30 minutes or more are employed. In the absence of oxygen DTNB can be added at any time during the hydrolysis, but in the procedure recommended here it is present throughout. This modified DTNB method was used to determine rate constants for auto-oxidation of thiocholine and for spontaneous alkaline hydrolysis of acetyl- and benzoylthiocholines. Unlike acetylthiocholine, benzoylthiocholine proved to be so unstable in various buffered solutions at pH 8 that it appears to have only limited usefulness as an enzyme substrate.