Quantifying sulfhydryl oxidation rates using Ellman's procedure

• DOI: 10.1063/5.0246333
• Publication Date: 2025-01-07T14:38:11Z
• Authors: Wei Cao; Gurpreet S. Matharoo; M. Shajahan Gulam...; Erzsebet Szabo; Katarin A. MacLeod; Iris J. Joye; David A. Pink

Ellman's procedure has been used to study the oxidation rates of cysteine (CSH) and glutathione (GSH) in aqueous solutions, and it was reported that, for CSH, the number of sulfhydryl molecules not oxidized became zero at a specific time, called tc, where it was reported to be finite. We point out that under very general considerations, we should observe tc to be unbounded, and it becomes infinite. The reason is that as the process of forming a disulfide bond proceeds, the probability of two CSH molecules finding each other eventually becomes vanishingly small so that the number of unoxidized molecules approach zero only as tc becomes infinite. We used a Smoluchowski equation to model the process of disulfide bond formation in order to understand how a finite tc can be observed. In addition, atomic scale molecular dynamics simulations were carried out in order to study the spatial distributions of CSH and GSH in aqueous solutions. It was found that electrostatic interactions bring about aggregation of these molecules, and we conclude that this aggregation “hides” unoxidized sufhydryl moieties from interacting with (5,5′-dithiobis-(2-nitrobenzoic acid) DTNB of Ellman's reagent, thereby remaining undetected. It will thus appear as if the number of unoxidized moieties has become zero. In order that all sulfhydryl moieties be detected, it is necessary to disrupt the aggregate, as has been carried out for proteins, so as to expose those moieties to be oxidized and be detected.