The effect of denaturants on the quantitation of free sulfhydryl groups in the enzyme rhodanese (thiosulfate sulfurtransferase, EC 18.104.22.168) has been reinvestigated in some detail. The sulfhydryl assay with the colorimetric reagent 5,5′-dithio-bis(2-nitrobenzoic acid) Nbs2 shows four sulfhydryl groups per enzyme molecule (mol. wt. 32 600) when the colorimetric reagent is added to the assay mixture before the denaturant, sodium dodecyl sulfate. On the other hand, only two sulfhydryl groups per molecule are observed when Nbs2 is added after denaturation has been initiated. The time dependence observed in this latter procedure indicates that the loss of the two groups is rapid and permanent. The results depend on the denaturant used: urea acts like sodium dodecyl sulfate while guanidine reveals four sulfhydryl groups independent of reagent order. The assay also gives four sulfhydryl groups independent of reagent order with urea or sodium dodecyl sulfate under conditions which are expected to limit metal ion-catalyzed oxidation of sulfhydryl groups (e.g. oxygen exclusion or metal ion chelation). Recent studies have shown that rhodanese has a molecular weight of 32 600, no disulfides and four sulfhydryl groups per molecule. These results together with the observations reported here are taken to indicate that a disulfide can be formed during denaturation of rhodanese and that the pathway of denaturation determines the result obtained.
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