Identification and qualitative evaluation of sulfhydryl-or disulfide-containing compounds, on paper or thin-layer plates, by spot test is usually performed with the nitroprusside method (1) or with the modified Folin-Marenzi method (2), using 2,2'-dithiobis(5nitropyridine)
(3), 5,5'dithiobis(2-nitrobenzoic acid) (4) or 4,4'-bis(dimethylamino)diphenylcarbinol (5) as spray reagents. Other techniques are also reported ( 6), but they are of less general applicability.
Some of these methods can also be performed in the presence of cyanide or bisulfite in order to split the disulfide bond, the spot reaction being, in any case, specific for sulfhydryl groups only. The sensitivity ranges up to about 0.2 pg for the most sensitive method, but it greatly depends upon the thiol tested. Sometimes, the procedure is not very simple and requires reagents which are unstable or not commercially available. The time of appearance and of persistence of the colored spot can vary greatly. Interferences by thiosulfate or bisulfite, among other reagents, are occasionally reported.
We propose herein a new spot test on paper which does not seem to have the previously mentioned deficiencies, is easy to perform, and has a high sensitivity.
It is based on the appearance of a strong color (red or violet, depending upon the compound tested), stable for many days, when phenazine methosulfate (PMS) reacts with free sulfhydryl groups; the reaction is given by disulfides only after they have been treated with cyanide or bisulfite.
The interaction between PMS and -SH groups has been observed occasionally (7) but has never been fully studied: The red color, observed in solution also, is probably due to the appearance of phenazine methosulfate-2-one or -3-one, the absorption spectra of which show a maximum around 500 nm (8).
The spray test is performed with a freshly prepared 0.12% solution of PMS (a Sigma product) in 10e3 M phosphate buffer, pH 7, or in water; the color develops in a few minutes at room temperature (higher temperatures do not hasten the reaction), and it is stable for many days; the useful pH range is 7-8. The paper can also be sprayed before applying the PMS reagent, with a 1% solution of cyanide or a 10% solution of bisulfite in water, in order to split the disulfide bonds. Before being developed with PMS, the paper should be carefully dried. In any case, the intensely colored spot shows up against a pale green background in the presence of PMS reagent, a yellow one when bisulfite is also used, 610