Polyacrylamide gels which contain a novel mixed disulfide compound can be used to detect enzymes that catalyze thiol-producing reactions.
The synthesis of N-[5-(hydroxyethyl)dithio-2-nitrobenzoylaminoethyl] acrylamide (I) is described. If the disulfide bond in this compound is reduced with thiol reagents, an intense yellow color develops (epsilon 412 V 13,600 at pH 7.4) due to essentially the same chromophore as 5-thio-2-nitrobenzoic acid, the reduced form of 5,5'-dithiobis(2-nitrobenzoic acid)(Ellman's reagent). Polyacrylamide gels were prepared that were crosslinked with N,N'-methylenebisacrylamide and which contained I as an integral part of the polymerized acrylamide chain. Acetylcholinesterase (from electric eel and human brain tissue slices) and alkaline phosphatase (from Escherichia coli and calf intestine) were subjected to electrophoresis and then the gels were immersed in an appropriate thiol-substrate buffer (acetylthiocholine and cysteamine-S-phosphate, respectively). A yellow band developed rapidly in the acrylamide gel at the site of enzyme activity. Electrophoresis on the mixed disulfide-polyacrylamide gel proved to be a rapid and sensitive technique to detect very small amounts of enzyme (approximately 0.02 fmol acetylcholinesterase) and should have wide application for detecting other enzymes that hydrolyze thiol substrates.
D000110:Acetylcholinesterase / Q000032:analysis*; D000469:Alkaline Phosphatase / Q000032:analysis*; D000818:Animals; D001921:Brain / Q000201:enzymology; D002384:Catalysis; D002417:Cattle; D055598:Chemical Phenomena; D002621:Chemistry; D004220:Disulfides; D004557:Electric Organ; D004591:Electrophoresis, Polyacrylamide Gel; D004593:Electrophorus; D004926:Escherichia coli / Q000201:enzymology; D006801:Humans; D007422:Intestines / Q000201:enzymology; D013053:Spectrophotometry; D013438:Sulfhydryl Compounds / Q000378:metabolism*
D004220:Disulfides; D013438:Sulfhydryl Compounds; D000110:Acetylcholinesterase; D000469:Alkaline Phosphatase