Fluorescent thiol reagents. XII. Fluorescent tracer method for protein SH groups using N-(7-dimethylamino-4-methyl coumarinyl) maleimide. An application to the proteins separated by SDS-polyacrylamide gel electrophoresis

Abstract

A new fluorescent thiol reagent, N-(7-dimethylamino-4-methyl coumarinyl) maleimide (DACM), had a high molar extinction coefficient and a high quantum yield when reacted with protein SH groups. We could see directly the position of the labeled proteins in the polyacrylamide gel under ultraviolet illumination, since the maximum emission wavelength of DACM was about 480 nm. In addition, the quantum yield of DACM attached to the denatured proteins was almost identical regardless of the protein species. The properties enabled us to measure by fluorometry the amount of DACM, on a picomole level, incorporated in each protein band separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE). © 1977.