A sensitive assay for maleimide groups.
A sensitive spectrophotometric assay has been developed for maleimide groups incorporated into proteins. The assay involves the reaction of maleimide with an excess of cysteine and quantitation of the remaining cysteine using an enzymatic assay that is based on the stoichiometric conversion of an inactive disulfide form of papain to the active enzyme. The restored enzymatic activity is then measured using a chromogenic substrate. The amount of maleimide is calculated as the difference between the amounts of initial cysteine and assayed remaining cysteine. The limits of detection of this assay are about 0.1 nmol maleimide in a final assay volume of 1.2 mL. This assay is about 100-fold more sensitive than a similar assay using Ellman's reagent.
Singh
R
R
ImmunoGen, Inc., Cambridge, Massachusetts 02139.
D003545:Cysteine / Q000737:chemistry; D004228:Dithionitrobenzoic Acid; D005033:Ethylmaleimide / Q000737:chemistry; D007700:Kinetics; D008301:Maleimides / Q000737:chemistry*; D010206:Papain / Q000737:chemistry; D011506:Proteins / Q000737:chemistry*
D008301:Maleimides; D011506:Proteins; D004228:Dithionitrobenzoic Acid; D010206:Papain; D003545:Cysteine; D005033:Ethylmaleimide