Maleimides can be directly assayed spectrophotometrically at 302 nm. However, the small extinction coefficient of 620 M^-1cm^-1 renders this assay insensitive, and the assay is further complicated by the protein absorbance at the same wavelength. Although the enzyme-based maleimide quantification is more sensitive, the method is expensive and extremely time-consuming.

Our Amplite^® Colorimetric Maleimide Assay Kit quantifies maleimide groups by first reacting a sample with a known amount of thiol present in excess and then assaying the remaining unreacted thiol using 4,4'-DTDP with a molar extinction coefficient of 19,800 M^-1cm^-1. The amount of maleimide is calculated as the difference between the initial amount of thiol and the amount of unreacted thiol after the complete reaction of all maleimide groups. This spectrophotometric assay for the determination of maleimide groups is a reverse GSH assay. It takes advantage of the high reactivity of thiols of GSH with the maleimide moiety. Maleimide of the sample is allowed to form a stable thiosuccinimidyl linkage with GSH. After the reaction is complete, the excess GSH, i.e., the remaining thiols of GSH in the reaction mixture, is estimated by using 4, 4'-DTDP. The amount of GSH reacted with the sample is titrated to determine the extent of maleimide.