Microfluorometric analysis of protein thiol groups with a coumarinylphenylmaleimide

Characteristics of thiol adducts formed by the fluorogenic maleimide, N-(4-(7-diethylamino-4-methylcoumarin-3-yl) phenyl) maleimide (CPM), (Sippel: J Histochem Cytochem 29:314, 1981) were determined in solution, on the epithelia in beef cornea paraffin sections, and with an egg white model. Absorption was found maximal at 385-390 nm with a molar absorbancy of not less than 30,000 cm2/mmol, while emission peaked at 465 nm; the spectra and output were hardly affected by hydrolytic opening of the succinimide ring. Fluorescence measured in an epi-illuminating microfluorometer faded rapidly at high magnifications, but the initial output from sections of increasing thickness under a 10 x objective was proportional to the thiol density (concentration x thickness) up to a limit equivalent to an absorbance of nearly 0.5 at 387 nm. The staining included less than 5% nonspecific fluorescence, as determined on duplicate sections blocked by 2,2'-dithiopyridine. Factors affecting the use of CPM for quantitation of both thiols and disulfides are discussed.