N-(1-Pyrene)maleimide: a fluorescent crosslinking reagent

Publisher Website
Google Scholar
Abstract
N-(1-Pyrene)maleimide (PM) is nonfluorescent in aqueous solution but forms strongly fluorescent adducts with sulfhydryl groups of organic compounds or proteins. The conjugation reactions of PM are relatively fast and can be monitored by the increase in fluorescence intensity of the pyrene chromophore. In cases where primary amino groups are also present in the stem, a red shift of the emission spectra of the fluorescent adducts subsequent to the initial conjugation is observed, as characterized by the disappearance of 3 emission peaks at 376, 396 and 416 nm, and the appearance of 2 new peaks at 386 and 405 nm. Model studies with adducts of L-cysteine and cysteamine indicate that the spectral shift is the result of an intramolecular aminolysis of the succinimido ring in the adducts. Evidence from both chemical analysis and NMR studies of the addition products supports this reaction scheme. PM adducts of N-acetyl-L-cysteine and .beta.-mercaptoethanol, which have no free amino group, do not exhibit a spectral shift. Among several protein conjugates only the PM adduct of bovine serum albumin (PM-BSA) shows the spectral shift resembling that of PM-cysteine. PM reacts with the sulfhydryl group of the single cysteine residue at position 34 in BSA. The finding that the .alpha.-amino group of the N-terminus in PM-BSA is blocked after the spectral shift is completed strongly suggests that PM cross-links the N-terminus and the cysteine residue in BSA. The relative proximity of the sulfhydryl and amino groups is very critical in the cross-linking as demonstrated by the observation that the spectral shift observed with PM-BSA can be prevented by addition of denaturating reagents such as 1% sodium dodecyl sulfate immediately after labeling and by the failure of PM-glutathione to undergo the intramolecular aminolysis. Since the intramolecular rearrangement of PM adducts is associated with characteristic fluorescence changes, PM can serve as a fluorescent cross-linking reagent which provides information about the spatial proximity of sulfhydryl and amino groups in proteins.