Fluorescent probes as a measure of conformational alterations induced by nucleophilic modification and proteolysis of bovine .alpha.2-macroglobulin

Publisher Website
Google Scholar
Abstract
Conformational alterations occurring in bovine .alpha.2-macroglobulin (.alpha.2M) resulting from proteolysis and nucleophilic modification were monitored by UV difference spectra, circular dichroism and changes in the fluorescence of 6-(p-toluidino)-2-naphthalenesulfonate (TNS) and bis(8-anilino-1-naphthalenesulfonate) (Bis-ANS). These 2 dyes appear capable of differentiating between conformational changes induced by proteolysis and those induced by methylamine treatment. It appears that TNS is a sensitive probe for monitoring protease-induced but not methylamine-induced conformational changes in bovine .alpha.2M. Bis-ANS appears suitable for monitoring conformational changes induced by methylamine treatment or proteolysis of the molecule and was used as a probe to monitor the kinetics of the conformational change induced by methylamine treatment. The conformational change did not occur simultaneously with cleavage of the thiol ester bonds by the nucleophile, measured by titration of free SH groups with 5,5''-dithiobis(2-nitrobenzoate). The data are consistent with a model in which initial nucleophilic attack results in exposure of SH groups, resulting in a conformational change measured by an increase in fluorescence. This event is followed by a unimolecular step respresenting a conformational change in the protein that results in a further increase in the fluorescence signal. The second-order rate constant for hydrolysis of the thiol ester bonds was 3.4 .+-. 1.0 M-1 s-1, while the rate constant for the conformational change was (4.4 .+-. 0.8) x 10-4 s-1.