Local environment perturbations in alpha1-antitrypsin monitored by a ratiometric fluorescent label

Abstract

The complex multistep inhibition of proteinases by alpha₁-antitrypsin (α₁-AT) was investigated by covalently labeling its unique Cys residue with a ratiometric environment-sensitive fluorescent dye, 6-bromomethyl-2-(2-furanyl)-3-hydroxychromone (BMFC). The binding of BMFC-labeled α₁-AT with pancreatic elastase led to significant changes in the dual emission of BMFC. The 8 nm blue shift of one of the bands and ca. 65% change in the intensity ratio of the two emission bands suggested an increased exposure of the labeled Cys-232 residue to the bulk water on complex formation. In contrast, the bacterial V8 proteinase-induced cleavage of the reactive center loop of BMFC-labeled α₁-AT did not generate any significant change in the Cys-232 region. Similar experiments with elastase and α₁-AT conjugated to the classical environment-sensitive dye, IANBD, confirmed these results but led to much smaller modifications in the emission spectrum. Stopped-flow investigation of the reaction between BMFC-labeled α₁-AT and elastase showed both a well-described fast and a new slow step of the inhibition process. The latter step is probably associated with the structural reorganization aimed at stabilizing the final complex. These results present a convenient fluorescence ratiometric approach based on the BMFC label for studies of protein conformational changes.