The mechanism of alpha 1-antitrypsin polymerization probed by fluorescence spectroscopy

Abstract

The polymerization of alpha1-antitrypsin within the hepatic cell leads to alpha1-antitrypsin deficiency. Both the conformational changes and the kinetics of the polymerization process are poorly understood. Here we describe fluorescence experiments investigating the polymerization reaction using the fluorescent probe4, 4'-dianilino-1,1'-binaphthyl-5,5'-disulfonate (bis-ANS) which bound to both native and polymerized alpha1-antitrypsin. Biphasic changes in bis-ANS fluorescence were observed during formation of alpha1-antitrypsin polymers. Initially a rapid increase in fluorescence signal was observed; it was followed by a gradual reduction in fluorescence signal. The first phase is a conformational change in which the A beta-sheet of alpha1-antitrypsin opens, whereas the second phase represents the insertion of the reactive center loop into the A beta-sheet of another molecule and therefore determines the rate of the polymerization process.