The mechanism of alpha 1-antitrypsin polymerization probed by fluorescence spectroscopy.
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.
Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria, 3168, Australia.
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James
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Bottomley
D001704:Biopolymers / Q000737:chemistry / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D005456:Fluorescent Dyes / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D013050:Spectrometry, Fluorescence; D000515:alpha 1-Antitrypsin / Q000737:chemistry / Q000378:metabolism*
D001704:Biopolymers; D005456:Fluorescent Dyes; D000515:alpha 1-Antitrypsin