THE MECHANISM OF ALPHA(1)-ANTITRYPSIN POLYMERIZATION PROBED BY FLUORESCENCE SPECTROSCOPY

The polymerization of alpha(1)-antitrypsin within the hepatic cell lea ds to alpha(1)-antitrypsin deficiency. Both the conformational changes and the kinetics of the polymerization process are poorly understood. Here we describe fluorescence experiments investigating the polymeriz ation reaction using the fluorescent probe 4,4'-dianilino-1,1'-binapht hyl-5,5'-disulfonate (bis-ANS) which bound to both native and polymeri zed alpha(1)-antitrypsin. Biphasic changes in bis-ANS fluorescence wer e observed during formation of alpha(1)-antitrypsin polymers. Initiall y a rapid increase in fluorescence signal was observed; it was followe d by a gradual reduction in fluorescence signal. The first phase is a conformational change in which the A beta-sheet of alpha(1)-antitrypsi n opens, whereas the second phase represents the insertion of the reac tive center loop into the A beta-sheet of another molecule and therefo re determines the rate of the polymerization process. (C) 1998 Academi c Press.