Folding pathway of human alpha(1)-antitrypsin: Characterization of an intermediate that is active but prone to aggregation

The folding-unfolding kinetics of human alpha(1)-antitrypsin (alpha(1)-AT) were examined by monitoring intrinsic Trp fluorescence and extrinsic ANS fluorescence. While the unfolding of alpha(1)-AT followed a single exponential phase, refolding exhibited three exponential phases. The fast phase (tau(1,r) < 40 sec), which was independent of urea concentration, appears to be hydrophobic collapse that may be limited by cis-trans isomerization of prolyl residue. The medium phase (tau(2,r) = 200 sec) yielded an intermediate (I-N), which is capable of elastase binding. The slowest (tau(3,r) = 1000 sec) phase completes refolding to the native protein, which intersects with the unfolding kinetics at the same urea concentration (1.9 M) as the equilibrium midpoint. Concentration-dependence of the amplitude of major refolding phases indicated that I-N is prone to kinetic competition between the on-pathway to native protein and aggregation. (C) 1996 Academic Press, Inc.