Protein misfolding plays a role in the pathogenesis of many diseases. ␣ 1 -Antitrypsin misfolding leads to the accumulation of long chain polymers within the hepatocyte, reducing its plasma concentration and predisposing the patient to emphysema and liver disease. In order to understand the misfolding process, it is necessary to examine the folding of ␣ 1 -antitrypsin through the different structures involved in this process. In this study we have used a novel technique in which unique cysteine residues were introduced at various positions into ␣ 1 -antitrypsin and fluorescently labeled with N,N-dimethyl-N-(iodoacetyl)-N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-ethylenediamine. The fluorescence properties of each protein were studied in the native state and as a function of guanidine hydrochloride-mediated unfolding. The studies found that ␣ 1 -antitrypsin unfolded through a series of intermediate structures. From the position of the fluorescence probes, the fluorescence quenching data, and the molecular modeling, we show that unfolding of ␣ 1 -antitrypsin occurs via disruption of the A and C ␤-sheets followed by the B ␤-sheet. The implications of these data on both ␣ 1 -antitrypsin function and polymerization are discussed.Protein folding is the process by which the primary sequence is translated into tertiary structure. Modification of this sequence via in vivo mutation or in vitro mutagenesis often alters the ability of the protein to fold correctly. Misfolding of proteins during their synthesis within the cell can lead to loss of protein function (1), an example being ␣ 1 -antitrypsin (␣ 1 -AT) 1 deficiency (2-4). Many well characterized ␣ 1 -AT variants are associated with an ␣ 1 -AT plasma deficiency; in these cases, the variants aggregate at their site of synthesis within the liver cell (5, 6). This aggregation is via an ordered polymerization process that is initiated by protein misfolding. In the case of the ␣ 1 -AT Z mutation (Glu-342 3 Lys), the rate of protein folding is much slower than in the native state, leading to the accumulation of an intermediate, which then polymerizes (4). It has recently been shown that the heat-induced polymerization of ␣ 1 -AT also involves the formation of an unfolding intermediate (7). The actual process of polymerization has been well studied, and two mechanisms have been proposed: the loop-A-sheet and loop-C-sheet mechanisms (8 -11). Both processes involve the insertion of the reactive center loop residues of the donating ␣ 1 -AT molecule into either the A ␤-sheet (loop-A-sheet) or the C ␤-sheet (loop-C-sheet) of the acceptor molecule (11). There is evidence for the occurrence of both of these mechanisms; indeed, in vitro ␣ 1 -AT has been shown to undergo both depending upon the buffer used (12-14).␣ 1 -AT is a member of the serine proteinase inhibitor (serpin) superfamily and is composed of 394 amino acid residues arranged into three ␤-sheets (A, B, and C) and nine ␣-helices (A-I). X-ray crystallographic and biochemical data have shown that ␣ 1 -AT can adopt a number of ...

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