Proteinase inhibitor members of the SERPIN superfamily are characterized by the presence of a proteolytically sensitive reactive-site loop. Cleavage within this region results in a conformational transition from an unstable "stressed" native protein to a more stable "relaxed" cleaved molecule. In order to identify the principal molecular aspects of this transition, 1H nuclear magnetic resonance (n.m.r.) and FT-IR spectroscopy were applied to the study of four SERPINs. 1H n.m.r. spectra of approximately 20 high-field ring-current-shifted methyl signals exhibited slightly different chemical shifts in the native and cleaved forms of α1-antitrypsin (α1-AT), α1-antichymotrypsin (α1-ACT) and C1̄ inhibitor (C1̄-INH), but not ovalbumin, between 20 °C and 90 °C. Ring current calculations based on crystal co-ordinates for cleaved α1-AT and α1-ACT and native ovalbumin showed that these signals originate from highly localized interactions between different buried residues corresponding to α-helix and β-sheet segments of the SERPIN fold. The small shift changes correspond to small relative conformational side-chain rearrangements of about 0.01 nm to 0.05 nm in the protein hydrophobic core, i.e. the tertiary structure interactions in the two forms of the SERPIN fold are well-preserved, and changes in this appear unimportant for the stabilization found after reactive centre cleavage. Fourier transform infrared (FT-IR) spectroscopic studies of the amide I band showed that the native and cleaved forms of α1-AT, α1-ACT and C1̄-INH contain 28% to 36% α-helix and 38% to 44% β-sheet. Second derivative FT-IR spectra using H2O and 2H2O buffers revealed very large differences in the amide I band between the native and cleaved forms of α1-AT, α1-ACT and C1̄-INH, but not for ovalbumin. The α-helix band was most sensitive to 1H-2H exchange, while the β-sheet bands were not, and greater amounts of antiparallel β-sheet were detected in the cleaved form. 1H n.m.r. showed that polypeptide amide 1H-2H exchange was greater in the native forms of α1-AT, α1-ACT and C1̄-INH than in their cleaved forms, whereas for ovalbumin it was unchanged. The FT-IR and 1H-2H exchange data show that alterations in the secondary structure are central to the stabilization of the cleaved SERPIN structure. The basis of the energetically stressed native SERPIN structure can be attributed to a large number of weakened hydrogen bonds in α-helix and β-sheet secondary structure components, not to alterations in its tertiary structure. This stress is relieved upon peptide cleavage at the reactive centre. © 1992.