The two forms of chicken cystatin, with different isoelectric points, that have been described previously were indistinguishable in analyses of amino- and carboxy-terminal residues, amino acid composition, and peptide maps. The two forms thus are highly similar and most likely differ only in an amide group or in a small charged substituent. The binding of either cystatin form to highly purified, active papain was accompanied by the same pronounced changes in near-ultraviolet circular dichroism, ultraviolet absorption, and fluorescence emission. These changes were compatible with perturbations of the environment of aromatic residues in one or both proteins of the complex, arising from local interactions or from a conformational change. Modification of the single tryptophan residue of cystatin, at position 104, with N-bromosuccinimide resulted in considerably smaller spectroscopic changes on binding of the inhibitor to papain, indicating that the environment of this residue is affected by the binding. Analogous modification of Trp-69 and Trp-177 of papain markedly affected the fluorescence changes observed on binding of cystatin to the enzyme, similarly suggesting that these two residues of papain are involved in the interaction. The fluorescence increase of papain at alkaline pH, arising from Trp-177 and due to deprotonization of the adjacent His-159, was abolished on binding of cystatin to the enzyme, further supporting the proposal that this region of papain participates in the interaction with the inhibitor. A stoichiometry of binding of either cystatin form to papain of 1:1 and a lower limit for the binding constant of 10(9) M-1 were determined by titrations monitored by either the ultraviolet absorption or fluorescence changes induced by the interaction.

The interaction between recombinant human cystatin C and the cysteine proteinases papain and actinidin was studied by spectroscopic, kinetic and equilibrium methods. The absorption, near-u.v.c.d. and fluorescence-emission difference spectra for the cystatin C-proteinase interactions were all found to be similar to the corresponding spectra for chicken cystatin. The kinetics of binding of cystatin C to the two enzymes were best described by a simple reversible one-step bimolecular mechanism, like the kinetics of the reaction of chicken cystatin with several cysteine proteinases. Moreover, the second-order association rate constants at 25 degrees C, pH 7.4 and I0.15, of 1.1 x 10(7) and 2.4 x 10(6) M-1.s-1 for the reactions of cystatin C with papain and actinidin respectively, were similar to the corresponding rate constants for the chicken inhibitor and close to the value expected for a diffusion-controlled rate. The dissociation equilibrium constants, approx. 11 fM and approx. 19 nM for the binding of cystatin C to papain and actinidin respectively, were also comparable with the dissociation constants for chicken cystatin. The affinity between cystatin C and several inactivated papains or actinidins decreased with increasing size of the inactivating group in a manner similar to that in earlier studies with the chicken inhibitor. Together, these results strongly indicate that the mechanisms of the reactions of cystatin C and chicken cystatin with cysteine proteinases are identical or highly similar, but differ from that of reactions between serine-proteinase inhibitors and their target enzymes. The model for the proteinase-inhibitor interaction, based on the X-ray structure of chicken cystatin, therefore should be largely applicable also to human cystatin C.

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The interaction between five N-terminally truncated forms of chicken cystatin (starting at Leu-7, Leu-8, Gly-9, Ala-10 and Asp-15) and the cysteine proteinases papain and actinidin was studied by spectroscopic, kinetic and equilibrium methods. The u.v. absorption, near-u.v. c.d. and fluorescence emission difference spectra for the interactions with papain were all similar to the corresponding spectra for intact cystatin. The second-order association rate constants at 25 degrees C, pH 7.4, I 0.15, for the binding of the truncated forms to papain varied about 2-fold, from 6 x 10(6) to 1.5 x 10(7) M-1.s-1, and were comparable to the value of 9.9 x 10(6) M-1.s-1 for intact cystatin. In contrast, the rate constants for the dissociation of the complexes with papain increased markedly with increasing extent of truncation, from 7.5 x 10(-6)s-1 for Leu7 cystatin (a truncated form of cystatin having Leu-7 as its N-terminal amino acid) to 1.6s-1 for Ala10-cystatin, whereas the dissociation rate constants for the latter form and Asp15-cystatin were similar. Consequently, the binding affinities between the truncated cystatins and papain decreased in an analogous manner, as was also shown for the interaction with actinidin by equilibrium measurements. Studies of the binding of the truncated cystatins to inactivated papains indicated that small substituents on the active-site cysteine of the enzyme can be accommodated in the complex without any loss of affinity when the N-terminal segment of the inhibitor is removed. Taken together, the results suggest that in the N-terminal region of chicken cystatin only residues preceding Ala-10 participate in the interaction with proteinases. Of these residues, Leu-7 and Leu-8 together account for about two-thirds of the unitary free energy of binding contributed by the N-terminal region, the relative importance of the two residues being dependent on the target proteinase. Both Gly-9 and residues N-terminal of Leu-7 further stabilize the interaction but contribute substantially smaller binding energies than do the two leucine residues.

The conformational changes around the thioester-bond region of human or bovine alpha 2M (alpha 2-macroglobulin) on reaction with methylamine or trypsin were studied with the probe AEDANS [N-(acetylaminoethyl)-8-naphthylamine-1-sulphonic acid], bound to the liberated thiol groups. The binding affected the fluorescence emission and lifetime of the probe in a manner indicating that the thioester-bond region is partially buried in all forms of the inhibitor. In human alpha 2M these effects were greater for the trypsin-treated than for the methylamine-treated inhibitor, which both have undergone similar, major, conformational changes. This difference may thus be due to a close proximity of the thioester region to the bound proteinase. Reaction of trypsin with thiol-labelled methylamine-treated bovine alpha 2M, which retains a near-native conformation and inhibitory activity, indicated that the major conformational change accompanying the binding of proteinases involves transfer of the thioester-bond region to a more polar environment without increasing the exposure of this region at the surface of the protein. Labelling of the transglutaminase cross-linking site of human alpha 2M with dansylcadaverine [N-(5-aminopentyl)-5-dimethylaminonaphthalene-1-sulphonamide] suggested that this site is in moderately hydrophobic surroundings. Reaction of the labelled inhibitor with methylamine or trypsin produced fluorescence changes consistent with further burial of the cross-linking site. These changes were more pronounced for trypsin-treated than for methylamine-treated alpha 2M, presumably an effect of the cleavage of the adjacent 'bait' region. Solvent perturbation of the u.v. absorption and iodide quenching of the tryptophan fluorescence of human alpha 2M showed that one or two tryptophan residues in each alpha 2M monomer are buried on reaction with methylamine or trypsin, with no discernible change in the exposure of tyrosine residues. Together, these results indicate an extensive conformational change of alpha 2M on reaction with amines or proteinases and are consistent with several aspects of a recently proposed model of alpha 2M structure [Feldman, Gonias & Pizzo (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 5700-5704].