Interaction of the cysteine proteinase inhibitor chicken cystatin with papain.
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.
Lindahl
P
P
Department of Veterinary Medical Chemistry, Swedish University of Agricultural Sciences, Uppsala Biomedical Center.
Alriksson
E
E
Jörnvall
H
H
Björk
I
I
D000818:Animals; D002645:Chickens; D002942:Circular Dichroism; D015891:Cystatins; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D010206:Papain / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism*; D013056:Spectrophotometry, Ultraviolet; D014364:Tryptophan
D015891:Cystatins; D011506:Proteins; C100158:cystatin, egg-white; D014364:Tryptophan; D010206:Papain