ASSESSING THE STABILITY OF CYSTATIN CYSTEINE PROTEINASE COMPLEXES USING MILDLY-DENATURING GELATIN-POLYACRYLAMIDE GEL-ELECTROPHORESIS

A method for assessing the stability of cystatin/cysteine proteinase c omplexes using mildly-denaturing gelatin-polyacrylamide gel electropho resis (gelatin-PAGE) is described. As suggested by the use of well-kno wn cystatins (human stefins A and B, and oryzacystatins I and II) and the plant cysteine proteinase papain, the ability of cystatin/cysteine proteinase complexes to remain stable during electrophoresis is assoc iated with the degree of affinity between the enzyme and the inhibitor (and inversely associated with the K-i values), at least with the dis ulfide bond-lacking cystatins. Complexes with K-i values greater than or equal to 10(-8) M (weak interactions) are partly or completely diss ociated under the conditions used, while those with lower K-i values ( strong interactions) remain stable. As shown by the differential effec ts of two plant cystatins, oryzacystatins I and II, against a cysteine proteinase present in crude (complex) extracts from a plant pest - th e two-spotted spider mite (Tetranychus urticate Koch). the gelatin-PAG E procedure is suitable for studying the ability of cystatins to form highly stable complexes with cysteine proteinases. without the need fo r prior purification steps. Considering the well-recognized potential of proteinase inhibitors for pest and pathogen control, this analytica l approach will be useful for rapidly assessing the respective potenti al of various cystatins for protection of plants, animals, and humans.