Assessing the stability of cystatin/cysteine proteinase complexes using mildly‐denaturing gelatin‐polyacrylamide gel electrophoresis

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Abstract
A method for assessing the stability of cystatin/cysteine proteinase complexes using mildly‐denaturing gelatin‐polyacrylamide gel electrophoresis (gelatin‐PAGE) is described. As suggested by the use of well‐known cystatins (human stefins A and B, and oryzacysatins I and II) and the plant cysteine proteinase papain, the ability of cystatin/cysteine proteinase complexes to remain stable during electrophoresis is associated with the degree of affinity between the enzyme and the inhibitor (and inversely associated with the Ki values), at least with the disulfide bond‐lacking cystatins. Complexes with Ki values ≥ 10−8M (weak interactions) are partly or completely dissociated under the conditions used, while those with lower Ki values (strong interactions) remain stable. As shown by the differential effects of two plant cystatins, oryzacystatins I and II, against a cysteine proteinase present in crude (complex) extracts from a plant pest — the two‐spotted spider mite (Tetranychus urticae Koch), the gelatin‐PAGE procedure is suitable for studying the ability of cystatins to form highly stable complexes with cysteine proteinases, without the need for prior purification steps. Considering the well‐recognized potential of proteinase inhibitors for pest and pathogen control, this analytical approach will be useful for rapidly assesing the respective potential of various cystatins for protection of plants, animals, and humans.