Isoelectric focusing of subtilisin inhibitors: Detection and partial characterization of cereal inhibitors of chymotrypsin and microbial proteases

Inhibitors of subtilisin and other microbial serine proteases were detected after isoelectric focusing of cereal extracts in polyacrylamide gels by a negative staining technique based on the chromogenic substrate acetyl-d,l-phenylalanine-2-naphthyl ester. The 10 chymotrypsin isoinhibitors identified in experiments with barley extracts were also inhibitors of subtilisin, and sensitivity of detection was about 25 times higher when gels were stained for inhibitors of this enzyme. In addition, one specific subtilisin inhibitor zone was detected. These 11 barley subtilisin isoinhibitors and similar inhibitors in wheat, triticale, rye, oats, sorghum, rice, and maize were further characterized and classified by isoelectric focusing. Techniques used for this purpose included application of microbial enzymes with different specificity of inhibition, comparison of cereal varieties, pretreatment of extract with monospecific antibodies toward a purified inhibitor from the same or a related species, and pretreatment of extract with inhibitor affinity gel. The procedure used for detection of subtilisin inhibitors was also effective after separation of isoinhibitors by sodium dodecyl sulfate-gel electrophoresis.