Quantification of Ca2(+)-dependent protease activities by hydrophobic and ion-exchange chromatography

Hydrophobic and ion-exchange chromatography were compared for yield of Ca2(+)-dependent proteases and their inhibitor in studies designed to quantify Ca2(+)-dependent proteases activity for comparative purposes. Ion-exchange (DEAE-Sephacel) proved superior to hydrophobic chromatography (Phenyl-Sepharose). Under the proper conditions, DEAE-Sephacel effectively separated low-calcium-requiring form of Ca2(+)-dependent protease (CDP-I) and CDP inhibitor. Characterization of the assay system for components of the Ca2(+)-dependent proteolytic system separated by ion-exchange chromatography indicated that proteolytic degradation of casein by Ca2(+)-dependent proteases was linear with time for up to 60 min at 25 degrees C and that it was linear up to .4 to .45 units of activity. Therefore, we recommend that, after identification of fractions containing Ca2(+)-dependent protease (CDP-I or CDP-II), these fractions be pooled, and reassayed at a volume that yields values of less than .45 units of activity. Unlike CDP-I and CDP-II, CDP inhibitor lost its activity rapidly with frozen storage (frozen in liquid nitrogen, then stored at -70 degrees C); therefore, inhibitor should be assayed in fresh (unfrozen) samples only.