Rapid purification of calcium‐activated protease by calcium‐dependent hydrophobic‐interaction chromatography

Abstract

Both low Ca²⁺- and high Ca²⁺-requiring forms of Ca²⁺-activated protease (calpains I and II) were found to bind to phenyl-Sepharose in a calcium-dependent manner, suggesting that both enzymes expose a hydrophobic surface region in the presence of Ca²⁺. Inclusion of leupeptin in column buffers prevented the loss of activity during hydrophobic-interaction and substrate-affinity chromatography. Under these conditions calpain II (high calcium-requiring form) was rapidly purified from bovine brain and rabbit skeletal muscle using successive phenyl-Sepharose and casein-Sepharose columns.