Covalent chromatography. Preparation of fully active papain from dried papaya latex

Abstract

1. A Sepharose–(glutathione–2-pyridyl disulphide) conjugate has been prepared. 2. Its use in a new type of chromatography, covalent chromatography by thiol–disulphide interchange, is described. 3. With this technique, papain containing 1 intact catalytic site [thiol with high reactivity towards 2,2′-dipyridyl disulphide (2-Py-S-S-2-Py) at pH4] per mol of protein is readily prepared both from dried papaya latex and from commercial 2×crystallized partially active papain. 4. The catalysis of the hydrolysis of α-N-benzoyl-l-arginine ethyl ester at pH6.0, 25.0°C, I=0.3 by fully active papain thus prepared is characterized by Km=18.2±<0.1mm and kcat.=16.4±0.5s-1.