Covalent chromatography. Preparation of fully active papain from dried papaya latex.
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 2xcrystallized partially active papain. 4. The catalysis of the hydrolysis of alpha-N-benzoyl-l-arginine ethyl ester at pH6.0, 25.0 degrees C, I=0.3 by fully active papain thus prepared is characterized by K(m)=18.2+/-<0.1mm and k(cat.)=16.4+/-0.5s(-1).
D002845:Chromatography; D002846:Chromatography, Affinity; D004220:Disulfides / Q000378:metabolism; D005978:Glutathione; D006868:Hydrolysis; D007700:Kinetics; D008722:Methods; D010206:Papain / Q000302:isolation & purification*; D011134:Polysaccharides; D012408:Rubber / Q000032:analysis*; D013438:Sulfhydryl Compounds / Q000378:metabolism
D004220:Disulfides; D011134:Polysaccharides; D013438:Sulfhydryl Compounds; D012408:Rubber; D010206:Papain; D005978:Glutathione