Many methods have been described in the literature for determining the activity of ribonuclease (McDonald, 1955); most of these employ ribonucleic acid as substrate and are not suitable for kinetic investigations because it is not clear which
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).
1. The kinetics of the hydrolysis of benzoylarginine ethyl ester in packed columns of CM-cellulose-70-ficin and CM-cellulose-90-ficin were studied. 2. The apparent Michaelis constant, K'(m), of these preparations was calculated and shown to be dependent on the flow rate at low rates of perfusion through the columns. 3. The values for k(3) of these preparations were calculated and shown to be nearly independent of flow rate. 4. A modified form of the integrated Michaelis rate equation was used to describe the action of these materials and its limitations are discussed. 5. The hydrolysis of solutions of casein by these columns was studied.
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