Covalent coupling of calf brain prolidase

Calf brain prolidase covalently bound to CNBr‐Sepharose 4B, retained about 32% of the activity of the uncoupled enzyme. The free enzyme showed slightly greater stability than the bound preparation when stored at 20°C or at 0°C. However, in either case the free and bound enzymes were more stable at the lower temperature. Greater thermal stability was shown by the free enzyme than by the bound preparation over a temperature range of 25°C–60°C. The free and bound prolidase, with and without Mn +2 , had maximal activity at pH 4.0. Although the bound enzyme showed a single maximum, the free preparation exhibited three pH maxima of 4.0, 9.0, and 66.8, in decreasing order of activity. The ions Ag + , Cu + , Hg +2 , and Zn +2 were strongly inhibitory on the free enzyme, whereas inhibition of the bound enzyme, with the exception of Zn +2 , was less. Unlike the coupled enzyme, a stimulatory effect was obtained on the free preparation with Co +3 , Mg +2 , and Mn +2 . Various other compounds were studied and their effects were noted.