Clark D S, Bailey J E
Department of Chemical Engineering, California Institute of Technology, Pasadena, California 91125.
Biotechnol Bioeng. 1984 Sep;26(9):1090-7. doi: 10.1002/bit.260260912.
Electron paramagnetic resonance (EPR) spectroscopy has been applied in concert with measurements of catalytic activity and the quantity of active immobilized protein to study the deactivation in 50% n-propanol of alpha-chymotrypsin immobilized on CNBr-Sepharose 4B. These analyses focus on the behavior of two distinct active forms of immobilized enzyme, designated here A and B, identified in previous studies. Raw data provided by EPR spectroscopy clearly show that the relative quantities of active chymotrypsin-A and active chymotrypsin-B change as a result of exposure to alcohol, with the relative quantity of the B form increasing with time. These and additional results provide evidence that the distribution of A and B forms is a function of active enzyme loading but independent of the means used to obtain the loading. Different kinetic models in conjunction with experimental observations consistently indicate that the activity of enzyme form B, by far the more active enzyme form, does not change significantly during the initial 60 min of catalyst deactivation but then decreases appreciably.
