Trypsin, in powder form and in frozen D2O-glucose solutions, at temperatures from 100° to 300°K, was excited with vacuum ultraviolet and near ultraviolet radiation to determine how absorbed photon energy is partitioned into radiative, nonradiative and/or inactivating processes; at 300°K most of the absorbed energy is not reemitted, so that it (0.98-0.99 for excitation at 120 nm and 0.75-0.90 at 280 nm) is potentially available for inactivation. Since the effects of excitation wavelength and temperature on the emission quenching yields are generally different from those on the inactivation yields of dry trypsin, the mere retention of quenched energy by an enzyme does not necessarily lead to its inactivation. Thus, as predicted previously, the radiation inactivation of trypsin must proceed by rather specific mechanisms which undoubtedly depend upon environment-sensitive processes, since the nature of the molecular environment can modify the partitioning of energy so significantly; for example, there are differences in the phosphorescence-to-fluorescence ratio, in the activation energy for quenching, and in the lifetimes and kinetics of the decay of phosphorescence when trypsin in frozen glasses and dry trypsin are excited by various wavelengths of ultraviolet radiation.
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