Differentiation between type I and type II substrate binding to cytochrome P-450 by variation of temperature

The binding affinities of type I- and type II-substrates to cytochrome P-450 solubilized from phenobarbital induced rat liver microsomes in dependence on the temperature have been determined. Both classes of substrates have been found to exhibit different temperature behaviour. The tendency of type I-substrates (benzphetamine and hexobarbital) to form complexes increases with increasing temperature; type II-substrates show the inverse tendency. From the van't Hoff plot the binding enthalpy was calculated and discussed in connection with the entropy and free enthalpy values. These data ascertain the suggestion of different binding sites for both classes of substrates.