1978 Volume 84 Issue 5 Pages 1195-1202
It was found that an increase in fluorescence intensity at 340 nm is observed on the binding of Streptomyces subtilisin inhibitor (SSI) with subtilisin BPN' in the pH range 6-10.
The dissociation constant, K1, of the enzyme-inhibitor complex was determined as a function of pH and temperature by direct fluorometric titration utilizing the single photon counting technique in the protein concentration range of 10-9M. K1 values as low as 10-10M could be obtained with reasonable accuracy by this high-sensitivity detection method.
From the temperature dependence of K1, it was found that the binding is endothermic, and is entirely “entropy-driven” in nature.
The effect of pH on K1 suggested the participation of an ionizable group with pKapp=8.5 in the binding.