Direct fluorometric determination of a dissociation constant as low as 10(-10) M for the subtilisin BPN'--protein proteinase inhibitor (Streptomyces subtilisin inhibitor) complex by a single photon counting technique.

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, Ki, 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(-9) M. Ki values as low as 10(-10) M could be obtained with reasonable accuracy by this high-sensitivity detection method. From the temperature dependence of Ki, it was found that the binding is endothermic, and is entirely "entropy-driven" in nature. The effect of pH on Ki suggested the participation of an ionizable group with pKapp = 8.5 in the binding.