title: 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.
abstract: 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.
mesh_terms: D001426:Bacterial Proteins / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009994:Osmolar Concentration; D011480:Protease Inhibitors / Q000378:metabolism*; D011485:Protein Binding; D013050:Spectrometry, Fluorescence; D013381:Subtilisins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D013696:Temperature; D013816:Thermodynamics
chemical_list: D001426:Bacterial Proteins; D011480:Protease Inhibitors; D013381:Subtilisins