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 b...

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Bibliographic Details
Published in:Journal of biochemistry (Tokyo) 1978-11, Vol.84 (5), p.1195-1202
Main Authors: Uehara, Y, Tonomura, B, Hiromi, K
Format: Article
Language:English
Subjects:
Bacterial Proteins - metabolism
Hydrogen-Ion Concentration
Kinetics
Osmolar Concentration
Protease Inhibitors - metabolism
Protein Binding
Spectrometry, Fluorescence
Subtilisins - antagonists & inhibitors
Subtilisins - metabolism
Temperature
Thermodynamics
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Summary: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.
ISSN:0021-924X
1756-2651