COOPERATIVE IONIZATION OF ASPARTIC-ACID-158 AND HISTIDINE-159 IN PAPAIN - EVIDENCE FROM F-19 NUCLEAR-MAGNETIC-RESONANCE AND FLUORESCENCE SPECTROSCOPY

The chemical shift of the single resonance in the 19F NMR spectrum of papain which was irreversibly inhibited by 3-bromo-1,1,1-trifluoropropanone exhibits pH-dependence. The fluorescence intensity of this papain derivative shows pH-dependence on 2 groups which exhibit co-operative ionization. This co-operative behavior is probably a function of the probe since the fluorescence intensity of S-ethene-thio-papain is dependent on a single ionization constant, whereas that of S-(2-hydroxyethane)-thio-papain is dependent on 2 ionizable groups again acting co-operatively. The 1,1,1-trifluoroketone probe will be hydrated in aqueous solution and would be capable of H-bonding with the protein. The 2 groups detected are considered to be aspartic-acid-158 and histidine-159. The co-operative ionization of these groups in substrate hydrolysis is discussed.