Active-site ionizations of papain. An evaluation of the potentiometric difference titration method.
The underlying assumption of the potentiometric difference titration method, as applied to the evaluation of the sulfhydryl-dependent ionizations in the active site of papain, is that the pKa of His-159 is independent of whether the neighboring sulfhydryl (Cys-25) is protonated or methylthiolated. That this idealized assumption may not strictly apply is indirectly indicated by the larger pKa of His-159 in S-methylpapain versus that in S-methylthiopapain, as determined from fluorometric titrations (delta pKa = 0.32 +/- 0.05, 25 degrees C). On the basis of the Wegscheider principle of the equivalence of protons and methyl groups, the potentiometric difference titration method will underestimate the concentration of thiolate-imidazolium ion pair in the active site versus that of the thiol-imidazole tautomer, provided that there is no significant H-bonding interaction in the latter species.
D001665:Binding Sites; D006863:Hydrogen-Ion Concentration; D007477:Ions; D009994:Osmolar Concentration; D010206:Papain / Q000032:analysis*; D011199:Potentiometry; D011522:Protons; D013050:Spectrometry, Fluorescence; D013438:Sulfhydryl Compounds / Q000032:analysis*; D013696:Temperature
D007477:Ions; D011522:Protons; D013438:Sulfhydryl Compounds; D010206:Papain