DIRECT OBSERVATION OF THE TAUTOMERIC FORMS OF HISTIDINE IN N-15 NMR-SPECTRA AT LOW-TEMPERATURES - COMMENTS ON INTRAMOLECULAR HYDROGEN-BONDING AND ON TAUTOMERIC EQUILIBRIUM-CONSTANTS

The individual tautomeric forms of histidine have been directly observ ed in N-15 NMR spectra at -55-degrees-C in ethanol/water solution. The chemical shifts of the nitrogen atoms in the tautomers agree well wit h values previously estimated for them from N-15 NMR spectra of N(delt a1) and N(epsilon2) methyl histidines and of histidine in the dry powd er state. A comparison of the chemical shift position of N(delta1) in the amphion and anion forms of histidine shows that N(delta1) of the N (epsilon2)-H tautomer forms at most only a very weak hydrogen bond wit h the a-amino group. The existence of this hydrogen bond has been prop osed previously to explain the effect ionization of the amino group ha s on the tautomeric equilibrium constant. The pH dependence of the N-1 5 chemical shifts of histidine at 25-degrees-C in the ethanol/water co solvent closely matches that observed in water alone, showing that the ethanol cosolvent solution used in the low-temperature work has littl e or no effect on the intrinsic N-15 shifts of the individual tautomer ic forms or on the acid-base and tautomeric equilibria of the imidazol e ring. The significance of these results for determining tautomeric e quilibrium constants of imidazoles and for interpreting N-15 chemical shift data from histidyl residues in proteins is discussed.