Determination of acid dissociation constants of histidine-containing peptides by proton magnetic resonance spectroscopy

The acid dissociation constant of the imidazolium ring of the decapeptide luliberin has been determined by ¹H NMR-followed titration in D₂O. The normal procedure for the analysis of the titration curve, i.e. direct use of the Henderson-Haselbalch equation, is still applicable in this case, but for more complex peptides a modified calculation procedure is proposed. Results obtained when both methods were applied to luliberin are compared. The influence of D₂O when used as the solvent in this type of determination has been studied using N^α-acetyl-L -histidine methyl ester as a model compound. The difference between the acid dissociation constant of this molecule determined in H₂O and in D₂O implies that a correction of ?.25 unit is needed for those pK_a values calculated by plotting the chemical shifts in D₂O vs the apparent pH meter readings. The pK_a found for N^α-acetyl-L-histidine methyl ester, 6.30 ± 0.04, can be taken as a standard value for histidine-containing peptides.