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

Abstract

AbstractThe 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 Nα‐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 pKa values calculated by plotting the chemical shifts in D2O vs the apparent pH meter readings. The pKa found for Nα‐acetyl‐L‐histidine methyl ester, 6.30 ± 0.04, can be taken as a standard value for histidine‐containing peptides.