Determination of the dissociation constants of the lysine residues of lysozyme by proton-magnetic-resonance spectroscopy.

Abstract

Amino groups of a series of model compounds and proteins are methylated by reductive condensation with formaldehyde and borohydride to provide observable resonances for proton magnetic resonance experiments. The extent of methylation varies from 92% for ribonuclease A to 66% for bovine serum albumin. As observed by PMR difference spectroscopy, the conformation of most of these proteins is unaltered by the modification. Retention of native conformation by methylated lysozyme is also shown by enzymatic activity measurements and PMR observation of thermal denaturation.Resonances corresponding to each of the six Nɛ‐dimethyllysine residues in methylated lysozyme are resolved at 270 MHz and titrated to obtain individual pK values. A correction factor of 0.6 pH units obtained from model compound studies is applied to obtain apparent pK values ranging from 10.1 to 10.7 for the ɛ‐amino groups of lysozyme. Assignment of resonances to specific residues is accomplished using the selective broadening effect of Gd3‐. These results indicate the utility of the N‐dimethyl group for probing the environment of lysine residues in proteins.