In Vacuo Esterification of Carboxyl Groups in Lyophilized Proteins

Abstract

A new method is described for the esterification of carboxyl groups in proteins by reaction of the lyophilized protein in vacuo with gaseous alcohol and HCl catalyst. Carboxyl groups are rapidly esterified with no protein degradation. ¹³C-Methyl or ¹³C-ethyl esters of the α-, γ- and δ-carboxyl groups could be distinguished by the distinct chemical shifts of their resonances. Within the class of γ- or δ-esters, the chemical shifts have little variation; however, the chemical shift of a C-terminal esterified α-carboxyl group shows a strong dependence on the nature of the C-terminal amino acid and sequence. Iodomethane reacts with deprotonated carboxyl groups in lyophilized proteins to form methyl esters, but unlike the reaction with gaseous methanol/HCl, it does not selectively methylate carboxyl groups. The procedure permits the cost-effective incorporation of isotopic labels and provides a new approach using ¹³C-NMR spectroscopy for determining the number of different C-termini present in a protein preparation.