Use of the pH memory effect in lyophilized proteins to achieve preferential methylation of alpha-amino groups

It is demonstrated that the pH memory effect can be used to control the ion ization state of amino groups in lyophilized proteins and hence their chemi cal reactivity toward modifying reagents. When proteins were lyophilized fr om aqueous solutions at pH values between 6 and 7 and reacted in vacuo with iodomethane, the alpha-amino groups were found to be either preferentially or selectively trimethylated. Reaction with C-13-labeled iodomethane permi tted detection and identification of individual trimethylated alpha-amino g roups by C-13-NMR spectroscopy as distinct peaks in the spectral region bet ween 52 and 57 ppm, There was adequate sensitivity to detect minor resonanc es of free alpha-amino groups arising from proteolysis of the major protein or from protein impurities. The resonances of the trimethylated alpha-amin o groups in standard amino acids and peptides are sufficiently close to tho se in the derivatized protein to make a tentative identification of the N-t erminal amino acid. It is also demonstrated that advantage can be taken of the pH memory effect to use the preferential C-13-methylation of amino grou ps to verify whether a protein has a free or blocked amino terminus.