13C‐n.m.r. characterization of formaldehyde bonds in model mixtures and proteins containing lysine

Reactions involving formaldehyde, lysine and another amino acid (tyrosine, arginine, asparagine, glutamine or cysteine) were studied by 13 C‐n.m.r. analysis. The products of the crosslinking between lysine and tyrosine were found to be acid‐resistant and were isolated using ion exchange chromatography. With the other selected amino acids, the crosslinked products with lysine are acid‐labile and could not be isolated; however, n.m.r. allows the determination of their structure. In addition, the formation of methyllysine and of formyllsine have been observed. The 13 C‐n.m.r. chemical shifts of the formaldehyde derived hydroxymethyl, methylene, methyl and formyl carbons with the amino acids side chains help in the interpretation of 13 C‐enriched formaldehyde treated protein 13 C‐n.m.r. spectra. With BSA, we have observed the formation of hydroxymethyllysine, hydroxymethylhistidine, hydroxymethyl‐asparagine or ‐glutamine, methyllysine, and a crosslink between lysine and arginine. These results show the great importance of lysine in the treatment of protein with formaldehyde. Use of 13 C‐n.m.r. is considered highly suitable for obtaining more knowledge about formaldehyde binding to peptides and proteins.