Identification of formaldehyde- induced modifications in proteins: reactions with diphtheria toxin

Diphtheria toxoid, the principle component of diphtheria vaccines, is prepared by inactivating diphtheria toxin with formaldehyde and glycine. The treatment introduces intramolecular cross-links and intermolecular formaldehyde/glycine adducts in diphtheria toxin. The purpose of the present study was to elucidate the nature and location of formaldehyde-induced modifications at two functional sites of diphtheria toxin: the NAD + -binding cavity and the receptor-binding site. Therefore, diphtheria toxin was chemically modified using five different reactions: (1) dimethylation by formaldehyde and NaCNBH3, (2) acetylation by acetic acid N-hydroxy succinimide ester, (3) formaldehyde treatment, (4) the standard detoxification by formaldehyde and glycine, and (5) dimethylation followed by formaldehyde and glycine treatment. The modifications in these experimental diphtheria toxoids were studied by SDS-PAGE, primary amino group determinations (TNBS assay), and/or by liquid chromatography-mass spectrometry (LC-MS) analysis of enzymatically cleaved toxoids. LC-MS analysis confirmed that all but one lysine residue in diphtheria toxin were dimethylated by the reaction with formaldehyde and NaCNBH 3 (reaction 1). According to the TNBS assay, four amino groups per toxin molecule were on average not dimethylated. The reaction of formaldehyde with lysine residues is the first step in the formation of a stable intramolecular methylene bridge. Thus, the formation of intramolecular cross-links only depends on the close proximity of a second reactive residue. Indeed, LC-MS analysis revealed nine intramolecular cross-links between lysine and a nearby reactive residue in formaldehyde-treated toxin (reaction 3). Two masses were ascribed to peptide fragments with an intramolecular cross-link originating from the NAD + -binding cavity, and two masses for the receptor-binding site. It was assumed that formaldehyde-glycine adducts are connected mainly to the reactive residues at the surface of the protein. As a simplified model, acetylation of lysine residues of diphtheria toxin was investigated (reaction 2). LC-MS analysis demonstrated that buried residues are less reactive to the acetic acid N-hydroxy succinimide ester than the residues at the surface of the toxin. This result indicates that formaldehyde-glycine adducts will be only be attached to the more accessible residues. The TNBS assay also demonstrated partial modification of lysine residues (36% unmodified). Finally, the presence of formaldehyde-glycine attachments was studied at the NAD -binding cavity and the receptor-binding site (reaction 5). Five peptide fragments with formaldehyde-glycine modifications were observed from the NAD-binding cavity, and three fragments from the receptor-binding site. In conclusion, the functional sites of diphtheria toxin are affected by the formaldehyde and glycine treatment.