New insights into protein crosslinking via the Maillard reaction: structural requirements, the effect on enzyme function, and predicted efficacy of crosslinking inhibitors as anti-ageing therapeutics.

Protein crosslinking via the Maillard reaction with alpha-dicarbonyl compounds has been the subject of intense literature scrutiny. We report here a systematic study of three previously-neglected aspects of the reaction. Firstly, structural requirements were probed. An arginine-free peptide that contains two lysine residues, and a lysine-free peptide that contains arginine, were reacted with glyoxal, methylglyoxal and biacetyl. Methylglyoxal was able to crosslink in the absence of arginine residues, but glyoxal and biacetyl were not. Glyoxal crosslinked the lysine-free peptide via the N-terminus, but methylglyoxal and biacetyl could not. In this study, crosslinking did not require the presence of arginine but did require a free amino group, from a lysine residue, or the N-terminus. Thus specificity in structural requirements for protein crosslinking by alpha-dicarbonyls has been demonstrated. Secondly, protein function following glycation was examined by treating ribonuclease A with the three alpha-dicarbonyls, which were shown both to crosslink the enzyme and impair enzymatic activity. Thirdly, the effects of two reported Maillard reaction inhibitors, aminoguanidine and 3,5-dimethylpyrazole-1-carboxamidine on the crosslinking reaction were assessed, with a parallel measurement of the effect on enzyme activity. The results demonstrate that preventing protein crosslinking does not necessarily preserve enzyme activity. These results cast doubt on the likely efficacy of some purported anti-ageing compounds in vivo.