Background
Understanding peptide stability is a key factor underpinning the use of peptides as drugs and for following changes to long-lived proteins in the human body. Physiologically, polypeptides are subject to a range of non-enzymatic modifications including truncation, deamidation and racemisation, however, it is likely that other modifications remain to be elucidated.
Methods
Peptides were exposed to prolonged incubation at 37oC, as well as to elevated temperatures, at pH 7 and the products characterised using MALDI and ESI mass spectrometry and NMR spectroscopy.
Results
Incubation of peptides resulted in racemisation of the N-terminal amino acid. The rate varied depending on the N-terminal amino acid with 38% racemisation of PFHSPSY occurring after 2 weeks at 60 °C compared with 25% for SFHSPSY and 5% for AFHSPSY. Incubating PFHSPSY at 37oC resulted in ~5% racemisation after 8 weeks [1].
The penultimate residue also influenced racemisation, with the extent varying between 15 to 40%. A mechanism is proposed to explain this modification that involves formation of a Schiff base intermediate which may decompose via one of two pathways: one yielding a diketopiperazine and one producing a racemised N - terminus.
Conclusions
This study reports for the first time, spontaneous racemisation of the N-terminal amino acid in a peptide. While the implications for peptide structure are unknown, it is likely that having a D-amino acid at the N-terminus of long-lived proteins will inhibit cleavage by exopeptidases
Given the decades that some long-lived proteins such as those found in the brain, heart and lens are exposed to in a similar environment, it is proposed that this is a new modification to which long-lived proteins are subject. Analysis of one such long-lived protein (aquaporin 0) from aged human lenses revealed 28% conversion of the N-terminal amino methionine to the D-form.