Tritium labelled peptides
The simplest route to tritium labelled peptides involves the catalytic hydrogenation or hydrogenolysis of a peptide analogue containing unsaturated or iodinated amino acid derivatives. These peptides are assembled, purified and characterised in the same way as other peptides and then treated with tritium gas to generate the tritium labelled peptide. After removal of the labile radioactivity the tritium labelled peptide is purified, usually by reverse-phase HPLC.
The four amino acids (structures) most commonly used in this procedure are summarised below:
|Amino acid incorporated into peptide||Amino acid finally present in peptide||Specific activity range of peptide|
There are occasions when it is not appropriate to generate tritium labelled peptides via reduction of a precursor peptide:
- The peptide contains a cysteine residue or another thiol group – the sulphur of the thiol group can inhibit the reduction process by damaging the heterogeneous catalyst and additionally, the cysteine can be converted to dehydroalanine which is then capable of reduction to a mixture of D- and L- 2,3-3H-alanines. Peptides that contain methionine do not present this problem but can be prone to oxidation once isolated
- The desired peptide may not contain a proline, leucine, tyrosine or phenylalanine that can be used as a vehicle for tritium incorporation
- There may be a requirement for the tritium label to be present in a particular amino acid that has no suitable precursor
In these cases it is necessary to prepare the tritium labelled amino acid in advance and incorporate this into the peptide. This approach requires the application of appropriate skills and techniques since the syntheses are often conducted on very small scales – for instance, the incorporation of 50mCi of Fmoc-2,3-3H-alanine at a specific activity of 50Ci/mmole into a peptide involves the manipulation of 0.3mg of Fmoc amino acid.
Carbon-14 labelled peptides
The simplest way of introducing carbon-14 into a peptide is via a glycine residue or, if appropriate, at an N-acetyl group. The chemical syntheses of carbon-14 labelled glycine and acetic acid are relatively straightforward and high yielding and, since neither is optically active as with the nineteen other amino acids, no loss of radioactive material occurs on resolution. Both glycine and acetic acid can be obtained labelled on one or both carbon atoms to yield specific activities in the range 50-120mCi/mmole.
Where the use of glycine or acetic acid is not applicable it will be necessary to choose other amino acids for labelling. In these cases those amino acids that do not require side-chain protection should be considered first as the synthetic procedures are significantly less involved. In these instances we invite you to contact us to discuss your requirement in detail.
Dual label peptides
Dual label experiments are useful for determining the fate of different regions of a peptide during metabolism. A dual labelled peptide can be prepared either by preparing both a carbon-14 labelled peptide and a tritium labelled peptide and mixing as required or by preparing a single species labelled with both carbon-14 and tritium.