Tritium is the isotope of choice for studying native peptides in any biological or biochemical system, as valid receptor binding studies require labelled ligands with physical properties and biological activities identical to those of the unlabelled ligand. This can be achieved by substitution of hydrogen atoms by those of tritium. This leads to a molecule possessing virtually identical properties to those of the parent compound, as there has been no gross modification of its primary structure, whilst affording a material of appreciable specific radioactivity. A further advantage of using tritium as a radiolabel is that the ß-particle emissions are of low energy and thus their penetrating range in matter is very short. This makes tritium an ideal isotope for use in high resolution autoradiographic applications. Tritium must therefore be seriously considered the isotope of choice for use in receptor binding studies.
Tritiated analogues of peptides have proven to be of great value in studies of the modes of action, storage, release and degradation of biologically active peptides. Their use facilitates experimentation at low concentrations, such as those often found in normal physiological conditions, and thereby obviates the need for raising the concentration of the compound being studied to that of the minimum sensitivity of the mode of analysis. Unnaturally high concentrations may cause swamping effects and thereby alter the physiological role and metabolic fate of the compound studied.
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.
Tritium has a specific activity of 28.6Ci per matom (1.07TBq per matom) and therefore the incorporation of one tritium atom per peptide molecule will yield material with a specific activity of 28.6Ci per mmole. The specific activity of tritium labelled peptide can be increased by increasing the number of tritium atoms present per molecule. Higher specific activities can be achieved by labelling multiple amino acids within a peptide. Conversely, the specific activity of the labelled peptide can be reduced to any desired value by diluting the material with unlabelled material.
The specific activities that can be achieved with various amino acids (structures) 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.