Production and purification of mixed 14C-labelled peptides derived from plant biomass

Procedures are described for the production and purification of 14C-labelled peptides of mixed composition, derived from phytomass. Barley seeds (Hordeum vulgare) were germinated and grown in the dark for 6 days. On day 7, the seedlings were exposed to light in a 14CO2 atmosphere for 24 h. The plant leaves were harvested and their water-soluble 14C-labelled proteins extracted. These 14C-proteins were partially digested by sequential incubation with pepsin, α-chymotrypsin and trypsin. The resulting 14C-labelled peptides were separated from contaminating amino acids by elution from columns of copper-Chelex resin, and finally fractionated by gel-filtration chromatography and assigned to groups according to molecular size. The purified 14C-peptides ranged in relative molecular mass up to approximately 5,000, possessed a purity in excess of 97%, and were radiolabelled in all amino acid residues with an average specific radioactivity of 450 Bq/μmol. The methods described can be readily adapted to produce not only mixed 14C-labelled peptides of any required attribute, such as molecular size or ionic charge, but also mixed 14C-proteins of 14C-amino acids.