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

AbstractProcedures 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.