RELIABILITY OF THE RADIOAUTOGRAPHIC TECHNIQUE FOR THE DETECTION OF NEWLY SYNTHESIZED PROTEIN

Abstract

It is widely believed that the radioactivity detected by radioautography after administration of a labelled amino acid is due to newly-synthesized protein. The validity of this postulate was examined in work done on the organs of mice injected with leucine-C14 and sacrificed one-half or 24 hours later. Pancreas, liver, kidney and cerebellum were removed and divided into 3 aliquots to determinse (a) the total radioactivity, (b) the biochemical distribution of the radioactivity in the fresh organs, (c) the losses of radioactivity occurring after Bouin fixation and in the course of histological processing as well as the biochemical distribution of the radioactivity retained in histological sections. In addition, an attempt was made to extract radioactivity from mounted sections by various reagents. It was found that 10-50% of the total radioactivity is lost in Bouin's fluid (presumably as free leucine) and in ethanol and dioxane (presumably as lipid-linked metabolites). As for the radioactivity persisting in the processed organs, only insignificant amounts were found in the nucleic acid fraction, while most of it was associated with protein. The possibility that the radioactive amino acids are bound to protein by ionic linkage was examined by extracting sections with strong acids and bases, but only insignificant amounts were thus lost. It is concluded that at least 91-97% of the radioactivity retained in histological sections after labelled amino acid injection is firmly bound to protein (presumably by peptide bonds). Thus, radioautography under these conditions is a reliable method to detect and trace newly-synthesized protein.