Sources of error in the determination of specific radioactivity of amino acids isolated by ion-exchange chromatography

Abstract

1. 1. The specific radioactivity (dpm/μmole) of individual 14C-labeled amino acids isolated by ion-exchange chromatography was different, in contrast to being constant, in the consecutive fractions of the same amino acid. This phenomenon was attributed to the resolution of 14C- and 12C-amino acids on ion-exchange resin. The specific radioactivity of the amino acid increased during progressive elution from a cation-exchange resin but it decreased during elution from an anion-exchange resin. Therefore, the specific radioactivity of the amino acid cannot be determined at the point of its elution peak; the true specific radioactivity of the amino acid is obtained as a mean specific radioactivity from the total radioactivity and the amino nitrogen content of pooled fractions of that amino acid. The implication of this finding is discussed in the context of compartmentation resulting from the probable competition between 14C- and 12C-amino acids for the enzyme-protein in the course of their metabolism in vivo. 2. 2. The recovery of radioactivity (expressed as dpm after correcting for quenching) of [ 14C]-γ-aminobutyrate eluted from ion-exchange columns by citrate buffers was not quantitative. This was attributed to the adsorption of the isotope onto the wall of the vial, of variable amounts of the [ 14C]amino acid in citrate buffers of different molarity at pH 5.1, 5.28, 5.84, and 6.5. The determination of specific radioactivity of [ 14C]-γ-aminobutyrate under these conditions was shown to give false low values. A method is given for obtaining quantitative recovery of radioactivity of [ 14C]-γ-aminobutyrate in citrate buffers by liquid scintillation spectroscopy.