Standardization of the ultraviolet spectrophotometric determination of ribonucleic acid.

' Several perplexing questions involving standardization techniques surface when ultraviolet spectrophotometry is used for the determination of unknown ribonucleic acids isolated from tissue sources. One must consider the effect of hydrolysis on total absorbance, nucleotide makeup of a ribonucleic acid versus molar absorptivity and additivity of riucleotides on total measured absorbance. Experimental evidence indicating how these factors may affect quantification is described here, and it favors the use of nucleotide mixtures as standards for the determination. In view of the variations in molar absorptivities which are possible for the different ribonucleic acids, it seems feasible that calibration with a selected mixture of nucleotides is suitable if one first determines the distribution of nucleotide concentrations in the unknown ribonucleic acid after total hydrolysis before preparing calibration standards made up from nucleotides in those same concentrations. Alternatively, the molar absorptivity of the mixture could be calculated from the contributions of the percentages of nucleotides found.