Appropriate conditions have been found for the charging of all 20 amino acids to unfractionated transfer ribonucleic acid from Neurospora crassa. A single set of reaction conditions employed for the charging of 14 amino acids is not suitable for charging alanine, glutamic acid, glutamine, glycine, methionine, and serine. The time course of aminoacyl transfer ribonucleic acid formation, with transfer ribonucleic acid limiting, for alanine, glutamic acid, glutamine, and methionine, using standard Conditions, reaches a peak in 5-10 min and rapidly declines instead of leveling off. This decline is apparently the result of two factors: (1) instability of the aminoacyl transfer ribonucleic acid in Tris buffer (pH 7.5) and (2) loss of charging capacity of the reaction mixture. By varying the ratio of the concentration of Mg2+to adenosine triphosphate from the standard 20 down to 10 for methionine and alanine, 2 for glutamic acid, and 1 for glutamine and lowering the pH of the reaction to 7.0 for alanine, the loss of charging capacity during the course of the reaction can be prevented. The attachment to transfer ribonucleic acid of glycine and some, but not all, of the other amino acids is inhibited by certain anions, especially phosphate. The transfer ribonucleic acid acceptors for methionine and phenylalanine can be fully charged with the analogs ethionine and p-fluorophenylalanine, respectively. In each case the difference in structure of the amino acid and its analog is sufficient to cause a difference in the chromatographic mobility of their respective complexes with identical transfer ribonucleic acid molecules. © 1969, American Chemical Society. All rights reserved.
