Bender and Krebs (1) developed the manometric technique for the determination of n-amino acids and for an assay of n-amino-acid oxidase (DAAO) activity. However, their procedure does not determine which of the amino acids are of the n-form. Hedin and Schultze (2) separated the amino acids by ion-exchange chromatography and determined each of the amino acids present before and after DAAO treatment using a dialyzed crude enzyme preparation. The amount of each amino acid destroyed was assumed to be of the n-form. Recently several methods were published for the determination of small amounts of n-amino acids in the presence of large quantities of the n-isomers. Halpern and Westley (3) separated the diastereoisomeric trifluoracetyl-dipeptide esters of neutral amino acids by gas chromatography. Schmitt and Zenk (4) determined the n-amino acids after stereospecific enzymic acetylation. Soda (5) presented a method based on the reaction of alpha-keto acids formed upon oxidation of n-amino acids with DAAO with 3-methyl-2benzothiazolone hydrazone hydrochloride. Manning and Moore (6) prepared diastereoisomeric dipeptides of the amino acids and separated them using column chromatography.
The method described here combines the use of purified DAAO with ion-exchange chromatography for the simultaneous determination of the stereoisomerism of the amino acids. It i's applicable to samples which contain relatively large amounts of the n-amino acids such as blood plasma and excreta of chicks fed n-amino acids (7).
Methods
Amino acid analysis of test solutions was performed on a singlecolumn amino acid analyzer4 as described previously (8).