IDENTIFICATION OF THE ELUSIVE AMINO ACID Gh 
physical, chemical and biological properties of the antipodes so secured compared with 
those of the natural material; in the case of amino acids with more than one asym- 
metric center, separation of the diastereomers should precede the resolution of each 
racemate into its optical antipodes. 
6) Determination of the optical configuration of the molecule should be accom- 
plished by any of a number of chemical, physical, optical or biological techniques. 
7) The natural occurrence of the new amino acid should be independently con- 
firmed by some investigator other than the discoverer. 
IDENTIFICATION BY CHROMATOGRAPHIC AND COLORIMETRIC MEANS 
At the present time, a facile and convenient means for establishing the identity of an 
amino acid from natural sources involves a comparison of its chromatographic be- 
havior with reference amino acids in a variety of solvent systems. Ofttimes, no other 
criteria are employed. Now it is well known that certain structurally related amino 
acids, such as leucine and isoleucine, reveal a chromatographic behavior in numerous 
solvent systems wherein appreciable differences in mobility are the exception rather 
than the rule. It is therefore evident that through the use of chromatographic pro- 
cedures alone, the danger is ever present that an amino acid of as yet unsuspected 
structure will remain hidden behind, or occupy the same position as a closely related 
amino acid of known structure, and hence will be overlooked. This probability was 
dramatically emphasized only recently with the announcement by OGLE, LOGAN AND 
ARLINGHAUS!® that a newdmino acid had been isolated from acid hydrolysates of 
collagen, a protein that had hitherto been the subject of chromatographic analysis 
by many investigators in many laboratories. In a private communication from Dr. 
OGLE, it was learned that during a study of the peptide sequence of collagen, a tri- 
peptide was isolated which, after acid hydrolysis, displayed a previously unobserved 
peak running just before hydroxyproline upon passage of the hydrolysate through 
a Dowex-50 column employing 0.2 N sodium phosphate buffer as the eluant and a 
pH of 3.1. Analysis revealed that the new material was an imino acid and was of the 
same elemental composition as y-hydroxyproline. That the compound was neither 
proline nor y-hydroxyproline was indicated by a comparison of infrared spectra and 
the melting points of various derivatives, by the color reaction obtained with nin- 
hydrin in glacial acetic acid and by the fact that it failed to react with dimethyl- 
aminobenzaldehyde following oxidation with chloramine-T. As degradation of the 
material with permanganate yielded f-alanine, the new material was assigned the 
structure of B-hydroxyproline. It should be noted that the paper-chromatographic be- 
havior of this new material was identical with that of y-hydroxyproline in all solvent 
systems tested. 
That amino acids of varying structure may possess identical mobilities in a number 
of different solvent systems is an occurrence that is by no means rare. One such occur- 
rence that was noted in our laboratory some years ago involved a material which 
was obtained from the copper-catalyzed condensation of pyruvic acid and glycine 
in an alkaline medium (Fig. 2), and which possessed an empirical formula of C;HgNO;. 
This formula could correspond to p-hydroxy-f-methylaspartic acid if it were assumed 
that the condensation involved the a-carbon atom of the pyruvic acid reactant. If, 
however, the condensation involved the f-carbon atom of the pyruvic acid, then 
References p. 22/24 
