512 OCCURRENCE OF FREE AMINO ACIDS 
Round Table Discussion 
METHODOLOGY AND OCCURRENCE OF FREE AMINO ACIDS 
Session Chairman: KuRT SCHREIER 
Transcript Editor: MILTON WINITz 
SCHREIER: I should lke to ask Dr. Soupart to tell us about his new analytical method. 
Soupart: In this presentation, I want to draw your attention to a new device for the counting 
of analytical samples and the continuous recording of chromatographic effluents. This method 
has been worked out by my colleagues, Eric SCHRAM and ROBERT LOMBAERT, an electronic 
engineer, and permits the determination of tritium and C in aqueous solution by means of 
scintillating anthracene powder. 
The use of anthracene powder may be considered as an important improvement in the hetero- 
genous counting of weak / emitters. In the present method, the aqueous solution to be counted 
is run through a cell of constant volume, containing anthracene particles at calibrated size, with 
the following advantages: (a) Counting of individual samples or continuous recording of chroma- 
tographic effluents is possible with a single cell; (b) reproducibility of the results and excellent 
stability of the efficiency of the scintillator are ensured due to the constant characteristic of the 
cell and to the absence of quenching; (c) no problems are encountered resulting from the slow 
settling or creeping along the walls of the powder, as in the case with counting vials; (d) sample 
preparation and counting technique are much simplified as compared with methods involving 
precipitates in planchettes or incorporation of difficultly soluble substances in liquid scintillators. 
Moreover, the method is non-destructive and the counted sample may be recovered for other uses. 
H. ROSENBERG: Professor SOUPART, would the background you observe indicate any ireversi- 
ble adsorption of radioactive material on the anthracene? 
Soupart: Not in this case, because the background remains constant at least for a few weeks. 
SCHREIER: | will now ask Wrn1ItTz to tell us something about methods for the determination of 
D-amino acids in proteins. 
Winitz: What methods can be employed for the determination of D-amino acid residues in 
ploteins is, of course, a problem that has plagued biochemists for a good many years and is one to 
which there is, as yet, no satisfactory answer. My own speculations concerning a possible approach 
to this problem—and I should emphasize that they are merely speculations—arise from a 
question that was asked of me by Dr. ROSENBERG with regard to which of the presently available 
methods would offer some hope for unequivocally demonstrating the existence of D-amino acid 
residues in proteins. His concern with this problem arose from his own very excellent work on the 
isolation of D-serine from the serine ethanolamine phosphodiester of the turtle and from the 
possibility that D-amino acids might likewise reside in the proteins of this same species. 
In seeking a solution to this difficult problem, it would of course be most advantageous if one 
could completely hydrolyze a protein to its constituent amino acids, in the absence of accompany- 
ing racemization or decomposition of the amino acids, and then use some very sensitive assay 
method, such as oxidation with p-amino acid oxidase in a Warburg respirometer, in order to 
ascertain whether there are actually one or more D-amino acid residues present amongst the 
several hundreds or thousands of L-amino acid residues which constitute the protein molecule. 
However, if you attempt to completely hydrolyze the protein molecule with acid, racemization 
occurs in the case of certain residues, such as glutamic acid, and partial or complete destruction 
occurs in the case of others, such as serine, threonine and tryptophane. If you attempt to use 
alkaline hydrolysis, residue destruction and racemization pose even more severe problems. And 
if you attempt to use the less drastic enzymatic approach—the usual enzymatic hydrolysis em- 
ploying trypsin, pepsin, pancreatin or some other mixture of proteolytic enzymes—it is reason- 
able to assume that any of the resulting smaller peptides that might contain D-residues would be 
highly resistant to complete hydrolysis by virtue of the L-directed stereospecificity of the pro- 
teases employed. The failure to detect D-amino acids in these hydrolyzates with p-amino acid 
oxidase would not eliminate the possibility of their presence in a peptide-bound form. 
References p. 524 
