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ELECTROLYTIC DESALTING OF AMINO ACIDS. CONVERSION OF ARGININE TO ORNITHINE * BY WILLIAM H. STEIN AND STANFORD MOORE (From the Laboratories of The Rockefeller Institute for Medical Research, New York, New York) (Received for publication, November 27, 1959) In order to circumvent the difficulties occasioned by high concentra- tions of inorganic salts, the electrolytic desalting method of Consden, Downloaded from www.jbc.org by guest, on September 29, 2011 Gordon, and Martin (1) has been widely used to prepare samples of blood plasma and urine for paper chromatography (cf. (2)). With a view to- wards employing the technique in quantitative work with starch col- umns,’ the present experiments were undertaken in order to determine the extent to which various amino acids could be recovered after submission to the electrolytic desalting pro>edure. Inorganic salts, similar to those found in urine, were added to a known mixture of amino acids to give a salt-amino acid ratio of 10: 1. The solution was desalted in the manner described by Consden, Gordon, and Martin (l), the resulting mixture con- centrated to a known volume, and an aliquot chromatographed on starch (3). The results of three such experiments are given in Table I. It will be noted that the recoveries of leucine-isoleucine-phenylalanine, glutamic acid-alanine, and threonine average about 95 per. cent. The slightly low figures in these instances are probably indicative of a small manipulative loss incident to the desalting procedure. There are evi- dences of specific losses of histidine, proline, and methionine or tyrosine (valine presumably being stable). By far, the most strikingly low recov- ery is that of arginine, which is accompanied by more than theoretical recovery of material in the cystine range. Since ornithine is known to move at the same rate as cystine on starch (3), it was logical to suppose that part of the arginine might have been converted to ornithine. Evi- dence in support of this supposition was provided by the calorimetric ninhydrin procedure of Chinard,2 which is quite specific for ornithine in the presence of cystine or any other amino acid except proline. When alter- nate fractions of the cystine peak were examined by the Chinard procedure, the characteristic red color given by ornithine was observed. An orni- thine peak could thus be plotted and integrated without interference from 1 Experiments have demonstrated that the high inorganic salt content of blood plasma or urine interferes with the separations of amino acids on starch columns. 2 We wish to acknowledge the generous cooperation of Dr. F. P. Chinard, who placed this procedure at our disposal prior to its publication. 103 104 ELECTROLYTIC DESALTING cystine. In the first chromatogram, for example, the ornithine found was equivalent to 40 per cent of the arginine originally present. This quantity of ornithine, when added to the amount of unchanged arginine, accounted for 87 per cent of the arginine submitted to the desalting process. Thus the chromatographic behavior, the calorimetric reactions, and the quanti- tative data all support the conclusion that arginine is transformed to a considerable extent to ornithine by the desalting procedure. Further evidence was provided by desalting experiments in which ar- ginine was the only amino acid added to the salt mixture. At the be- TABLE I Downloaded from www.jbc.org by guest, on September 29, 2011 Recovery of Amino Acids from Known Mixture after Electrolytic Desalting The desalted amino acid mixture was chromatographed on starch according to the nrocedure already described (cf. Fig. 1 (3) ). - Constituent < I - Per cent recovery T- ‘C -- Leucine-isoleucine-phenylalanine. ............. 95 94 94 Valine-methionine-tyrosine .................... 88 88 84 Proline ...................................... 81 83 92 Glutamic acid-alanine ........................ 95 90 94 Threonine ................................... 94 96 95 Aspartic acid ................................ 92 87 86 Serine ....................................... 103 98 105 Glycine ...................................... 105 106 111 Arginine ..................................... 47 18 48 Lysine ....................................... 89 86 99 Histidine .................................... 89 67 69 Cystine (with ornithine) ..................... 140 171 115 ginning of the experiment, the salt mixture plus arginine gave no color in the Chinard procedure. Aliquots withdrawn during the desalting process gave a constantly increasing ornithine color which reached a maximum at the end of the experiment, when about 75 per cent of the arginine had been converted to ornithine. When an arginine solution containing no salts is submitted to the procedure, only a negligible amount (3 per cent) of orni- thine is formed. The extent of the conversion was not influenced by vari- ation of the rate of circulation of mercury in the desalting apparatus or by attempts to stir the mercury surface mechanically. The mechanism of the conversion of arginine to ornithine has not been investigated. The process may be analogous in some respects to the con- version of guanidine to ammonia during electrolysis over a mercury cath- ode, as reported by Davis, Yelland, and Ma (4). W. H. STEIN AND S. MOORE 105 The nature of the substances responsible for the definitely high yields in the serine and glycine peaks (Table I) has not been ascertained. Meth- ionine sulfoxide and citrulline, however, are two compounds known to travel in this range on starch chromatograms. EXPERIMENTAL Desalting Procedure-The synthetic mixture of amino acids simulated the composition of a hydrolysate of bovine serum albumin and was pre- pared in the manner described previously (3). A volume of 0.15 cc. of this solution (about 15 mg. of amino acids) was added to 37.5 cc. of water and 12.5 cc. of a salt mixture of the following approximate composition: Downloaded from www.jbc.org by guest, on September 29, 2011 200 mg. of (NH&SO+ 900 mg. of NaCl, 275 mg. of KZHPOI, 20 mg. of CaC&, and 50 mg. of MgS04 made to a volume of 100 cc. with water. The entire mixture was placed in an electrolytic desalting apparatus construc- ted exactly as that described by Consden, Gordon, and Martin (1). A 110 volt direct current line was employed as a power source. The use of a 220 volt source (for chromatogram No. 672, Table I) accelerated the desalting procedure somewhat, but otherwise did not appear to influence the results significantly, except that the cystine plus ornithine recovery was lower. A 100 ohm, 3 ampere slide wire resistance was employed to maintain the power consumption below 100 watts during the early stages of the desalting, as recommended by Consden, Gordon, and Martin. Af- ter 10 to 20 minutes of operation, the current fell below 1 ampere with no resistance in the line. Desalting was continued for an additional period of 2.5 to 3.5 hours, at which time the current had reached a steady mini- mum of about 0.2 ampere. In the experiment employing 220 volts, de- salting was completed in 1 hour. The solution was washed from the ap- paratus, filtered, and concentrated to dryness under reduced pressure. The residue was taken up in 1 cc. of 0.1 N HCl, and 0.2 cc. of this solution was diluted with 0.5 cc. of the solvent to be used on the chromatogram. Exactly 0.5 cc. of this solution was placed on the starch column. Chro- matography was performed in the manner already described (3), except that the change of solvent to 2: 1 n-propyl alcohol-O.5 N HCl was made after the emergence of glycine (at about 140 cc.). Alternate 0.5 cc. fractions of the cystine peak were analyzed for orni- thine by the calorimetric procedure of Chinard. The fractions were neu- tralized with 2 drops of 0.8 N HCl, and 1.5 cc. of glacial acetic a.cid and 1 cc. of ninhydrin solution were added. The ninhydrin solution was com- posed of 625 mg. of ninhydrin and 10 cc. of 6 M H3P04 made to a volume of 25 cc. with glacial acetic acid. The photometer tubes were covered with aluminum caps (cf. (3)) and heated for 1 hour at 100” in a boiling water bath. After being cooled, 4 cc. of glacial acetic acid were added and 106 ELECTROLYTIC DESALTING the tubes were read at 505 rnp in a Coleman junior spectrophotometer. The quantity of ornithine present was determined by comparison with a standard curve. Solutions containing arginine alone were also desalted. The salt mix- ture employed had the composition indicated previously, except that an equal amount of Na&Os was substituted for the (NH&Sod. To 12.5 cc. of salt mixture were added 37.5 cc. of water and 5 cc. of a solution con- taining about 2 mg. of arginine. At various times the current was inter- rupted, the solution removed from the apparatus, and its volume measured. Three 2 cc. aliquots were removed and concentrated to dryness in photome- Downloaded from www.jbc.org by guest, on September 29, 2011 ter tubes in a vacuum desiccator. After the addition of 0.5 cc. of water, the tubes were analyzed for ornithine by the Chinard procedure. The percentage of arginine transformed to ornithine at various times was as follows: 0.5 hour, 21 per cent; 1 hour, 38 per cent; 2 hours, 57 per cent; 3.5 hours, 74 per cent. The exact extent of the transformation was not repro- ducible from one experiment to the next. In a second experiment, a 75 per cent conversion occurred in 1 hour, whereas a Bhird gave 84 per cent in 3.5 hours. The authors wish to acknowledge with appreciation the expert technical assistanceof Mrs. Gertrude C. Carey. SUMMARY Starch chromatography has revealed that large lossesof arginine and slight lossesof some other amino acids occur during the electrolytic desalt- ing procedure of Consden, Gordon, and Martin. The low recoveries of arginine have been found to result from the conversion of a major part of this amino acid to ornithine during the desalting process. BIBLIOGRAPHY 1. Consden, R., Gordon, A. H., and Martin, A. J. P., Biochem. J., 41, 590 (1947). 2. Dent, C. E., Symposia Biochem. Sot., 3, 34 (1949). 3. Moore, S., and Stein, W. H., J. Biol. Chem., 1’78, 53 (1949). 4. Davis, T. L., Yelland, W. E., and Ma, C. C., J. Am. Chem. Sot., 69, 1993 (1937).
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