Docstoc

ELECTROLYTIC DESALTING OF AMINO ACIDS. CONVERSION OF

Document Sample
ELECTROLYTIC DESALTING OF AMINO ACIDS. CONVERSION OF Powered By Docstoc
					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).

				
DOCUMENT INFO
Shared By:
Categories:
Tags:
Stats:
views:9
posted:10/2/2011
language:English
pages:4