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                     COMPOUNDS  AT 45’.
                            BY CARL        L. A. SCHMIDT.
   (From   the   Division   of Biochemistry,       University     of   California   Medical
                                    School,    Berkeley.)

                    (Received for publication,            April   1, 1929.)

    In his original communication dealing with the reaction between
amino acids and nitrous acid, Van Slyke (1) showed that while the

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majority of the naturally occurring amino acids when treated with
nitrous acid yield their nitrogen quantitatively    at room tempera-
tures, glycocoll and cystine yield more than the theoretical amount
of nitrogen.      Later, Levene and Van Slyke (2) demonstrated that
the amount of gas given off by glycocoll increased slowly with time,
so that at the end of about half an hour the estimated amount of
nitrogen was found to be 112 per cent. On the other hand, Van
Slyke (3) found that the amount of gas given off by leucine in 10
minutes did not differ materially from the amount which was
evolved in 4 minutes. Van Slyke (1) also showed that as much as
135 per cent of nitrogen was obtained when glycyl-glycine was
treated with nitrous acid for 1 hour. Leucyl-glycine and leucyl-
leucine did not react abnormally.      No nitrogen was evolved when
glycolic acid was treated with nitrous acid. With the exception
of guanosine, the purine and pyrimidine derivatives were found
to react normally.
    Wilson (4) studied more extensively the reaction between purines
and pyrimidines and certain related compounds and nitrous acid,
and found that certain of them are abnormal with respect to their
expected behavior towards nitrous acid. Skraup and Hoernes (5),
Lewis and Updegraff (6), and others (7) believe that under certain
conditions the reaction between certain amino acids and nitrous
acid may proceed further than the stage of deaminization.
    In attempting      to explain the abnormal behavior of glycocoll
588         Nitrous     Acid Effect on Amino Acids
and glycyl-glycine,      Van Slyke (1) after ruling out the possibilities
that the extra gas may be due to the action of nitrous acid on
glycolic acid, the expected end-product            when nitrous acid acts
upon glycocoll, and from the peptide nitrogen of glycyl-glycine            by
hydrolysis, concludes that the diazo compounds first formed do not
decompose entirely in the normal way to yield glycolic acid or
glycollyl-glycine    but a portion of the reaction proceeds in such a
manner as to lead to a disintegration        of the molecule.    As a result
of the decomposition, some of the peptide nitrogen of glycyl-glycine
 is either set free or exposed so that it may be acted on by nitrous

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acid and eventually        contribute   to the gas which is measured.
 Van Slyke offers no explanation regarding the abnormal behavior
of cystine.      He states that by treating the gas with cuprous chlo-
ride to absorb a trace of carbon monoxide, he was unable to reduce
 the gas volume to the theoretical.
    It is evident that since the total nitrogenof glycocoll and of cys-
tine is in the form of free amino groups, any extra nitrogen which
 may be given off when they are treated with nitrous acid must come
from the decomposition          of the latter substance.      The fact that
 carbon dioxide is given off in the decomposition of glycocoll and
 glycyl-glycine suggests that a part of the action of nitrous acid may
 be one of oxidation.      Van Slyke (1) found that somewhat more gas
 is given off when cystine is treated with nitrous acid than when gly-
 cocoll is similarly treated.
     It interested us to study the reaction between certain amino
 acids and related compounds further in order to obtain more
 data relative to the compounds which were studied by Van Slyke.
 It also interested us to determine the effects of a higher tempera-
 ture on the reaction between nitrous acid and certain amino acids.
 The experiments were carried out at 45’. This temperature                was
 chosen in order to accelerate if possible any abnormal reaction
 which may take place. Lewis and Updegraff (6) showed that at
 this temperature practically all of the tyrosine content of casein is
 destroyed in an hour.        In their experiments the Millon reaction
 was one of the tests used to follow the destruction            of tyrosine.
 The disappearance of the Millon test may, however,                 indicate
 merely the introduction of nitroso groups into the phenyl ring rather
 than a complete destruction of the tyrosine molecule.              Thus, it
 has been shown by Wheeler and Mendel (8) that diiodotyrosine
                                                                        TABLE       I.

 Time        Effect        on the Reaction               between             Nitrous  Acid              and Certain             Amino       Acids
                                                     and Related              Compounds.
     Temperature,                   45”.

                                                                                             Per cent     of nitrogen         after:
                                                                                4 min.           15 min.           30 min.              60 min.
 Amino      acids.
    Alanine ........................                                               98                                   100               99
   @-Alanine         .......................                                       99               99                    99              99
    Arginine      .......................                                        108              118                   132              150
   Cysteine        .......................                                       113              122                   122              128
    Cystine     ........................                                         111              124                   127             131
    Glutamic          acid ..................                                    100               99                   101             101
   Glycocoll..           .....................                                  108               113                   115             117

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   Histidine        .......................                                     102                                     106             109
   Norleucine.              ....................                                100                                     100             100
   Omithine          ......................                                       98              101                   101             101
   Oxyproline            .....................                                      0                                      0               0
   Phenylalanine                  ..................                            100               100                    99             100
   Serine ..........................                                            105                                     108             105
   Tryptophane                ...................                               118               158                   168             194
   Tyrosine       .......................                                         99              103                   106             113
   Glycyl-glycine                    hydrochloride                ...           127               130                   130
   Leucyl-glycine                  ..................                           104               103                   105             110
Related       substances.
      acid .........................                                               3                    8            11                     13
  Betaine        of tryptophane.*.                         ......                  0.9                  1.6           2.2                    2.6
  Formic        acid ....................                                          0                                  0                      0
  Glucose ........................                                                 0                                  0                      0
  Glycolic        acid ...................                                         0                                  0                      0
  Oxyindole            Sample                 1.t. .........                      49               87               100                     99
         “                      “            Z.$. .........                                                           0                      0
                                                                                   0                0
  p-Hydroxyphenylacetic                               acid ....                    0                                  0                      0
  Sodium        oxalate .................                                          0                                  0                      0


590          Nitrous Acid Effect on Amino Acids
does not give the Millon reaction.           All apparatus and reagents
were kept in an air bath which was under thermostatic                control.
The permanganate solution was changed frequently to insure com-
plete absorption of the gases. The reacting solutions were shaken
continuously for the time indicated in Table I. The nitrogen con-
version factors were calculated with the aid of the equation given
by Sharp (9). All amino acids used were recrystallized              products
and whenever analyses were not available, estimations of amino
nitrogen were carried out at room temperatures.                All solutions
were made equivalent to a glycocoll solution containing 400 mg.

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per 100 cc. On account of the manipulations,           it was found impos-
sible to keep the temperature as constant as desired.           The analyti-
cal errors may be as great as 2 per cent.
    The results are presented in tabular form in Table I. The data
confirm the observations of Van Slyke that glycocoll, cystine, and
glycyl-glycine yield an abnormal amount of gas when treated with
nitrous acid. At the temperature           employed, tryptophane        yields
more nitrogen than can be accounted for on the basis of the amino
group.      In fact, the data indicate that in an hour’s time, trypto-
phane yields almost its total nitrogen content.           About 18 per cent
of the second nitrogen group is set free in 4 minutes.           At 22’ only
the expected amount of nitrogen was given off in 4minutes, while in
30 minutes the yield was 110 per cent and in 60 minutes, 116 per
cent. The unsaturated           oxyindole compound yields its nitrogen
completely within a period of 30 minutes, while the saturated
compound does not react.             The betaine of tryptophane        reacts
only slightly.      It is not surprising to find that the indole nitrogen
of tryptophane       and of the unsaturated oxyindole compound is set
free. Kendall and Osterberg (10) have shown that both indole
and isatin yield 7 to 12 per cent of their nitrogen in about 4 min-
utes. Arginine yields more nitrogen than can be accounted for on
the basis of the amino group.           The amount increases with time.
This indicates that the guanidine group is slowly attacked by ni-
trous acid. This was also noted by Sekine (11). Serine, and
particularly    tyrosine and histidine, yield slightly more than the
expected amount of nitrogen whereas alanine, glutamic acid,
norleucine, ornithine,        and phenylalanine      react normally.        No
nitrogen is given off by oxyproline.         Glycyl-glycine,    as noted by
Van Slyke, yields more than the expected amount of nitrogen.
                            C. L. A. Schmidt                               591
  Leucyl-glycine     yields slightly more than the theoretical amount
  of nitrogen; the value is much less than that of glycyl-glycine.
  It does not seem probable that the extra gas obtained from leucyl-
  glycine comes from the glycyl radical.          Since the amount increases
  slowly with time, it is possible that a slight amount of hydrolysis
  takes place. Fischer and Koelker (12) obtained more than the
  calculated amount of gas on treating leucyl-serine with nitrous
  acid at 60”. Under the same conditions                  of experimentation,
  glycyl-leucine    yielded considerably       more gas than the amount
 obtained from leucyl-serine.          Formic acid, glucose, sodium oxa-

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 late, and glyeolic acid yielded, within the limits of error, no gas.
      It appears that the abnormal behavior of cystine and cysteine
 can be connected with their sulfur radicals.                When cystine is
 shaken for several minutes either at room temperature                or at 45’
 with nitrous acid, several drops of barium chloride solution being
 added to the mixture, a precipitate of barium sulfate forms which
 increases in amount as the shaking is continued.             40 mg. of cystine
 were treated with nitrous acid in the presence of barium chloride
 and the mixture allowed to stand at room temperature for about
 20 hours.      56 per cent of the cystine sulfur was recovered as ba-
 rium sulfate.      The reagents were free from sulfate ions. In Table
 I data are given which show that gas is evolved when oc-dihydroxy-
 j%dithiodipropionic      acid is shaken with nitrous acid, the amount
 increasing with time during the period of 1 hour. This substance
 was prepared from cystine according to the directions given by
Westerman and Rose (13). The product is not entirely pure.                    It
is difficult to rid it of a small amount of yellow color.            The color
is indicative of the presence of a nitrogenous group, possibly the
nitroso.      Dunn and Lewis (14) believe that the light yellow coIor
of deaminized casein is due to the presence of nitroso groups.
     In attempting to account for the abnormal behavior of glycocoll,
a number of hypotheses might be advanced.               It is evident that the
extra gas cannot, as Van Slyke has shown, be due to the reaction
of nitrous acid on glycolic acid, the expected end-product               of the
reaction.      Neither is it due to the action of nitrous acid on formic
acid or oxalic acid, substances which might conceivably be formed
from glycocoll by oxidation.            Practically   the same amount of
gas was obtained from solutions containing 2 equivaIents of gly-
colic acid in addition to the glycocoll as was found for glycocoll
592         Nitrous     Acid Effect on Amino Acids
alone. It is not fully established that the gas which is measured
is wholly     nitrogen.     It is conceivable that in a reaction in-
volving decomposition of nitrous acid, nitrous oxide may be giv-
en off. Such reactions are known (15). However,                 it does not
appear probable that the extra gas measured is nitrous oxide
since this substance is quite soluble in water.           We found prac-
tically no difference in our glycocoll estimations when fresh per-
manganate solution and fresh water in the burette and level-
ing bulb were employed.          It is evident that when glycocoll is
treated with nitrous acid at least two reactions, one fast and one

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somewhat slower, take place. The first reaction probably involves
the formation of the diazo compound and its subsequent decom-
position, yielding glycolic acid and nitrogen.         A not considerable
portion of the reaction does not proceed altogether this way.              It
would be possible to postulate a number of intermediate                com-
pounds but in the absence of positive identification, this had better
not be done. The decomposition           of this intermediary     compound
which, for the sake of illustration    only, may be compared to nitro-
syl-sulfuric acid, results not only in the breakdown of the glycocoll
molecule with the formation of carbon dioxide but also in the set-
ting free of unabsorbed gas which probably is nitrogen.            The reac-
tion is one of oxidation involving the formation and subsequent
decomposition       of an intermediary        compound.      This is con-
ditioned upon the presence of an amino group in the molecule
which is acted on. Probably a similar type of reaction takes place
when cystine sulfur is oxidized by nitrous acid. When the
 amino group of glycocoll is protected, as in leucyl-glycine,           this
 intermediary compound probably does not form.
     In Fig. 1 the results which were obtained by Van Slyke and by
 ourselves with a number of substances have been plotted.            A num-
 ber of points have been included in certain of the curves, the data
for which are not given in Table I. It is evident that the amount
 of gas which is given off by glycyl-glycine        at any time after the
 first 2 minutes is considerably greater than that yielded by glyco-
 co11 in the same time. This is probably due, as Van Slyke has
 pointed out, to the decomposition            of some of the glycollyl
 radical, t,hus setting free a certain amount of nitrogen from the
 peptide linkage.       The evolution of gas from the cystine and cys-
 teine reactions proceeds much more slowly than in the case of
 glycocoll and glycyl-glycine.
                        C. L. A. Schmidt
   The contrast in the behavior of the two oxyindole compounds is
noteworthy.     The saturated compound is resistant to the action
of nitrous acid. If the breakdown      of the unsaturated  compound
and the liberation of the indole nitrogen from tryptophane    are due
to oxidation by nitrous acid it does not, however, result in the

          Glycyl-glycine       (Van Siyke)   W22’C.
        : Glycyl-glycine       45°C.
        x Cystine      45°C.    p Cystine  (Van Slyke)   WC.
        a Cysteine       45°C.

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                                FIG. 1.

setting free of any extra nitrogen as, for instance, in the decom-
position of cystine. It is also possible that the reaction is one of
hydrolysis. Since the betaine derivative of tryptophane is very
much more resistant to decomposition by nitrous acid, it appears
that deaminization is an essential prerequisite to further extensive
decomposition of the molecule.
594             Nitrous        Acid Effect on Amino Acids
   The oxyindole compounds were kindly supplied to us by Dr.
E. C. Kendall of the Mayo Foundation, and the betaine derivative
of tryptophane  by Mr. R. W. Jackson of Yale University.


   1. The reaction between nitrous acid and certain amino acids
and related compounds at 45’ has been studied.
   2. It is found that glycocoll, glycyl-glycine,     cystine, cysteine,
tryptophane,    arginine, and an unsaturated      oxyindole derivative
yield more nitrogen than can be accounted for on the basis of the

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free amino groups.       Other substances tested were found to react
   3. Explanations    as to the mechanisms involved in the reactions
which yielded abnormal amounts of gas are given.


 1.   Van Slyke,     D.  D., J. Biol. Chem., 9, 185 (1911).
 2.   Levene,   P.   A., and Van Slyke, D. D., J. Biol. Chem., 12,285 (1912).
 3.   Van Slyke,     D.  D., J. Biol. Chem., 12, 275 (1912).
 4.   Wilson,  D.    W.,  J. Biol. Chem., 66, 183 (1923). See also Russo, G., Boll.
         Sot. Ital. BioZ. Xper., 2, 177 (1927).
 5.   Skraup,    2. H., and Hoernes,     P., Monatsh. Chem., 27,631 (1906).
 6.   Lewis, H. B., and Updegraff,         H., J. BioZ. Chem., 66,405 (1923).
 7.   Skraup,    Z. H., Biochem. Z., 10,245 (1908).          Lampel,    H., Monatsh. Chem.
         28, 625 (1907).
 8.   Wheeler,     H. L., and Mendel,     L. B., J. BioZ. Chem., 7,1 (190%.10).
 9.   Sharp, P. F., J. Biol. Chem., 60,77 (1924).
10.   Kendall,    E. C., and Osterberg,       A. E., J. BioZ. Chem., 40,328 (1919).
11.   Sekine,    H., Nogakukaiho,       Tokyo,     1, 197 (1919);      quoted      from    Chem.
         Abst., 16, 243 (1921).
12.   Fischer,    E., andKoelker,     W. F., Ann. Chem., 340,179 (1905).
13.   Westerman,       B. D., and Rose, W. C., J. BioZ. Chem., 79,413 (1928).
14.   Dunn,     M. S., and Lewis, H. B., J. BioZ. Chem., 49,327 (1921).
15.   Houben,      J., Die Methoden       der organischen      Chemie,      Leipsic,    2nd edi-
          tion, 2, 39 (1921).

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