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					                     STUDIES             ON RACEMIZATION
  XII.   ACTION      OF   ALKALI       ON POLYPEPTIDES                       COMPOSED        OF
                                    LEVO-ALANINE

                     BY P. A.      LEVENE           AND     P. S. YANG
(From th6 Laboratories      of     The   Rockefeller       Institute   for     Medical   Research,
                                         New York)

                  (Received for publication,              November     2, 1932)




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    The present communication deals in the first place with the
problem of the effect of the number of constituent amino acids
of a peptide on the degree of racemization through the action of
dilute alkali. In order to avoid the effect of differences in structure
of the amino acids, polypeptides were prepared composed of
alanine only. Di-, tri-, tetra-, and pentapeptides were used for
the experiments. These peptides were allowed to stand at 25” for
periods of 2, 8, and 16 days, the concentration of alkali being
0.2 and 1.0 N and the proportion of alkali to peptide being 2 : 1 and
1O:l. The results of these experiments are given in Table I.
In Column 4 is given the racemization of the amino acids of each
p&i&       calculated on the basis of the total number of amino acids
 entering into the structure of the peptide, and in Column 5 the
 average racemization of racemizable amino acid calculated on the
 basis of the total number of amino acids lessthe two terminal ones,
 inasmuch as it may be accepted that these two are not susceptible
 to racemization.
    It is evident that the differences in the degree of racemization
 calculated by one or the other method must differ with the length
 of the chain of the peptides. Referring to the degree of racemiza-
 tion calculated on the basis of the total number of amino acids
 as the over-all racemization, and to that calculated on the total
 number less the two terminal ones as the true racemization, the
 ratio between the first and the second values will increase with the
 increase of the length of the chain. In fact, by this ratio it is
 possible to establish the length of the chain. In the case of
                                              405
406                    Studies on Racemization.                                            XII
proteins, the ratio may be regarded equal to unity in view of the
length of the chain.
   From the data reported in Table I, it may be concluded that the
rates of racemization of the amino acids in the tetra- and penta-

                                                    TABLE         I
Summary          of Racemization        of Polypeptides                    Composed           of Levo-Alanine                   by
                                    Sodium    Hydroxide
                                                                      -    at 26”
                                                                                                            -

                            T  Concentration         of NaOH
                                                                       I                         Total
                                                                                                                  Average
                                                                                                                r acemisation




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                                               -7                          Duration                              ,Df racemk-
          Substance                                 Equivalent                             rs v2emization        able amino
                                                    3 substance                                                      acid
                                    (1)                  (2)                    (3)                  (4)              (5)
                                                                                      _-
                                     iv                                       days             per cent            per cent
1. Levo-alanyl-                    0.2                    2                       2              0
      levo-alanine                 0.2                    2                       8              0
                                   0.2                    2                     16               0
                                   1.0                   10                       2             4 (3
                                   1.0                   10                       8             4 (9
                                   1.0                   10                     16              4 (3
2. Di-levo-alanyl-                 0.2                    2                       2             4                    12
       levo-alanine                0.2                    2                       8             9                    27
                                   0.2                    2                     16             11                    33
                                   1.0                   10                       2             8                    24
                                   1.0                   10                       8            19                    57
                                   1.0                   10                     16             19                    57
3. Tri-levo-alanyl-                0.2                    2                       2             6                    12
       levo-alanine                0.2                    2                       8            21                    42
                                   0.2                    2                     16             21                    42
                                   1.0                   10                       2            22                    44
                                   1.0                   10                       8            32                    64
                                   1.0                   10                     16             32                    64
4. Tetra-levo-al-                  0.2                    2                       2             8                     13
      anyl-levo-                   0.2                    2                       8            23                    38
       alanine                     0.2                    2                     16             29                    48
                                   1.0                   10                       2            24                    40
                                   1.0                   10                       8            37                    61.5
                                   1.0                   10                      16            37                    61.5
                                               -                                                            -

peptides are of the same order of magnitude.      In the tripeptide
it is markedly lower in the experiments with 0.2 N alkali and less
marked in the experiments with higher concentration;   in the latter,
in the 8 and 16 day experiments, the values are not much different
from those of the higher peptides.   That the rate of racemization
                        P. A. Levene and P. S. Yang                                                                    407
 of a tripeptide should be lower than that of the higher peptides
is to be expected in view of the fact that as soon as one amino acid
is hydrolyzed off, the remaining dipeptide is no longer racemizable
under the conditions of the experiment.
   In Table II are given data previously published on racemization
of several other tri- and tetrapeptides    containing one or two leucine
residues susceptible to racemization.         Again it is seen that the
degree of racemization of the tripeptides is lower than that of the
tetrapeptides.    Furthermore,    it is seen that the degree of racemiza-




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tion of the tripeptides is of the same order of magnitude whether

                                          TABLE             II

Summary    of   Previous   Work Done      on Racemization    of Polypeptides                               by Sodium
                             Hydroxide      at 25” for 8 Days
                                                                                                           -

                                                                 [al;    of amino    acid                         Average
                                                                 in hydrolysate      with                       r acemiza-
                                          ChlX?Il-                                                 Total
                                                                    20 4”;.t$“’                 ~acemim            tion of
            Substance                    t&ion     of                           a               ,ion afta .      1racemiz-
                                           NaOH                                                                      able
                                                                                                  8 days
                                                                                                                    amino
                                                                   RXX-                                               acid
                                                                                Control
                                                                   mised
                                                                                                           --
                                             N                    degrees       degrees         per cent         per cent
1. Dextro-leucyl-dextro-                    0.2                  -15.5                             0               0
                                                                                    15.5
     leucyl-dextro-leucine                  1.0                  -14.9                             4              12
2. Levo-alanyl-dextro-leucyl-               0.2                  -14.0                             0               0
                                                                                -13.9
     dextro-leucine                         1.0                  -13.2                             5              13.5
3. Dextro-leucyl-dextro-
     leucyl-glycyl-glycine                  1.0                  -13.0              16.1         19               38
4. Glycyl-dextro-leucyl-
     dextro-leucyl-dextro-
     leucine                                1.0                  -12.3              16.1         23.5             35
                                                        -                                   -              -


both terminal components are leucine or whether one is leucine
and the other alanine.      Also in the two tetrapeptides    differing in
their composition     but both having leucine as the racemizable
component, the degree of racemization under identical conditions
is of the same order of magnitude.
   On comparing the data given in Tables I and II, it may be seen
that the degree of racemization       of peptides composed of alanine
is higher than that of those composed of leucine and yet the
stability of leucine peptides is greater than that of those composed of
alanine. The conclusion thus may be arrived at that the amino acids
408             Studies on Racemization.               XII

which confer stability on a peptide with respect to hydrolytic agents
do the same with respect to racemization.      Thus, taking alanine as
the parent substance of all other optically active aliphatic a-amino
acids (which may be regarded as derived from alanine by the
substitution     of the methyl group by other radicles, or by the
substitution   of the 2nd hydrogen atom by a second alkyl group),
it was found that peptides composed of alanine alone were the
least stable, and those of the type of isovaline were the most stable.
All other peptides composed of amino acids containing a hydrogen




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atom on carbon atom (2) but differing from alanine occupied in
the scale of stability a position intermediate    between the two first
named.’      The same relationship has been observed with regard to
ketopiperazines.      Thus, the relative degree of racemization       of
polypeptides higher than tripeptides seems to depend principally on
the nature of the amino acids, all external conditions being the same.
It is realized that the experimental observations        need to be ex-
tended before this conclusion is firmly established.
   But even the limited data available at this date are of consider-
able significance for the explanation of the observations         made
on the racemization of proteins.       It was found that the action of
alkalies on proteins was different from that on ketopiperazines.
In the latter case dilute alkalies (0.1 or 0.2 N) produced exten-
sive racemization of not less than about 70 per cent,2 whereas
 1.0 N alkali causedhydrolysis of the ketopiperazine to the dipeptide
without racemization of the latter. On the other hand, proteins
showed a degree of racemization higher with the stronger alkali
than with the more dilute.3 Another peculiarity in regard to
racemization of proteins was the fact that under identical condi-
tions individual proteins suffered different degreesof racemization.
Are these peculiarities of the behavior of proteins consistent with
the theory of the polypeptide structure?
   The experiments on the alanine peptides reported in this com-
munication clearly show that, similarly to proteins, they are
racemized to a higher degree with 1.0 N alkali than with 0.2 N.
Furthermore, the degree of racemization attained in 8 days by the
   1 Levene, P. A., Steiger, R. E., and Rothen, A., J. Biol. Chem.,       97,
719 (1932).
   2 Levene, P. A., and Steiger, R. E., J. Biol. Chem., 86, 703 (1930).
   3 Levene, P. A., and Bass, L. W., J. Biol. Chem., 82,171 (1929).
                        P. A. Levene and P. S. Yang                                        409

racemizable components of these peptides is of the same order of
magnitude as that of the amino acids of albumin, fibrin, and
gelatin.     The degree of racemization of edestin and casein under
the same conditions is somewhat higher, but this difference may be
explained on the assumption that there are amino acids which
racemize at a higher rate than alanine.
    Summarizing from the data reported in this communication                 it
follows:
    1. The degree of racemization         of polypeptides under identical




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conditions is dependent upon the structure of the amino acid.
    2. In polypeptides higher than tripeptides the degree of racemi-
zation of the racemizable amino acids seems to be independent
of the number of constituent amino acids.
    3. Under identical conditions,         the course of racemization       of
individual amino acids in polypeptides is similar to that in proteins.
    4. The progress of racemization          of proteins is consistent with
the theory of the polypeptide structure and not with the theory of
the ketopiperazine      structure.
    It is realized that thus far these conclusions are based on limited
experimental material.         Unfortunately,     the preparation    of suffi-
cient quantities of the higher peptides is beset by many difficulties.
An important       question which has not yet been sufficiently           in-
vestigated is the mutual effect of neighboring amino acids with
respect to racemization.

                                     EXPERIMENTAL

                               Preparation          of Material
   1. Dextro- and Levo-Alanine-        Dextro-alanine   was obtained
by hydrolysis   of silk fibroin.  The hydrochlorides    of the esters
were converted into the free esters by the barium hydroxide and
chloroform method.      It was found advantageous     to initiate the
reaction by the addition of a small quantity of 40 per cent sodium
hydroxide to the ester hydrochlorides.     The esters obtained from
1 kilo of silk fibroin were worked      up in one operation.      The
reaction mixture was kept in an ice-water bath during the entire
operation.
   The alanine obtained in this manner had the following rotation.
            +   1.15O    x 5
   rLY1;=                      = +    14.33”   (in 20 per    cent   hydrochloric   acid)
            2 x    0.2006
410                    Studies on Racemization.                       XII
   (b) Levo-alanine  was prepared   by aminating     levo-bromo-
propionic acid which was obtained from d-alanine.   The rotation
of the levo-bromopropionic   acid thus prepared ranged within
-43” to -45” and that of the levo-alanine was as follows:
               -     1.24’   X 5
    [a]:   =                       = - 14.51” (in 20 per cent hydrochloric acid)
                   2 x 0.2136

   9.    Levo-Bromopropionyl      Chloride-Levo-bromopropionyl chlo-
ride    was prepared in the usual way by means of thionyl chloride.




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The     rotation of the substance (homogeneous) was between -36”
and      -38”.
   S.    Levo-Alanyl-Levo-Alanine
                                         HOHHO
                                         I II I           I II
                                CHx--C-C-N-C-C-OH
                                        I         I
                                       NH2        CH,
   (a) Levo-Bromopropionyl-Levo-Alanine-           gm. of freshly pre-
pared levo-bromopropionyl       chloride were condensed in the usual
way with 12.5 gm. of levo-alanine.        The yield was 20 gm. or 64
per cent of the theory.       The crude product had the following
specific rotation.
                                   + o.50°   x    5
                         [& =                         = + 14.79” (in water)
                                   2 x   0.0845

     (b) Amination-10     gm. of the crude levo-bromopropionyl-levo-
alanine were readily dissolved in 111 cc.of concentrated ammonium
hydroxide (sp. gr. 0.90). The solution was allowed to stand with
occasional shaking at room temperature          for 3 days.     It was then
evaporated under reduced pressure to dryness.             A little absolute
alcohol was added and the solution again evaporated to dryness.
The dry residue was refluxed with 100 cc. of methyl alcohol for
about 30 minutes to remove the ammonium bromide formed.
After cooling, the refluxed mixture was filtered and washed with a
little alcohol.     For purification the crude dipeptide was dissolved
in twice its weight of boiling water.         Absolute alcohol (200 cc.)
was added to the solution, which became milky immediately.               On
cooling, the dipeptide         crystallized in glistening    plates.    The
                P. A. Levene and P. S. Yang                             411

crystals were filtered off, washed with a little alcohol, and dried in a
vacuum oven at 50”. The yield was 5.5 gm. or 74 per cent of the
theory.   This dipeptide had the following rotations and com-
position.
                      + 1.02O x 5
               Ial; = 2 x 0.1169 = + 21.8’ (in water)

               + 0.73" x 5
        r& =               = + 36.5’ (in 2   N   hydrochloric   acid)
                2 x 0.05




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   4.681 mg. substance: 7.739 mg. COz and 3.125 mg. Hz0
   4.800 “       “      : 0.74 cc. N at 24" and 749 mm.
  20.20 “        “      : 3.18 ‘I I‘ ‘I 19” ‘I 737 “
       GHIsNzOS. Calculated.     C 44.97, H 7.55, N 17.50, NH2 8.75
       160.09       Found.        “ 45.08, “ 7.47, “ 17.46, “ 8.72

  4. Di-Levo-Alanyl-Levo-Alanine
                     HOHHOHHO
                      I II I I II I I II
               CHa-C-C-N-C-C-N-C-C-OH
                      I            I              I
                     NH2          ‘3%            C&

   (a) Levo-Bromcpropionyl-Levo-Alanyl-Levo-AEanine-40       gm. of
levo-bromopropionyl chloride were condensed in the usual way
with 20 gm. of the dipeptide. The levo-bromopropionyl-levo-
alanyl-levo-alanine was obtained as a thick suspensionwhich did
not settle after standing overnight in the cold. It was filtered by
suction, washed thoroughly with ice water and a little alcohol,
and then dried in a vacuum oven at 50”. The yield of crude
product was 30 gm. or 83 per cent of the theory.
   (b) Amination-30     gm. of the crude levo-bromopropionyl-
levo-alanyl-levo-alanine were treated as in the case of levo-
bromopropionyl-levo-alanine.     The yield was 20.5 gm. or 87 per
cent of the theory. The tripeptide showed a bluish pink biuret
reaction, and had the following rotation and composition.

               + 1.55O x 5
        I& =               = + 77.5” (in 2   N   hydrochloric   acid)
                2 x 0.05
412                Studies on Racemization.                            XII
   Abderhalden4 reported the corresponding                        tripeptide   of the dex-
tro series to have the following rotation.
                   [aI:    = -72.2’      (in 2 N hydrochloric      acid)

     4.046 mg. substance:        7.696 mg. COz and 2.750 mg. Hz0
     4.090   “       “         : 0.623 cc. N at 25”      and 763 mm.
   18.40     “       “         : 1.94      “ ‘I (‘ 24.6”   “ 762   “
 (CgHITN304)   + +HzO.         Calculated.      C 44.98, H 7.56, N 17.50,      NH2   6.10
 240.10                        Found.           “ 45.13, “ 7.60, “ 17.51,        “   5.87




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  5. Tri-Levo-Alanyl-Levo-Alanine




    (a) Levo-Bromopropionyl-Di-Levo-Alanyl-Levo-Alanine-18                  gm.
of levo-bromopropionyl         chloride were condensed in the usual way
with 17.5 gm. of di-levo-alanyl-levo-alanine.             The crude product
weighed 22.5 gm. or 80 per cent of the theory.
    (b) Amination-10        gm. of finely powdered levo-bromopropionyl-
di-levo-alanyl-levo-alanine        were dissolved in 165 cc. of concentrated
ammonium hydroxide (sp. gr. 0.90).               It formed a cloudy solution
after shaking for some time. After standing at room temperature
for 3 days, the solution was evaporated to dryness under reduced
pressure.     The evaporation          was repeated by the addition of
about 50 cc. of absolute alcohol.             The residue was refluxed with
150 cc. of methyl alcohol for about 30 minutes.                  After cooling,
the refluxed mixture was filtered and washed with a little absolute
alcohol.    For purification       the crude tetrapeptide       was suspended
in 150 cc. of boiling water.            On adding 2 cc. of concentrated
ammonium hydroxide,            it dissolved.      The solution was shaken
with about 5 gm. of norit and filtered.             To the filtrate 500 cc. of
boiling absolute alcohol were added. On cooling and gentle
shaking, the tetrapeptide         crystallized in glistening plates.     After
standing overnight in the cold, it was filtered, washed with a little
alcohol, and dried in a vacuum oven at 50-60’.                  The yield was
6.50 gm. or 74 per cent of the theory.              The t,etrapeptide is very
  4 Abderhalden,     E.,    Fermentforschung,         13, 55 (1931).
                 P. A. Levene and P. S. Yang                                  413

insoluble in water and gives a purplish          biuret       reaction.     It has
the following rotation and composition.
                + 2.30” X 5
        kg =                = + 115’ (in 2 N hydrochloric           acid)
                 2 x 0.05

   The corresponding tetrapeptide of the dextro series was reported
by Abderhalden4 to contain 1 molecule of water and to have the
following rotation,




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               [al:: = -120.5”   (in 2 N hydrochloric      acid)

   4.480 mg. substance: 7.530 mg. CO2 and 3.060         mg. Hz0
   4.365 “        “    : 0.701 cc. N at 25’     and     753.4 mm.
  19.00 “                        66 6‘ 88 23.50 u       754
 (CI~H~SN,OS)++&O.     ’ d~klated.      C 46.27 H     7.77 N ‘i7.99 NH2 4.49
 311.18                  Found.         “ 45.83: ”    7.64, “ 18.30; “ 4.54;
                                    ash, none

  6. Tetra-Levo-Alanyl-Levo-Alanine




   (a) Levo-BromopropionyETri-Levo-Alanyl-Levo-Alunine-To                  a
solution of 14.4 gm. of tri-levo-alanyl-levo-alanine      in 92 cc. of 0.5 N
sodium hydroxide, kept below 0” in an ice-salt mixture, were added
15.5 gm. of levo-bromopropionyl          chloride and 200 cc. of 0.5 N
sodium hydroxide alternately         in small portions with vigorous
shaking.    During the process of treatment, which occupied about
60 minutes, the temperature was never allowed to rise above 0”.
Gelatinous levo-bromopropionyl-tri-levo-alanyl-levo-alanine             sep-
arated. When the coupling was complete, 30 cc. of 5 N hydro-
chloric acid were added together with 100 cc. of water to make the
reaction mixture fluid. After standing overnight in the cold, it
was filtered and washed thoroughly with water. The residue was
voluminous when moist but shrank to a hard small mass after
drying in a vacuum oven at 50’. The crude product weighed
16.3 gm. or 83 per cent of the theory.
   (b) Amination-16.3      gm. of the finely powdered levo-bromo-
414               Studies on Racemization.                                 XII

propionyl-tri-levo-alanyl-levo-alanine            were suspended in 300 cc. of
concentrated        ammonium       hydroxide       (sp. gr. 0.90) by shaking
mechanically        for about an hour.         After standing for 4 days at
room temperature,          the suspension appeared crystalline and was
evaporated under reduced pressure to dryness.                 50 cc. of absolute
alcohol were added and the evaporation                     repeated.   The dry
residue was refluxed with 250 cc. of methyl alcohol for about half
an hour.       After cooling, the refluxed mixture was filtered, washed
with a little methyl alcohol, and dried.               The crude pentapeptide




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weighed 14 gm. or 96 per cent of the theory.
   For purification 7 gm. of the crude pentapeptide were suspended
in 340 cc. of boiling water.           It dissolved on adding 4 cc. of con-
centrated ammonium hydroxide.                 The solution was shaken with
about 5 gm. of norit and filtered.                To the filtrate were added
700 cc. of boiling absolute alcohol.                 On heating the alcoholic
solution on a steam bath for about 30 minutes to drive off the
ammonia, the pentapeptide separated as an amorphous powder.
It was filtered off, washed with a little alcohol, and dried in a
vacuum oven at 50-60”.            The pure pentapeptide weighed 5.3 gm.
and gave a purplish biuret reaction.             It had the following rotation
and composition,
                 + 2.65”       X 5
        [a]; =                           =   +   132.5”   (in 2   N hydrochloric   acid)
                   2 x 0.05

  The corresponding pentapeptide of the dextro series was re-
ported by Abderhalden* to have the following rotation.
                  Ial:     =   -136.4”       (in 2    N hydrochloric       acid)

    4.141 mg. substance:         7.024 mg. COz and 2.760 mg. Hz0
    4.955      “    “          : 0.787 cc. N at 29”     and 760 mm.
                               : 1.705 (‘ (‘ “ 23.5”
  26.00        “    “                                     ‘I 754  ‘I
    8.480      “               : 0.025 mg. ash
  (CI&Ix,N,O~)+~~O.            Calculated.    C 45.98 H 7.46 N 17.89                NH2    3.60
  391.22                       Found.         Ii 46.25: “ 7.45: I1 17.94,             “    3.54

Racemization with Sodium Hydroxide at 25” of Polypeptides
                  Composedof Levo-Alanine
  Samples of peptides were weighed into Pyrex test-tubes (18 X
150 mm.) and dissolved in 7 cc. of standard alkali (0.2 and 1.0 N)
and allowed to stand for periods of 2, 8, and 16 days. At the
                    P. A. Levene and P. S. Yang                                                              415
end of each period 7 cc. of concentrated        hydrochloric  acid were
added. The tubes were then sealed and heated at 125-130” for
6 hours in a glycerol bath.      Optical rotations were made on the
hydrolysate    in 4 dm. tubes.
   For analysis, 5 cc. in the experiment with the di- and tripeptide,
and 3 cc. with the tetra- and pentapeptides            were withdrawn,
                                           TABLE       III

Racemization   of   Levo-Alanyl-Levo-Alanine                       and       of Di-Levo-Alanyl-Levo-
                      Alanine      by Sodium           Hydroxide            at 25”




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                                               After   acid    hydrolysis     at 125~130°
                     I                I                                                           I




                               Levo-alanyl-levo-alanine
                            daus            ml.            degrees           ‘C.       degmes          per cent

Racemization   by        Control          1.381            -0.48             26        -13.68
  0.2 N NaOH                  2           1.381            -0.48             28        -13.68            0
                              8           1.381            -0.48             27        -13.68            0
                            16            1.381            -0.48             28        -13.68            0
Racemization   by        Control          1.381            -0.48             26        -13.68
  1.0 N NaOH                  2           1.381            -0.46             28        -13.11            4 (3
                              8           1.381            -0.46             27        -13.11            4 (‘0
                            16            1.381            -0.46             28        -13.11            4 (?I

                             Di-levo-alanyl-levo-alanine

Racemization   by        Control          2.071            -0.72             27        -13.67
  0.2 N NaOH                  2           2.071            -0.69             25        -13.10           4
                              8           2.071            -0.65             27        -12.53           9
                            16            2.071            -0.64             28        -12.34          11
Racemization   by        Control          2.071            -0.72             26        -13.67
  1.0 N NaOH                  2           2.071            -0.66             27        -12.53           8
                               8          2.071            -0.58             27        -11.20          19
                             16           2.071            -0.58             28        -11.20          19


neutralized to phenolphthalein   with 5 N sodium hydroxide, and
diluted to 15 cc. Total nitrogen (Kjeldahl)     and amino nitrogen
(micro-Van Slyke, 15 minutes shaking) were made on 10 cc. and
2 cc. respectively.
   Control experiments   were carried out in each instance in the
same manner as described for the racemization,      except that the
substance was dissolved in 7 cc. of concentrated hydrochloric   acid
416                   Studies on Racemieation.                                         XII
to which were then added the 7 cc. of standard alkali and the
solution heated at 125-130” for 6 hours.
   The weights of each peptide taken were as follows :
           1.   Levo-alanyl-levo-&nine                       0.0007 mol = 0.1121 gm.
           2.   Di-levo-alanyl-levo-alanine                  0.0007 “ = 0.1681 “
           3.   Tri-levo-alanyl-levo-alanine                 0.0007 “ = 0.2178 “
           4.   Tetra-levo-alanyl-levo-alanine               0.0007 “ = 0.2738 “
                                           TABLE       IV
Racemization        of Tri-Levo-Alanyl-Levo-Alanine                         and        of Tetra-Levo-Alanyl-




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                       Levo-Alanine by Sodium                Hydroxide             at 25”
                                              After   acid     hydrolysis         at 125430”

                              Time




                                Tri-levo-alanyl-levo-alanine
                              day8          ml.        degrees                “C.              degrees     per cent
Racemization by             Control       2.807        -0.96                  27              -13.45
  0.2 N NaOH                    2         2.807        -0.90                  26              -12.61            6
                                8         2.807        -0.76                  27              -10.65           21
                               16         2.807        -0.76                  28              -10.65           21
Racemisation by             Control       2.807        -0.96                  27              -13.45
  1.0 N NaOH                    2         2.807        -0.75                  26              -10.51           22
                                8         2.807        -0.65                  27                -9.10          32
                               16         2.807        -0.65                  28                -9.10          32
                              Tetra-levo-alanyl-levo-alanine

Racemization by             Control       3.451        -1.20                      27          -13.67
  0.2 N NaOH                    2         3.451        -1.10                      26          -12.53            8
                                8         3.451        -0.92                      27          -10.48           23
                               16         3.451        -0.85                      28           -9.68           29
Racemization by             Control       3.451        -1.20                      27          -13.67
  1.0 N NaOH                    2         3.451        -0.91                      26          -10.37           24
                                8         3.451        -0.75                      27            -8.57          37
                               16         3.451        -0.75                      28           -8.57           37

   The degree of total racemization was calculated from the
following formula.
                   14; - 14;
                             X 100 = per cent
                      b&l
where [cY~];is the specific rotation of the control experiments and
[al]; the specific rotation of the racemized amino acids. The
obtained values are recorded in Tables III and IV.

				
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