Document Sample
					 A NEW         DECOMPOSITION                     PRODUCT     OF KERATIN
              WHICH   GIVES                 MILLON’S   REACTION.
                                  PRELIMINARY           NOTE.

                             BY ROSS       AIKEN        GORTNER.

(From   the Biochemical  Laboratory   of the Station for Experiment&          Evolution,
                   the Carnegie Institution    of Washington.)

                                                                                           Downloaded from by guest, on September 21, 2009
                      (Received    for   publication,    April   14, 1911.)

    The red coloration produced by the interaction          of an aromatic
phenol and Millon’s reagent (mercuricnitrate        indilutenitrous      acid)
is one of the most characteristic     of the color reactions of the pro-
teins.    Among the nineteen known compounds which form the
protein molecule only one, p-oxy-a-amino          phenyl propionic        acid
 (tyros’n) is of such a nature as to give a coloration with Millon’s
reagent and therefore      a positive reaction is taken to prove the
presence of tyrosin.
    In a recent article’ I have shown that the coloration of the in-
teguments of the meal worm (Tenebris molitor) is due to the presence
of a tyrosinase, acting upon a ch. omogen.         In many respects this
chromogen resembles tyrosin, in that it is not precipitated                 by
phosphotungstic      acid or by basic lead acetate and in that it gives
a strong Millon’s reaction.        It differs from tyrosin, however, by
being extremely soluble so that when evaporation            was carried al-
most to dryness and a few drops of water were added no tyrosin-
like crystals could be obtained, neither did anyotherof           the tyrosin
tests, excepting the coloration with tyrosinase, produce positive
    While this was a solitary instance no special attention was paid
to it, the supposition     being that the chromogen was present in
 exceedingly small amounts and was probably tyrosin.
  1 This   Journal,      vii, p. 365, 1910.
356      A New       Decomposition           Product      of Keratin

    In a later study of the pigment of black wool’ a melanin was
isolated by the action of 0.2 per cent sodium hydroxide which was
soluble in alkalies, dilute mineral acids and strong acetic and formic
acids. Further study of this body has shown that it is probably a
melano-protein,     i.e., an acid albumin      in which a portion of the pro-
tein molecule is so modi$ed as to possess the properties usually as-
cribed to melanins; that is, the pigmented portion of the molecule is
readily soluble in dilute acid while combined in the protein molecule,
but, when hydrolyzed by strong mineral acid or acted on by strong
alkali, a pigment is obtained which is insoluble in mineral acids.

                                                                                     Downloaded from by guest, on September 21, 2009
    In my earlier paper, I define melanin as “those dark pigments
which occur normally or pathologically             in the animal body, skin,
hair or feathers,”       and I still adhere to this nomenclature          and call
the “melano-protein”         a melanin, since it is doubtless in this form
that the pigment occurs in the wool.2
    Approximately        20 grams of this melanin were hydrolyzed by
boiling for 30 minutes with fuming hydrochloric             acid. The solution
was evaporated        to dryness on a water-bath,       distilled water added
and the black melano-humin            filtered off. The remainder           of the
hydrochloric     acid was then precipitated        by addition of silver sul-
phate and the sulphuric acid quantitatively              removed by baryta.
The clear filtrate was concentrated almost to dryness and allowed
to crystallize.      Then a small amount of cold water was added and
the solution filtered from the crj stals. Thesewere well washedwith
 cold water and dried in vacua over concentrated                 sulphuric acid.
The dry crystals weighed about 0.3 gram, were of the character-
istic tyrosin shape and gave all of the usual tyrosin tests (Millon’s,
 Pyria’s, DenigW and Mijrner’s and the coloration with tyrosinase).
 To my surprise, however, the mother liquor still gave Millon’s re-
 action with undiminished intensity. Thinking that perhaps hydroly-
 sis was incomplete and tyrosin polypeptides might be present, the
 solution was diluted to 150 cc. and sulphuric acid added to 25

   1 Gortner: This Journal, viii, p. 341, 1910.
   2 Although I havetested many varieties of keratin, including    four samples
of human hair, horn, black and white feathers, albino hair and feathers,
negro hair, horse hair, black and white wool, etc., “melano-proteins”      have
been obtained only from black wool, red and brown horse hair and auburn
human hair.    The detailed report on these pigments, alkali albumins,     etc.,
will shortly be ready for publication.
                        Ross Aiken   Gortner                    357
per cent by weight, and the mixture boiled for 22 hours.         The
acid was then neutralized with baryta and tyrosin, determined
in the usual manner. No tyrosin was present, but the Millon’s
reaction was still very intense. A difference was, however, observed
in the reaction, for if a large excess of the reagent were added or
if the heating had been too vigorous the coloration did not appear
or else was rapidly decolorized.
    The solution containing this material was evaporated to dryness
on a water bath and insufficient water was added to dissolve the
residue. On filtering the most soluble portion was found to contain

                                                                        Downloaded from by guest, on September 21, 2009
the Millon reacting material showing beyond doubt that the substance
could not be tyrosin.
    Abandoning hope of obtaining the pure compound from this
small quantity of material I hydrolyzed 300 grams of air-dried
black wool with two liters of 25 per cent sulphuric acid, boiling the
mixture for 24 hours. After separating tyrosin (6.1 grams) in the
usual manner, I found the Millon’s reaction of apparently un-
diminished intensity and, although I was able to concentrate the
reacting material in the most soluble fraction (after 20 grams of
leucin (?) had been separated which gave no coloration with the
reagent and a third fraction was removed which only gave a faint
color). I was, however, unable to obtain the substance in any
semblance of purity by this method. It is very evident, however,
that some aromatic phenolic body is present in this keratinmolecule
which is not identical with any of the known products of protein
    I expect to take up the study anew in a short time and with the
 aid of a Geryk vacuum pump it may be possible to separate this

Shared By:
Tags: A-NEW, -DECO