C
O
SIX UNKNOWN GREEN PIGMENTS
FROM
MOLLUSKS
PATRICIA MCGEE
JUNE 4, 1964
Dr. JOHN PHILLIPS
HOPKINS MARINE STATION
The unknown green pigment from Tegula funebralis and the similar
green pigments from Tegula brunnea, Tegula montereyi, Tegula pulligo,
the red and black abalone (Haliotis) are the pigments under study.
The unknown green pigments were extracted with methanol in all
cases. The procedure of extraction and removal of the carotenoid
contaminations are as follows:
The gonad of the organism is removed and placed in a waring
blender with 100% methanol. The supernatant from this process
is then centrifuged to remove all the tissue particles. This
solution is taken to dryness on a vacuum distiller at not more than
40°C. The pigments are then redissolved in water. The aqueous
solution is then partitioned with anhydrous ether; the green
pigment remains in the aqueous phase and the carotenoids move to
the ether phase. Thi process is repeated with fresh ether until
the yellow color in the ether has disappeared. The aqueous green
pigment is then taken to dryness and redissolved in distilled
water two times. The pigment in this state is what I refer to
as the crude pigment. The absorption spectra of the pigments in this
stage are provided in the attached sheets.
The pigments, in all cases, were observed to have the
characteristics of pH indicators after the pH was initially taken
to 11. At pH 11, a precipitation is formed. This precipitation
was found to be soluble in distilled water at pH 4 to 6. A similar
precipitation may also be obtatined by addition of acetone to an
C
aqueous solution of the crude pigment. This precipitate was also
soluble in water, and in Tegula funebralis, by a Folin protein test
it indicated that thereis 3.6mg of protein per ml of "standard'
solution of crude pigment.
The pigments were all pH indicators in the alkaline range.
They all appeared yellow to orange, depending on concentration,
at pH 9.5. The spectra of the pigment from Tegula brunnea in the
acid and alkaline states appears in the attached sheets. The
exact turning point of the T. brunnea was determined at 8.3 pH.
All the pigments were observed to be reduced by the addition
of Nag S2 O (sodium hyposulfite). The pigments, however, could
not be induced to reoxidize by addition of sodium peroxide or
auto-oxidation by bubbling with air. An attempt was also made to
reduce the pigments under the less severe conditions of anoxia by
nitrogen bubbling through them. This was unsucessful.
In the case, of Tegula funebralis it was calculated that it
takes 5.7x102 M sodium hyposulfite to reduce the pigment to the
yellow state. It is noted that upon reduction the visible peak
at 650mu is removed; however, the ultra violet peaks at 271 and 325mu
remain the same. (the absorption spectra are included).
The configuration of all the spectra of these various pigments
are similar; however, the absorption peaks vary within a range of
4Omu in the visible and 2 to 10mu for th UV peaks. It would seem
that perhaps the compounds are members of the same family. This
asumption is further supported by identical solubilities and
behavior toward hydrogen ion concentration.
Absorption Spectra
TEGULA FUNEBRALIS
TEGULA BRUNNEA
TEGULA MONTEREYI
PEGULA PULLIGO
RED ABALONE
BLACK ABALONE
Solubilities
TEGULA FUNEBRALIS
TEGULA BRUNNEA
TEGULA MONTEREYI
TEGULA PULLIGO
RED ABALONE
BLACK ABALONE
of Crude Pigments
650 325 271mu
640 330 271mu
610 330 271mu
610 330 271mu
610 330 270mu
610 320 270mu
Ho0, acet., MEOH., ETOH
SAME
SAME
SAME
SAME
SAME
153
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