Table Of ContentPURDUE UNIVERSITY
THIS IS TO CERTIFY THAT THE THESIS PREPARED UNDER MY SUPERVISION
John Robert Smyth. Jr.
by
Chrom&tograph!c Differentiation of the Pigments
e n title d
of Red and Buff Feathers
COMPLIES WITH THE UNIVERSITY REGULATIONS ON GRADUATION THESES AND IS APPROVED
BY ME AS FULFILLING THIS PART OF THE REQUIREMENTS FOR THE DEGREE OF
Doctor of Philosophy
Professor in Charge of Thesis
Head of S#hoe* Department
TO THE LIBRARIAN:—
THIS THESIS IS NOT TO BE REGARDED AS CONFIDENTIAL,
Professor in Charge
Registrar Form 10—2-39—IM
CHROMATOGRAPHIC DIFFERENTIATION OF THE PIGMENTS
OF RED AND BUFF FEATHERS
A Thesis
Submitted to the Faculty
of
Purdue University
by
John Robert Smyth, Jr*
In Partial Fulfillment of the
Requirements for the Degree
of
Doctor of Philosophy
August, 1949
ProQuest Number: 27712233
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ACKNOWLEDGEMENT
Sincere appreciation is expressed to Dr. B. B* Bohren, of the
Department of Poultry Husbandry, and to Dr. J. W* Porter, of the
Department of Agricultural Chemistry, for their counsel and guidance
in the collection of data and preparation of the manuscript. I am.
also indebted to Dr. J. Holmes Martin, of the Poultry Department, and
to Director H. J. Reed, of the Agricultural Experiment Station, for
the provision of the necessary funds, and for supplying laboratory
and other facilities needed in carrying on the study.
Appreciation is also extended to Mr. E. G. Buss and Mr. Edwin
Roth for valuable suggestions and interest shown in this investiga
tion. I am also indebted to Mrs. Louise Roth, of the Poultry Depart
ment, for her assistance in the laboratory.
ABSTRACT
Feather mélanine from dark red Exhibition Rhode Island Red, medium
red Production Rhode Island Red, light red New Hampshire, Buff Orping
ton, Dark Brown Leghorn and Dark Cornish breeds of domestic fowl. Callus
bankiva, bobwhite quail and from Jersey Buff and Bourbon Red breeds of
turkey were extracted with acid or alkali. Pigments from the hair of
a Red Setter male dog and from red human hâir were also extracted in
the same way.
Dilute acid extracts of the chicken feathers studied were fraction
ated into four different pigments by chromatography on talc-cellte
columns. One—tenth N acid extracts were found to contain two purplish
colored pigments; subsequent extraction of the feathers with 0.5 N acid
removed two brown pigments.
The two purple pigments showed similar physical adsorption and
light absorption properties. One pigment was dark purple colored in
acid solution and adsorbed slightly above the other on a chromatograph
ic column. The second pigment was red-purple colored in acid solutions.
Both pigments changed color when the pH of the solution was varied. The
dark purple fraction was yellow-peach colored in alkaline solution and
the red-purple pigment was yellow colored in alkaline solution. The
dark purple pigment showed a light absorption maximum at 575 milli
microns when dissolved in ethylene chlorohydrin and a maximum at 475
millimicrons when dissolved in a 0.5 N sodium hydroxide solution. The
red-purple pigment showed a light absorption maximum at 555 millimicrons
in ethylene chlorohydrin and at 450 millimicrons in alkali.
The two brown pigments (browns I and II) were also separated
chromatographically. In ethylene chlorohydrin, in acid and in alkali
the absorption of these pigments increased linearly with a decrease in
wavelength. Neither of these pigments changed color with a change in
pH of the solution.
Alkaline extracts of the acid insoluble pigments of the red fea
ther melanins yielded two brown pigments which could be separated
chromatographically. These pigments were apparently identical to the
two corresponding acid soluble brown pigments in physical adsorption
and light absorption properties. This result is interpreted to mean
that the acid insoluble pigments contain a group which determines the
solubility properties but does not contribute to the adsorption of the
pigments on talc or to their light absorption properties.
The four pigments reported were found in feathers from all breeds
of the domestic fowl studied and in those of the Gallus bankiva. The
other dark purple fraction was not found in any of the specie s in
vestigated. The red-purple pigment was not found in quail feathers or
in red dog hair, but it was found in both red and buff turkey feathers
and in human red hair. The two brown pigments were present in all
feathers and hair samples studied.
In chicken feathers the proportional quantity of the dark purple
fraction appeared to be inversely proportional to the total amount of
pigment present. Feathers from Exhibition Rhode Island Red males con
tained a greater quantity of the red-purple pigment than did feathers
from females of tills breed.
TABLE OF CONTENTS
Page
INTRODUCTION---------------------------------------------- --
A SURVEY OF THE LITERATURE------------------------------------3
Melanin Pigments ----- 3
Purification and Fractionation of Natural Melanin - - - - - - 11
Light Absorption Properties of Natural Melanin - -- -- --16
EXPERIMENTAL PROCEDURE-------------------------------------- 19
EXPERIMENTAL RESULTS---------------------- 24
Exhibition Rhode Island Red Chicken Feather Pigments - - - - 24
Comparison of Red Feather Pigments in the Domestic Fowl - - - 30
Red Feather Pigments from Other Species - ---- - - - - - - - 3 3
Red Hair Pigment ----------- -- - 36
Quantitative Differences in Red Feather Pigments - - - - - -
Between Sexes - - - - - - - - - - - - - - - - - - - - - - 3 8
DISCUSSION------------------------------------ 40
SUMMARY AND CONCLUSIONS------------------------------------ 44
BIBLIOGRAPHY 48
LIST OF TABLES
Table Page
1 — The pigments found in red feathers and hair - -- -- -- - 34
2 - Sexual differences in the quantity of the red-purple
pigment found in red chicken feathers — — — — — — — — — — — 39
LIST OF FIGURES
Figure Page
1 - Light absorption curves for the purple pigments
from red chicken feathers in ethylene chlorohydrin :
(1) Red-purple pigment; (2) Dark purple pigment - ----- 28
2 - Light absorption curves for the purple pigments
from red chicken feathers in 0.5 sodium hydroxidei
(1) Red-purple pigment; (2) Dark purple pigment-------- 28
3 - Light absorption curves for the acid soluble
brown pigments from red chicken feathers in
ethylene chlorohydrins (1) Brown I; (2) Brown II - - - - 29
4 - Light absorption curves for acid insoluble brown
pigments from red chicken feathers in ethylene chlorohydrin ï
(1) Brown I; (2) Brown I I - - - --- - 29
INTRODUCTION
The mechani by which the chromo some s and their genes are passed
on to successive generations is well known. However, much less is
known of the precise function of the gene in controlling developmental
and functional processes. Consequently increased interest has been
shown in recent years in the physiological processes initiated by the
genes which result in various phenotypes. Many of such studies have
been conducted on the development of pigments in various organisms.
The choice of pigmentation for studies of this type is probably due
to its striking phenotypic expression, relative freeness from en
vironmental effects and ease of visual classification. One approach
to the study of the gene physiology of pigmentation involves in
vestigations of the nature of the end-products of genic interaction.
The pigments found in the epidermal structures of most higher
vertebrates represent various forms of melanin. Melanin is a non
specific name for a large group of heterogeneous pigments which
absorb light in a uniform way» These stable compounds are probably
made up of a chromatic grouping attached to a complex protein and
do not lend themselves readily to chemical analysis. The pigment,
which occurs in the form of specific granules in hair and feathers,
is encased in the very stable protein, keratin. For these reasons
knowledge of the chemical and physical properties of the melanin
pigments is very limited. There is also only limited knowledge avail
able concerning the different pigment fractions which might con
stitute a certain phenotype. Before accurate genetic and physio
logical studies involving pigmentation can be conducted, better
techniques are necessary for the fractionation and purification of
