Chromatographic Differentiation of the Pigments of Red and Buff Feathers

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PURDUE UNIVERSITY

T H IS I S T O C E R T IF Y T H A T T H E T H E S I S P R E P A R E D U N D E R MY S U P E R V IS IO N

by

John Robert Smyth. Jr.

e n title d

Chrom&tograph!c Differentiation of the Pigments

of Red and Buff Feathers

C O M P L IE S W IT H T H E U N IV E R S IT Y R E G U L A T IO N S O N G R A D U A T IO N T H E S E S A N D I S A P P R O V E D

BY M E A S F U L F IL L IN G T H IS P A R T O F T H E R E Q U IR E M E N T S F O R T H E D E G R E E O F

Doctor of Philosophy

P rofessor in Charge of T h esis

H e a d of S#hoe*

D epartm ent

T O T H E L IB R A R IA N :— T H IS T H E S I S I S N O T T O B E R E G A R D E D AS C O N F ID E N T IA L ,

P ro fesso r in Charge

R eg istrar F orm 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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uest ProQuest 27712233 Published by ProQuest LLC (2019). C opyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States C o d e M icroform Edition © ProQuest LLC. ProQuest LLC. 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106 - 1346

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 ----3 Melanin Pigments Purification and Fractionation of Natural Melanin - - - - - - 11 Light Absorption Properties of Natural Melanin - - - - - - - 1 6

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 ------------Quantitative Differences in Red Feather Pigments - - - - - Between Sexes - - - - - - - - - - - - - - - - - - - - - - 3 8

DISCUSSION------------------------------------

40

SUMMARY AND CONCLUSIONS------------------------------------ 44

BIBLIOGRAPHY

48

36

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 - ----2

3

- 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

28

- 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 - 29 (1) Brown I; (2) Brown I I - - - ---

INTRODUCTION The mechani

by which the chromosome 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

a

melanin* With these facts in mind, the overall objective of this study was to clarify further the nature of the melanin pigments involved in the pigmentation of the epidermal structures of the domestic fowl.

An

attempt was made to develop© a technique involving the chromatographic fractionation of pigments of feather extracts.

After completion of a

technique an attempt was made to study the relationship between the pigments found in certain genotypes of the domestic fowl and to com­ pare the feather pigments of the fowl with those of other avian and mammalian species.

3

A SURVEY OF THE LITERATURE Melanin Pigments The occurrence of melanin, a pigment, is vide spread throughout the animal kingdom, being present at some stage of development in most invertebrates and vertebrates*

The term ^melanin" has been used by

investigators to describe many naturally occurring pigments and also certain colored compounds obtained by laboratory synthesis*

Figge

(1947) pointed out that chemists usually regard melanin as a polymer of oxidized tyrosine, vhile in biological vork, any black, brovn, red-brown, tan or amber pigment is likely to be called melanin*

There­

fore, melanin is a non-specific name for a large group of hetero­ geneous pigments which absorb visible light in a uniform way. Mason (1947) attempted to define and classify the various forms of melanin* His suggested classification is as follows: A*

Natural melanins* 1*

Native melanins*

These are in the form found in living

cells* (a) Animal melanins* (b) Plant melanins. 2*

Derived melanins.

These pigments are those which are

changed chemically due to extraction. (a) Simple* (b) Conjugate. B*

Synthetic melanins*

This group includes the polymeric pig­

ments obtained by in vitro oxidation of benzenoid amino acids and phenolic amines and amino acids.

4

I.

Photochemical oxidations.

-2. Chemical oxidations. 3.

Enzymatic oxidations.

As to just where melanin extracted from hair or feathers should be placed under this system is not dear.

Until more knowledge concern­

ing the chemical structure of the pigment is available, it seems plausible that these extracted pigments can be classified as native melanins. A number of workers have presented opinions concerning the number of chemically different melanin pigments present in mammals and birds. Ueumann (1937) found yellow, brown and black colored pigments in rabbit hair.

Serra (1947) also noted that at least these three differ­

ent pigments are present in rabbit hair.

Russell, Russell and Branch

(1943) after reviewing the situation, pointed out that at least three qualitatively different colors, sepia, brown and yellow exist in mammalian hairs.

In addition to these three pigments, Rothman and

Fie sch (1943) have isolated another pigment from red human hair, which is chemically different.

In the pigeon, Metzelaar (1926) first demon­

strated that a brown or chocolate pigment, separate from red and black, exists. Later Hawkins (1930) stated that all color combinations of pigeon feathers are due to three basic pigments, black, chocolate and red.

Bohren, Conrad and Warren (1943) working with the domestic fowl

reported that black, red and buff pigments are the three basic qualita­ tively different melanins present.

They believed black and blue chicken

feather pigments to be chemically identical.

Smyth (1947) confirmed

the presence of the three melanins which Bohren et al. reported, but pointed out that the blue pigment is a chemically separate melanin

5

as indicated by the light absorption properties in the ultra violet spectral regions for the black and blue pigments*

In addition to the

four basic feather melanins found by ânyth, Nickerson (1946) extracted a pigment from red and buff chicken feathers which is similar. If not identical to the fraction isolated from human red hair by Rothman and Fiesch.

In contrast to the above results, Lee and Penrose (1946) found

that they could extract red pigment from black human hair with dilute acid as well as from red hair*

This plus the fact that normal concen­

trations of pigment were obtained from albino hair led them, to suggest that they had measured an extraction-formed chromogen rather than actual hair pigment as Araow (1938) had assumed he was doing by use of the same technique.

However, digestion of black chicken feathers with

acids does not yield pigmented extracts*

Also no mention has been

made of pigmented extracts being produced by the numerous workers who have digested black hair in various normalities of acid.

In some birds

a non—melanic pigment, lipochrome, plays an important part and may even contribute the main coloration to the feather.

Test (1942)

demonstrated the presence of red, yellow and orange lipochrome in the woodpecker (genus Coloptes), and Kritzler (1943) found carotenoids in the display and eclipse feathers of Bishop birds. The fact that tyrosine may be converted through a chain of reac­ tions to artificial melanin has been throughly investigated and is generally conceded to represent the most logical means by which natural melanin is formed.

The artificial melanin produced is black

in color, although a reddish phase which dopa (3, 4 - dihydroxyphenylalanine) passes through has frequently been compared to natural red pigment*

For a detailed review of this reaction the reader

6

should see the paper by Eaper (1932)*

As pointed out by Nickerson

(1946), it is impossible to say at this time whether or not natural red mmlmniT, is more highly or less highly oxidized than black melanin* However, since this investigator could not oxidize red melanin in feathers to black, it is improbable that red melanophores represent blacks in which the formitive processes have been arrested at an in­ termediate stage.

Another approach to this problem has been through

the study of the pigment-cells or melanophores which deposit the pigment in the immature feather or hair follicle.

For a detailed

discussion of this work, the reader is referred to the papers of Willier and Rawles (1940, 1944)*

Hamilton (1940, 1941) studied

cultured tissue expiants of the domestic fowl where he was able to closely observe the role played by the melanophores.

This examina­

tion showed that two types of melanophores existed, one type pro­ ducing black pigment and the other yielding red.

Both red and black

melanophores were present in the cultures from red breeds, while those from black colored breeds contained only black melanophores. No granules were observed in the black melanophores which resembled those found in red melanophores, which indicated that the red granules did not represent an intermediate stage in the development of black.

In fact, Hamilton observed that the pigment granules

destined to be black changed in color during development from tan through brown to black*

The tan and brown phases were distinctly

different from the appearance of the red granules in red melanophores. From these observations Hamilton concluded that red pigment did not represent an arrested stage in the development of black pigment. Danforth (1937) had previously observed two types of melanophores

7

in reciprocal

grafts involving black and yellow chickens.

This

investigator and Hamilton both observed that black, yellow or red melanophores elaborated their respective pigments while lying side by side within the tissues.

Regardless of the evidence from the dopa

reaction, it would appear that natural red and black pigments differ in some way other than their degree of oxidation. Nickerson (1946) expressed the opinion that due to the lack of specificity of this enzyme, tyrosinase may utilize various substances other than tyrosine in the production of melanins. altogether specific for tyrosine.

Tyrosinase is not

It will also catalyze reactions

involving monohydric and polyhydric phenols resulting in various yellow, red or red-brown pigments.

Randall and Hitchings (1944)

found that tyrosinase will react with many of the phenethylamine derivatives.

That water-soluble esters of various estrogens can

also be oxidized by tyrosinase to yield red pigments has been demon­ strated by Graubard and Pineus (1942).

Spiegal-Âdolf (1937, 1939)

demonstrated that photosynthetic melanins which resemble natural sepia melanin may be produced from tryptophane and phenylalanine, as well as from tyrosine. Although a considerable amount of information has been accumulated concerning some of the chemical properties of natural melanins (for review, see Smyth (1947)); much less is known of their chemical com­ position.

The possibility that melanin might contain heavy metal ions

within their complex molecular makeup has been suggested many times* Gortner (1912) found two to three percent ash in black rabbit hair. The ash was mainly in the form of iron oxide.

Ladebeck (1921) also

believed that red chicken feather pigment contained some iron.

8

although less than black feathers.

île sch (1949) found higher iron

values in red-brown guinea pig hair than in white hair.

He also

studied the catalytic role played by copper in the fozmation of melanin and suggested that copper may remain attached to the melanin molecule# This investigator also pointed out that Giuliani found as much as two percent copper in the melanin ink of the octopus.

Fischer and Hilger

(1923) found a purplish copper porphyrin compound, turacin, in the feathers of the African Turcaco bird#

The exact relationship between

the heavy metals and melanin, if one exists, will have to await chemical analysis of more highly purified extracts of pigment and perhaps better assay techniques* Neumann (1937) made a chemical investigation of the yellow, brown and black melanin pigments of the rabbit.

He found that the black pig­

ment had the higher molecular weight and the greater nitrogen content, while the yellow pigment had the lower molecular weight and nitrogen content*

However, he noted that these differences were not marked*

St&ry and Eichtér (1938) state that melanin pigment is in the form of different keratin compounds.

They break this down into

^leucokeratides” (white hair), *melanokeratides* (black hair) and *rhodokeratides* (red hair)#

Browns and blondes result from mixtures

of *leucokeratiddb* and ^melanokeratides” in differing proportions. Serra (1943) hydrolysed the non-chromatic portion of melanin and compared the amino acid content with that of hydrolysed keratin*

He

found that they differed in amino acid content, and therefore, con­ cluded that keratin was not a part of the melanin molecule. Serra (1943# 1946, and 1947) stated that natural mammalian melanins were formed by a protein and a colored mel&noid, which were bound

9

together*

By hydrolysis of black rabbit melanin for forty-eight hours

vith twenty percent hydrochloric acid, he obtained protein breakdown products and a colored material which was believed to contain the melanoid and a little humin.

He assumed that the bond between the pro­

tein and the melanoid was broken by these means, and therefore, releas­ ed the melanoid fraction in a pure form*

The term ’"melanoid* has been

used by various authors to describe different things, but these should not be confused with the *chromatic grouping* idea of Serra*

For ex­

ample, Edwards and Duntley (1939) described a modified diffuse form of melanin which they called ^melanoid**, while Einsele (1937) used the term to describe humin produced by acid digestion of hair*

Serra

(1943) used the work of Mayer (1928) as evidence for his proteinmelanoid hypothesis*

Mayer synthesized a preparation similar to some

natural melanins by adding o - quinone to serum peptones and proteins* The colored products varied from black to brown depending on the pro­ teins used*

Greenetein, Turner and Jenrette (1940) used panereatin

to digest Harding-Passey mouse melanomas and thus free the pigment complex*

Their evidence indicated that melanin is a polymeric deriva­

tive of a polyphenol*

Greenstein (1947) stated that he was in accord

with the basic idea of Serra that melanin is combined with a protein carrier* The iron containing fractions of red human hair isolated by Rothman and Fiesch (1945) and Flesch and Rothman (1947), to which they have given the name *trichosiderin”, apparently contributes little or nothing to the red hair color*

Its relationship to the rest of the

hair and feather pigments is not understood, and for this reason the literature under this phase is being reviewed separately from the

10

general studies on melanin.

The above authors demonstrated that the

iron present in trichoeiderin is in the trivalent form.

They believe

that due to its purpiish-red color in acids, its reversible color change with the pH, its absorption spectrum, and the analysis of its cleavage products that it is a phenolic iron compound.

That the pig­

ment has a very high degree of stability is explained on the basis that the phenolic hydroxyl group is attached to a heterocyclic ring contain­ ing nitrogen*

Since Flesch and Rothman (1947) could not find tricho*

siderin in red hair samples from the horse, dog, eat and rabbit, they believe that it is a pigment peculiar to bright red human hair.

Arnow

(1938) extracted red hair from both hmans and horses with dilute acid by a technique similar to that used by Rothman and Flesch, but did not report finding a pigment resembling trichosiderin in either species. Nickerson (1946) obtained a purplish-red iron containing pigment from red and buff chicken feathers which appeared to be the same as trichosiderin.

From the light absorption properties he concluded that

much of the molecule is probably similar to that of the major portion of the red melanin, which he showed to be practically free of iron. The results of Flesch (1949) in obtaining iron from whole red guinea pig hair extracts are probably explained by the presence of trichosiderin. More evidence that trichosiderin is a phenolic iron compound is shown by the familiar reaction between ferric chloride and phenols pro­ ducing blue, violet or red colorations.

Wesp and Brode (1934) produced

over forty iron phenolic compounds which showed light absorption maxima very similar to that of the iron pigment reported by Rothman and Flesch. The maximum shifted with the phenolic source used, and Wesp and Brode

u obtained maxima over a considerable range in wave length, from below five hundred to well above six hundred millimicrons. Purification and Fractionation of Natural Melanin Since the earlier investigations of the melanin pigments, workers have attempted to secure melanin in a pure form, free from hair and fbather impurities such as keratin.

Keratin is the complex protein

in which the pigment is encased, and attempts to extract the pigment usually cause the break down of keratin, thereby resulting in a solu­ tion containing both materials.

Gomitz (1923) pointed out that the

need for the removal of keratin was recognized by Spottel, who reported that digesting the dark colored series of pigeon feathers in boiling concentrated sulfuric and nitric acids resulted in a breakdown of the keratin and left the pigment as a residue.

This method was of no

value in the light color series since these pigments are also soluble in acid.

Lloyd-Jones (1915) also working with pigeon feather pigments

reported that much of the keratin could be removed by boiling the feathers in 95 percent alcohol. In their study of the light absorption properties of red and black horse hair pigment Zwicky and Almasy (1935) claimed to have removed the effects of keratin by subtracting the light absorption for white horse hair from the corresponding absorption for red and black horse hair. Baker and Andrews (1944) reported that this method was not followed in their work as the light absorption curve of albinos had been found to be subject to considerable experiments! error.

Since non-albino whites

usually contained some dispersed black pigment granules, the absorption curves for white horse hair would probably show considerably more

12

experimental error than would result from the albino whites used by Baker and Andrews* Einsele (1937) noted that although the keratin from mouse hair was readily hydrolyzed by alkali allowing the pigment to go into solu­ tion, this method was unsatisfactory as the hydrolyzed keratin precipi­ tated by acid in the same pH range as did the pigment*

Since this

investigation involved only the dark color series of pigments, Einsele then successfully employed acid hydrolysis which left the pigment as a residue, and the keratin was removed by discarding the acid solution* Rothman and Flesch (1943) used glacial acetic acid to accomplish the keratolysis in red human hair*

Since the red hair contained an acid

soluble pigment, they evidently could not use anything but weak acids to hydrolyze the keratinous structure of the hair*

Further purifica­

tion was accompli shed by precipitation at the iso-electric point, which they found to be a pH of 7*0. In preparing solutions for a spectrophotometrie comparison of red and black chicken feather pigments, Nickerson (1946) removed the red pigment with cold dilute 0*01 N potassium hydroxide and the black by digestion with 6 N hydrochloric acid*

He stated that the readily al­

kali soluble red pigment went into solution while the keratin did not. The black pigment was obtained keratin—free by the same method as was used by Einsele (1937) and others. Êànyth (1947) studied the effects of proteolytic enzymes on alkaline extracts of white chicken feathers.

Pepsin, proteose, orthozyme and

trypsin were all found to hydrolyze the keratin precipitate from al­ kaline solutions.

However, some preparations of trypsin also hydro­

lyzed the pigment when employed on colored feathers, and so was of

13

little value as a means of purification*

The effects of the removal

and addition of keratin by pepsin treatment and addition of white feather extracts were studied on subsamples of a composite sample for each of black, blue, red and buff feathers*

From this work it was

concluded that variations in the pigment-keratin ratio in an average feather sample would not affect the absorption properties of the pig­ ment to a noticable degree*

There was also some evidence that pepsin

digestion might slightly alter the chemical structure of the pigment* In order to make an analysis of the pigments involved in a par­ ticular genotype, some method of fractionation is necessary*

Until

very recently the only means of separation has been based on differ­ ences in solubility*

One of the early pigment investigators, Gortner

(1911)> showed that melanin hair and feather pigments could be divided into two series depending on their solubility properties in acids* The dark series, including black and the grays were insoluble in acids, while the reds and buffs of the light series were acid soluble*

This

provided a rough separation of these two groups, which, although in­ adequate, has been used by many investigators* Buss (1949) devised a method for separating both red and buff feather extracts into three pigment fractions*

By extracting whole

washed feathers in 0*1 H hydrochloric acid a fraction which had the indicator properties of the acid soluble iron-containing pigment of Rothman and Flesch (1943) and Nickerson (1946) was extracted*

After

several digestions with 0*1 N acid the normality was increased to 0*5 N* A brown pigment was removed with this solvent and after several digestions appeared to be completely extracted from the feather*

By

increasing the normality of the acid to 6 N the keratinous structure

u of the feather was completely hydrolyzed and the remaining pigment left as an acid insoluble residue.

Thus three apparently different pigments

are involved in the phenotypic expression of red and buff feathers. Chromatography has in recent years been used extensively for quali­ tatively separating various colored materials into subfractions,

Wald

and Allen (1946) developed a chromatographic technique for the fractiona­ tion of Drosophila melanogaster eye pigments.

They adsorbed distilled

water solutions of wild type eye pigment on a column of talc and cellte, the latter being employed to promote more rapid flow of the solution. The chromatogram was developed by washing the column with 1 percent sodium carbonate, and the adsorbed pigments were eluted with a mixture of 1 percent sodium carbonate and 3 percent n-butyl alcohol in water. Since all of the eye pigments tended to fade in alkali, pigment solu­ tions were neutralized as soon as possible after exposure to alkaline compounds.

Four separate pigments were isolated from the wild type

eye extract by this technique. Attempts to apply chromatography to separation of melanin solu­ tions have been confined to the last few years*

Flesch and Rothman

(1945) made an attempt to purify and isolate trichosiderin by means of the chromatograph!c separation method.

They added methanol to acid

extracts of the pigment end passed the solution through a column of «1wmi nwm oxide.

The column was washed with water and acetic acid, and

the pigment was eluted with hydrochloric acid of various strengths. Although the pigment was well adsorbed on the column, elution did not give consistent results.

For this reason this approach was abandoned.

Serra (1946) developed a chromatographic technique for the purification of melanin extracts from rabbit hair and sheep wool.

He used calcium

15

carbonate as an adsorbent for black, gray and wild type extracts. Better results were obtained for the yellow genotypes, including yellow, orange, and sand colors by using magnesium carbonate columns.

In each

case the pigment was extracted and adsorbed from 0.5 N sodium hydroxide. The columns were washed with 1 N sodium hydroxide which removed the keratins and disintegrated pigment, while the melanin remained adsorbed at the top of the column.

Since the pigment could not be eluted from

the adsorbent, the carbonate column was treated with dilute hydrochloric acid and the melanin was then dissolved in dilute caustic. lieved this technique yielded purified pigment.

Serra be­

Pigment from black and

from yellow rabbits were mixed together by Serra (1947) and chromato­ graphed together on a calcium carbonate column.

The yellow pigment

passed down the column, while the black remained at the top.

An in­

vestigation of brown showed it to have adsorption intermediate between that of the black and yellow.

He pointed out that this is a simple

means of showing evident differences between at least three kinds of melanin - black, brown and yellow. Riley, Hesselback, Fiala, Woods and Burk (1949) have recently employed chromatography for purification of melanin pigment granules, varying in size from 0.2 to 0.6 microns or more, from Cloudman S 91 and Harding—Passey mouse melanomas.

Since these granules could be

reversibly adsorbed on Celite columns, they were subjected to chro­ matographic manipulation.

Adsorption of the granules was accomplished

from physiological saline (0.154 N sodium chloride) at a solution temperature of from 2 to 100 q# The melanin granules remained adsorbed at the top of the column while it was washed with 40 ml of cold physio­ logical saline.

Hemoglobin and other tissue components formed a pink

16

band and passed from the column.

The granules were then eluted with

distilled water at room temperature• These workers also found that when the above technique was modified so that elution was accomplished by flowing chromatography rather than extrusion and segmentation, two distinct black pigment zones were evident.

One black zone migrated

down the column at a much more rapid rate than the other.

The physi­

ological and chemical differences of the two zones of black melanin were not investigated. Light Absorption Properties of Natural Melanin A number of investigators have studied the light absorption prop­ erties of natural melanin.

This work covers hair pigments from mice,

guinea pigs, rabbits, horses and humans and feather pigments from numerous breeds of the domestic fowl.

That the light absorption curves

for mammalian hair melanin are essentially linear in nature has been shown by Zwicky and Almasy (1935), Daniel (1938), Araow (1938), Spiegel-Adolf (1939), Baker and Andrews (1944) and Ginsburg (1944). Determinations have been made in various strengths of acidic and alkaline solutions.

Bohren, Conrad and Warren (1943) and Smyth (1947)

have demonstrated the same type of absorption porperties for chicken feather melanin.

That the linear relationship extended into the

ultra violet spectral region was shown for horse hair melanin by Zwicky and Almasy (1935) and for chicken feather pigments by &nyth (1947).

Rothman and Flesch (1943) studied the light absorption prop­

erties of the iron pigment from red human hair and discovered a maximum at 535 millimicrons and a minimum at 465 millimicrons when readings were made in acid solution.

The general nature of the rest

17

of the absorption curve vas found to be similar to that reported by others in that the greatest absorption occurred at the lower wave lengths.

"When the light absorption readings were made in alkaline

solutions the maximum and minimum disappeared and the curves closely resembled those of other investigators*

Nickerson (1946) found the

same properties for the iron containing red pigment from chicken fea­ thers*

He pointed out that the maximum in the absorption curve of his

pigment samples agrees well with that of Rothman and Fleseh* Serra (1943) presented light absorption curves for black, brown, red and blonde human hair pigment extracted and read in *05 N sodium hydroxide*

In contrast to all other investigators, he found a maxtnmm

for each which occurred in the neighborhood of 560 millimicrons*

In

agreement with previous workers maximum absorption occurred toward the ultra violet region of the spectrum*

Although these maxima resembled

closely that of trichosiderin in acid solution, no one else has report­ ed similar results with alkaline solutions*

Rothman and Fleseh (1943)

and Nickerson (1946) have pointed out that the iron-containing red pig­ ment loses its maximum when light absorption determinations are made in alkaline solutions*

Fleseh and Rothman (1945) also showed that human

hair other than red did not contain trichosiderin* Light absorption studies have led to a controversy concerning whether different colored hair and feathers result from quantitative differences of one melanin or are due to the existence of chemically different pigments.

Zvicky and Almasy (1935)> Daniel (1938), Gremmel

(1939) and Serra (1943» 1946), all investigating mammalian hair melanins, have reported that different hair colors are due to quantitative vari­ ations of one basic pigment*

The proponents of qualitatively or

IS

chemically different melanins include Araow (1938), Baker and Andrews (1944) and Ginsburg (1944) working with mammalian hair pigment and Bohren, Conrad and Warren (1943) y Nickerson (1946) and JEknyth (1947) investigating chicken feather melanins* The evidence tends to substantiate the hypothesis that chemical differences do exist between different colored melanins within a species* Serra (1947) after extending his investigations to his ”pure melanoids® concluded that differences do exist after the protein carrier is hydro­ lysed*

These differences were greatest in the shorter wave length

spectral region*

Serra1s black, yellow and brown rabbit hair melanins

still contained maxima or a definite leveling off between 500 and 600 millimicrons wave length after hydrolysis of the protein carrier* Daniel (1938) studied genotypes of mice ranging from black through brown to light tan in color*

An examination of her data indicates

that tan does have different light absorption properties than does black*

She also noted that extreme dilute agouti and chinchilla brown

were possibly also slightly different*

This would be in accord with

the findings of other investigators that a qualitative difference exists between blacks and non-blacks*

The conclusions arrived at by Zwicky

and Almasy (1935) have also been questioned*

Araow (1938) pointed out

that the absorption curves of black and red horse hair pigment pre­ sented by Zwicky and Almasy cannot be made to superimpose one on the other when multiplied by a suitable constant to correct for differences in concentration*

Bohren, Conrad and Warren (1943) also pointed out

that a critical examination of Gremmel1s (1939) data showed that the colorimetric comparison of the different colored samples from horse hair gave exactly the results to be expected if the black pigment and red pigments differed qualitatively.

19

EXPERIMENTAL PROCEDURE Feather melanins were analyzed from dark red Exhibition Rhode Island Red, medium red production Rhode Island Red, light red New Hampshire, Buff Orpington, Dark Brown Leghorn and Dark Cornish breeds of domestic fowl. Callus bank!va, bobwhite quail and from the Jersey Buff and Bour­ bon Red breeds of turkey*

Pigment from the hair of a Red Setter male

dog and from red human hair was also studied*

All chicken feathers

utilized for this study were removed from the back or saddle region of the birds.

The feathers were carefully brushed to remove loose pieces

of foreign material, washed thoroughly in soap and water and rinsed in distilled water*

The samples were then placed in acetone for several

hours to remove any fatty or oily material on the surface of the feathers. After drying the feathers were again carefully examined for any foreign material which might still be present.

The red hair samples were pre­

pared for extraction by the method used by Fleseh and Rothman (1945)* The pigment was extracted from the feathers by modification of the technique employed by Buss (1949) * The washed feathers were di­ gested in boiling 0.1 N hydrochloric acid for fifteen minutes, and the resulting pigmented solution was decanted and saved for chromatographic purification.

The feathers were then placed in 0*5 N hydrochloric acid

and boiled for one half hour*

The resulting extract was also decanted

and saved, and the extraction was repeated several times until the acid solution was practically colorless after boiling.

The feathers, whose

structure was still relatively intact, were washed with distilled water to remove the acid, and then digested in boiling 0*5 N sodium hydroxide for one half hour*

The feathers were completely hydrolysed and the

20

remaining pigment was released by digestion in alkali.

Each of the

three pigment solutions was filtered through a fritted glass filter. The acid solutions were then ready for chromatographing.

The pigment

in the alkaline extract was then precipitated by the addition of hy­ drochloric acid.

After precipitation the pigment was removed by cen­

trifugation, washed with distilled water and taken up in ethylene chlorohydrin. All three extracts were chromatographed on columns of powdered talc and analytical celite (50 $ 50 by volume). flow of the solution.

Celite promotes the

"Shen the red 0.1 N hydrochloric acid extract

was introduced onto the column, the pigment remained adsorbed at the top of the column.

The chromatogram was then developed by washing the

column with a solution of sodium carbonate of pH 8 .6 , The talc column was then extruded and cut into sections to separate the various bands of adsorbed pigment.

These included two yellow bands which were saved

and a brown band which was discarded.

The pigment in the two bands

were eluted from the talc with cold 0.5 N sodium hydroxide, and then precipitated with hydrochloric acid, centrifuged, washed with distilled water and finally dissolved in ethylene chlorohydrin for spectrophotometric analysis. The pigment in the red-brown 0.5 H hydrochloric acid was also ad­ sorbed directly from the filtered acid extract on a chromatographic column.

As before, the chromatogram was developed with dilute sodium

carbonate and the bands were removed by extrusion and segmentation. Two bands appeared on these columns, a narrow yellow one, and a broad brown one.

The «mall yellow band was found to be identical with the

pigments extracted from feathers by 0.1 H acid.

This band was.

21

therefore, discarded, and the brown band was eluted with cold 0.5 N sodium hydroxide, precipitated and then dissolved in ethylene chloro­ hydrin for further chromatographic purification.

This pigment was

fractionated further on a column of talc and celite. hydrin was used to develop the column.

Ethylene chloro­

The two resulting pigment

bands were eluted by the same procedure as above, after removal of the ethylene chlorohydrin by several washings of the extruded column sec­ tions with distilled water.

The washed precipitate was then dissolved

in ethylene chlorohydrin for light absorption studies. The acid insoluble feather pigment fraction was dissolved in ethylene chlorohydrin and then chromatographed and treated in the same way as the acid soluble brown fraction. A good separation of the two brown fractions of both acid soluble and insoluble brown groups was accomplished more easily with certain concentrations of pigment.

It was found that it was sometimes desir­

able to rechromatograph the two brown pigments to obtain a more clean cut separation. The light absorption properties of all fractions of pigment iso­ lated by the chromatographic technique were studied by means of a Beckman Photoelectric spectrophotometer.

This instrument determines

light absorption as optical density and values are expressed as log lo. I Since this was a qualitative and not a quantitative study, log values were not corrected for concentration or depth of solution.

If two pig­

ments are of identical chemical and physical composition, their light absorption curves will be superimposable one over the other after ad­ justments for differences in concentration.

Following the report of

Lea (1945) that ethylene chlorohydrin (CH2 C1GS2 OH) is a neutral

22

solvent for black melanin, Smyth (1945) showed it to be a solvent for all known chicken feather pigments.

Since all of the pigments studied

are soluble in this solvent, distilled ethylene chlorohydrin was used as a. solvent for each pigment in light absorption determinations. Since it appeared that feathers of Rhode Island Red males contained more of one pigment fraction than did feathers of females, a quantita­ tive study was conducted to determine whether such differences really existed.

Duplicate 1.5 gram samples of saddle feathers from four

different males and four different females were weighed out on an ana­ lytical balance.

Each feather sample was prepared for extraction, and

then digested for one half hour in 100 cc. of 0.1 N hydrochloric acid. The extract from each sample was decanted and saved.

Following this,

each sample of feathers was redigested for another one half hour in fresh 0.1 N hydrochloric acid, and this extract was combined with the first.

The extracts were filtered through medium fritted glass filters

and made up to a volume of 100 cubic centimeters.

This solution was

introduced onto talc and celite columns, and the columns were developed with a solution of sodium carbonate of pH 8 .6 . The pigment involved was reddish—purple in acid but yellow in sodium carbonate.

The yellow

band of adsorbed pigment was removed from the talc column, and eluted with boiling 0.5 N sodium hydroxide. precipitated at pH 5*0*

After cooling the pigment was

An attempt was made to express the amount of

dried precipitate as a percentage of the original feather sample weight. The results were inconsistent, probably due to the presence of salts. Therefore, this method was abandoned.

Instead the dried pigment pre­

cipitate was dissolved in 0.5 N hydrochloric acid and made up to a volume of 50 cubic centimeters.

Each solution was filtered through a

23

fine fritted glass filter and the concentration of pigment in each solution was determined on the basis of the optical density at 550 millimicron wavelength.

Since numerous workers have shown that

melanin extracts follow Beer* s law, this method was considered a valid estimate of quantitative differences.

24

EXPERIMENTAL RESULTS Exhibition Rhode Island Red Chicken Feather Pianents Four chemically different pigments were found in the dark cushion and saddle feathers of Exhibition Rhode Island Red chickens*

Pigment

solutions from 0*1 N and 0*5 N hydrochloric acid extracts and 0*5 N sodium hydroxide extracts were studied*

After the acid soluble feather

pigments were extracted, the remaining pigments were removed by alkali* Thus all of the feather pigments were obtained in solution for investi­ gation*

A study of these pigmented extracts showed that the pigments

present could be divided into two general groups, two purples and two browns, on the basis of their chromatographic behavior, light absorp­ tion properties and pH determined color changes* W e n 0*1 N hydrochloric acid extracts of red feathers were chro­ matographed on talc and celite and developed with dilute sodium carbon­ ate, three bands separated and moved down the column.

A peach-colored

band located at the highest level on the column was the smallest in size.

The second and largest band of pigment was yellow in color.

The

third and lowest band on the column was brown in color and occurred only in «mail amounts in the 0.1 N acid extract*

Since the third band

was found in larger amounts in the 0*5 N acid extract of feathers, only the peach and yellow colored bands of the 0*1 N hydrochloric acid extract were retained for further study. W e n acid was added to the column, characteristic color changes occurred for the two top bands of pigment.

The top peach colored band

became dark purple in color, while the second band changed to a reddishpurple color*

Extrusion and segmentation of the columns was conducted

25

following acid treatment, as the color of the pigments was more distinct. After elution with 0.5 N sodium hydroxide, the dark purple pigment pro­ duced a yellow colored alkaline solution with a slight peach tint, while the red-purple colored band produced a bright yellow solution in alkali. Following iso-electric precipitation it was found that the two pigments were dark purple and red-purple in color respectively, in either acid or ethylene chlorohydrin. This was similar to their appearance when they were adsorbed on a chromatographic column and washed with acid* The pigment from, the small upper band on the column will be referred to, henceforth, as the dark purple fraction, and the fraction of purple adsorbed just below it on the column will be called the red-purple fraction.

If the chromatogram was allowed to develop for a number of

hours there was some tendency for another red-purple band to separate from the bottom of the main red-purple band of pigment.

Since attempts

to bring about this separation were inconsistent, a definite statement concerning a third member of the purple group cannot be made at this time. The presence of the dark purple pigment in feathers was the hardest to demonstrate.

If extractions were made for more than twenty minutes,

this pigment could not be located on a chromatographic column.

Although

this suggested that boiling in acid destroyed the pigment, purified precipitates were boiled for several hours in dilute acid and no change in the pigment was noted.

There appeared to be a high correlation

between the amount of brown pigment in the extract and the demonstra­ tion of the dark purple fraction on chromatographic columns.

The

exact relationships involved are not understood. In both ethylene chlorohydrin and acid solutions the two purple

26

pigments shoved characteristic light absorption curves with each having a definite mayiimim (Figure !)•

All light absorption curves shovn, rep­

resent average curves for a number of samples*

The dark purple pigment

showed a maximum at 575 millimicrons when readings were made in ethylene chlorohydrin, and a maximum at 567 millimicrons when readings were made in 0*5 N hydrochloric acid*

The red-purple fraction showed a maximum

at 555 millimicrons when dissolved in ethylene chlorohydrin, and this shifted to 535 millimicrons in acid*

At shorter wavelengths, below

470 millimicrons, both purple components showed an increase in light absorption.

The absorption maximum of each pigment remained the same

for different preparations, but the slope of the absorption readings at the lower wavelengths varied with the preparation*

Repeated chro­

matographic treatment reduced this slope for both purple pigments, but it was never completely lost.

Further absorption for these pig­

ments always increased at shorter wavelengths, but a maximum in the ultra violet spectral region was never observed*

The light absorption

curves for the purple pigments determined in alkali are presented in Figure 2*

These curves appeared similar to those for the acid solu­

tions, except that the maxima were shifted towards the lower wavelength region.

The red-purple pigment in alkali showed a maximum at 450

millimicrons, while the alkaline solutions of the dark purple pigment showed a maximum at 475 millimicrons* Nickerson (1946) described a pigment extracted from red and buff chicken feathers which appears to be similar to the red-purple fraction found in this investigation*

The light absorption maximum for each

pigment, when dissolved in dilute acid, is reported to be at 535 milli­ microns*

Both pigments also exhibited the same properties of color

27

change with pH*

The main difference in the light absorption of the

pigments in acid is found at the shorter wavelengths where Nickerson's pigment shows greater absorption*

This investigator also reported that

when the light absorption properties of this feather pigment were studied in alkali, the light absorption curve was essentially linear* Similar results were observed in this investigation for dilute acid extracts read in alkali before being subjected to chromatography*

How­

ever, after chromatographic isolation, a maximum appeared in the light absorption curves for each of the purple pigments.

The pigment solu­

tions studied by Nickerson were extracted in 0.1 N hydrochloric acid at 450 C. for several days.

Although the extraction method used differed

from that used in the present study, it would still appear that the pig­ ment solutions analyzed by Nickerson contained some of the brown pigments* These impurities could mask the appearance of maxima for the pigments studied in alkaline solutions.

The brown pigments might also result in

increased light absorption in the ultraviolet region. Vhen the precipitate from the 0.5 N hydrochloric acid extract of red feathers was dissolved in ethylene chlorohydrin and then chromato­ graphed on talc and celite, two bands of brown pigment appeared on the column.

The band which remained adsorbed at the top of the column

represented the greatest quantity of pigment. moved slowly down the column.

The other pigment band

Both pigments were removed from the

column, precipitated and then dissolved in a solvent.

Brown color

developed in either acid, ethylene chlorohydrin or alkali.

The light

absorption curves for both acid soluble brown pigments are presented in Figure 3.

The absorption of these pigments was essentially linear

in ethylene chlorohydrin, in acid and in alkali as the wavelength

28 2.0

1.0

.75

.25

.075-.05

- -

400

500 WAVE LENGTH IN

Figure

. Light absorption curves for the purple pigments from red chicken feathers in ethylene chlorohydrin: (1) Bed-purple pigment? (2) Dark purple pigment

2.0

1.0

.75

LOG .25 --

.05

- -

320

400

500

600

WAVE LENGTH IN Figure î • Light absorption curves for the purple pigments from red chicken feathers in 0.5 N sodium hydroxide : (l) Red-purple pigment? (2) Dark purple pigment

29 2.0

.75

LOG f -

.25

.05 320

400

500

WAVE LENGTH IN

Figure 3-

Light absorption curves for the acid soluble brown pigments from red chicken feathers in ethylene chlorohydrin: (1) Brown I; (2) Brown II

2.0

1.0

.75

LOG -r-

.25

075 —

.

.05

- -

320

400

500

600

MyH WAVE LENGTH IN Myn

Figure 4*

Light absorption curves for the acid insoluble brown pigments from red chicken feathers in ethylene chlorohydrin: (1) Brown I; (2) Brown II

30

decreased. The chromatographic and light absorption properties of the acid insoluble portion of red feathers were compared with those of the acid soluble brown pigments.

The acid insoluble fraction was dissolved in

ethylene chlorohydrin and then chromatographed on talc and celite.

Two

brown bands were formed at the same levels on the column as observed when the acid soluble fraction was chromatographed.

Mixtures of acid

soluble and acid insoluble pigments, dissolved in ethylene chlorohydrin, were also chromatographed.

Only two bands of brown pigment developed.

The light absorption curves for the acid insoluble brown pigments are presented in Figure U*

The variation found between light absorption

curves for duplicate preparations of each of the two pigment components of the brown group was much greater than was found between duplicate preparations of the pigments of the purple group*

Therefore, although

light absorption curves for corresponding pigments from the acid soluble group and the acid insoluble group were not completely superimposable one over the other, all of the evidence already presented plus the reasonably close similarity of the curves leads to the conclusion that the brown components of the acid soluble and insoluble groups are the same or very closely related.

Differences in solubility must then be

attributed to differences in achromatic portions of the molecule which are attached to identical chromatic groups. Comparison of Red Feather Figments in the Domestic Fowl After the methods for fractionating the red feather pigments of Exhibition Rhode Island Red Chickens had been established and the four

31

fractions already described had been studied, the investigation vas expanded to cover other breeds and strains of the domestic fowl whose feathers showed reddish coloration.

This investigation included four

breeds and two strains whose saddle or back feather region consisted of some shade of red.

Exhibition Rhode Island Red feathers are of a

very dark red color, while the other extreme was studied in feathers of the Buff Orpington which is light buff or gold in color.

The pro­

duction bred Rhode Island Rede had a plumage color slightly lighter than that of the Exhibition Reds, while the two strains of New Hamp­ shires, although widely unrelated, were both intermediate in their shade of red between that of the Production Rhode Island Reds and the Buff Orpingtons.

Parti-colored breeds of red or brown and black in­

cluded the Brown Leghorns and the Dark Cornish female.

The male of the

Cornish breed is practically solid black so it was not studied.

The

saddle feathers of the Brown Leghorn male had bright gold and black colors, but the female feathers were a drab colored mixture of brown and black and for this reason the two sexes were studied separately. The pigments found in each strain or breed are presented in Table 1.

The plus signs indicate the presence of a pigment, and the minus

signs indicate its absence.

The number of plus signs indicates the

relative quantity of that specific pigment found in each of the various breeds and strains studied.

The plus signs indicate only the relative

amounts for that particular pigment fraction, and do not indicate quantitative relationships between different pigments of any one breed or strain.

These quantitative relationships are based on observation

only. Feathers of all breeds and strains of chickens studied contained

32

the red-purple pigment.

It was present in approximately the same

amounts in all breeds, except for sex differences which will be dis­ cussed later, and the females of the Brown Leghorn, and Drak Cornish breeds.

These breeds had less red-purple pigment than expected on the

basis of the amount of reddish-brown color in the feathers.

One-tenth

N hydrochloric acid extracts were used to prove the presence of the redpurple pigment in each of the breeds studied.

In the case of Brown

Leghorn female feathers only a slightly reddish extract was obtained, but chromatography demonstrated the presence of the red-purple pigment, as well as the others in this extract.

Light absorption curves of the

chromatographically-separated red-purple fraction of each of the breeds and strains of chickens studied were compared.

All curves showed the

same maximum, and it appeared that the red—purple pigments present in the feather samples investigated were chemically identical. The dark purple pigment was found in all chicken feather samples analyzed except those from Brown Leghorn females.

It is probable,

however, that the dark purple pigment is also present in Brown Leghorn female feathers, but due to the very small amount of total pigment extracted this pigment could not be identified on the chromatograph!c column.

The various breeds end strains of chickens studied showed a

definite quantitative difference in the amount of the dark purple pigment present in their feathers.

It appeared that the shade of red

in the original feather sample was inversely proportional to the amount of the dark purple pigment found.

The Buff Orpington feathers con­

tained the most of this fraction, while the Exhibition Rhode Island Reds contained the least amount.

The Production Rhode Island Reds

were found to have slightly more of the dark purple fraction than

33

did the Exhibition Reds, while the feathers from the two New Hampshire strains contained an intermediate quantity.

The light absorption curves

for the dark purple fraction isolated from each of the breeds or strains of chickens were in agreement. The two brown feather pigments were found in all of the fowl stud­ ied.

Again, as in the Exhibition Rhode Island Red, the fraction ad­

sorbed at the top of the column (brown I) represented the bulk of the brown fraction in all cases.

The pigment adsorbed at the lower levels

of the column (brown II) also had the steeper slope in its light ab­ sorption curve.

The 0*5 N acid soluble pigments and the acid insoluble

pigments extracted in alkali of all breeds, except those of females of the Brown Leghorn and Cornish breeds were compared.

The light absorp­

tion curves for the two brown pigments of all breeds and strains in­ dicated that the same two pigments were present in each. In the case of the feathers from female Brown Leghorn and Cornish breeds little brown pigment was extracted with acid; and, therefore, it was necessary to use the alkaline extracted pigment in order to obtain sufficient quantities to demonstrate the presence of the brown II frac­ tion on a chromatographic column.

These feathers also contained a

considerable quantity of black pigment which was extracted in alkali with the brown group.

Nhen these pigments were introduced onto talc

and celite columns in ethylene chlorohydrin, the black pigment was adsorbed at the extreme top of the column and was easily discarded when the column was divided for elution of the pigments. Red Feather Pigments from Other Species The red pigments of feathers removed from the back or saddle region

3A

Table 1.

The pigments found in red feathers and hair

Species and breed or strain

RedPurple

DarkPurple

Brown I Brown II (top) (bottom)

Exhibition R.I.R. Ô and 9

m

/

////

u w

Production R.I.R. Ô and 9

m

//

////

////

Strain 1 Hew Hemps* d and 9

m

m

Strain 2 New Hamps* c? and 9

m

///

Buff Orpington 9

m

Domestic fowl (gallus domestieus)

u

///

///

u

Brown Leghorn d Brown Leghorn 9

m

—

m

//y

m

m

Dark Cornish 9

m

Gallus Bank!va d

///

m

m

m

Gallus Bank!va 9

#

—

tv

m

Bourbon Red d and 9

/

—

m

m

Jersey Buff d and 9

/

—

Bobwhite Quail d and 9

—

—

m

m

Red Human Hair 9

u-

Turkey

Red Setter Dog Hair d

35

of the Jungle Fowl or Gallus b&nkiva and the Bourbon Red and Jersey Buff breeds of turkeys were also studied. bobwhite quail were also analysed.

The red feather pigments of the In this species feathers were re­

moved from the whole body in order to secure an adequate sample*

The

plumage of the Gallus bankiva appeared similar in color to that of the Brown Leghorns, while the Bourbon Red and Jersey Buff turkey breeds had saddle feathers which were solid colored and differed only in that the Jersey Buff feathers were of a slightly lighter red shade*

The

bobwhite quail showed a parti-color combination of reddish-brown color with black and white areas*

A summary of the pigment analyses in the

species studied is presented in Table 1* The quantity of the red-purple pigment varied considerably between the species studied.

This pigment was present in both the Gallus

bankiva and the two turkey breeds, but appeared to be entirely absent in the bobwhite quail.

The amount of the red-purple pigment in the

Gallus bankiva feathers appeared to be similar to that of the Brown Leg­ horn,

Although the red-purple pigment was found in the feathers of

both red and buff breeds of turkeys, the quantity present was minute. The 0,1 N acid extracts showed only a slight yellow coloration, but chromatographic and speetrophotoaetric observations demonstrated the presence of a small amount of this pigment fraction in these extracts. The amount of the red—purple pigment found in the Jersey Buff turkey feathers was slightly less than that present in feathers from the Bourbon Red breed,

Spectrophotometrie analysis of the red-purple

pigments from the Gallus bankiva and turkeys indicated that they were the same as the pigment found in the red feathers of the domestic fowl, A maximum in each of the light absorption curves was obtained at 555

36

millimicrons* The dark purple feather pigment vas found only in the male Gallus bankiva*

The content of this pigment was similar to that found in

feathers of male Brown Leghorn Chickens.

The dark purple pigment was

not obtained from the feathers of the female Gallus bankiva, the two turkey breeds or the bobwhite quail*

Again, as in the case of female

Brown Leghorn chicken feathers, the absence of the dark purple fraction in the female Gallus bankiva may have been due to the small quantities of pigment extracted with acid*

The light absorption curve of the dark—

purple pigment of the male Gallus bankiva showed the same maximum at 575 milllmicrons wavelength as was found for the dark—purple pigment of the red feathers of the domestic fowl*

They were, therefore, assumed

to be the same pigment* The two brown pigments, brown I and brown II, were found in the Gallus bankiva, turkeys and bobwhite quail*

The brown pigment group

of turkey feathers was less easily extracted in dilute acid, however, than it was from chicken feathers. pigments of the bobwhite quail.

This was also true of the brown

Therefore, it was necessary to use

the acid insoluble portion extracted with alkali from these feathers in order to obtain sufficient quantities for chromatographic fractiona­ tion*

The light absorption curves of the brown feather pigments in­

vestigated appeared sufficiently similar to varient the conclusion that the same two pigments (brown I end brown II) are common to all the avian species studied* Bed Hair Pigment Since it appeared that all avian species, breeds and strains studied contained two or more of the same colored pigments in their

37

feathers, red hair samples from a human female and from a Red Setter dog were analyzed for pigments and the results compared with those ob­ tained from the birds studied.

The results are presented in Table 1.

The red hair of the dog did not contain either fraction of the purple pigment group*

The red human hair sample contained the red-

purple, but not the dark purple pigment.

This confirms the results

of Rothman and Fleseh (194-3) and Fleseh and Rothman (194-5) who found an iron-containing red-purple pigment in red human hair.

They also

reported that they were unable to isolate this pigment from red dog hair.

On the basis of light absorption analyses of the red-purple

fraction from human red hair, this pigment was concluded to be identi­ cal to the red—purple fraction of red avian feathers.

The light ab­

sorption curve of the fraction from human hair exhibited the same shape as the curve in Figure 1.. The maximum for ethylene chlorohydrin solutions was at 553 millimicrons wavelength as compared to the 555 millimicrons maximum found for the avian red-purple pigment.

"When

light absorption determinations were made on 0.5 N hydrochloric acid solutions of the pigments, both the avian red-purple pigment and that from red human hair showed a maximum at 535 millimicrons wavelengths. The conclusion that the red—purple pigments of avian feathers and red human hair are identical is contrary to the opinion stated by Fleseh and Rothman.

They stated that the red-purple pigment isolated from

red human hair is peculiar to that species.

However, their investi­

gation did not include a study of feather pigments.

Although no re­

sults were obtained concerning the iron content of the red-purple pigments isolated in this study, Nickerson (194-6) reported that red and buff chicken feathers have an iron containing pigment whose

38

light absorption properties are similar to the red-purple fraction reported by Rothman and Fleseh (1943)» Chromatographic separation of the brown components of the red hair samples showed the presence of two brown pigments in both species. Light absorption studies on these pigments showed the absorption of light to increase nearly linearly with increases in wavelength.

As

before, the light absorption curve for the brown II pigment had a steep­ er slope than that of the brown I fraction.

It also appeared that the

two brown pigments found in red dog hair and in human red hair are the as the pigments which appear to be common to all the other species studied. Quantitative Differences in Red Feather Pigments Between Sexes A given quantity of feathers from the back or saddle region of males was found to contain more of the red-purple fraction than a corresponding quantity of apparently similarly colored feathers removed from the same region of females.

The quantitative values for the pig­

ments were obtained with a photoelectric spectrophotometer and ex­ pressed as log lo values. These values are presented in Table 2. The I t test shows the quantitative differences between sexes to be statistically significant (P - 0.03).

Since some pigment was lost from the male A

sample, it is likely that an even greater difference was actually present. The red-purple pigment was the only pigment which obviously differ­ ed in quantity with sex; therefore, it was the only one studied.

An

investigation of the dark purple fraction might, however, also have shown a difference in the quantity of the pigment in the two sexes.

39

Table 2.

Sexual differences in the quantity of the red-purple pigment found in red chicken feathers

Males Amount of light absorption Females Amount of light absorption expressed as log lo expressed as log lo I I A

.191

E

.050

B

•312

F

*107

C

•185

G

•140

D

*426

H

•036

This investigation did not include any physiological studies on the causes of the difference in quantity of the red-purple pigment in the sexes, so it is not possible to state the relationship between this difference and the sex hormones* differences in feathers exist* feather structure.

It is a well known fact that sex

This is particularly noticeable in the

Hamilton (194-0, 1941) also demonstrated that sex

hormones increase the rate of differentiation of red melanophores. Testosterone appeared to stimulate the differentiation of red melano­ phores more than did the female sex hormones.

Although, experimental

proof is lacking, it is feasible to assume that sex hormones might initiate quantitative pigment differences in the sexes*

However, since

the structure of saddle feathers differs between males and females, the possibility that the structural differences are correlated with the amount of pigment extracted in a given period of time must be considered*

40

DISCUSSION Although a number of studies have been conducted on the natural melanins found within a species, only a few investigators have com­ pared the pigments of different species,

Arnow (1938) compared the

light absorption curves of dilute acid extracts of pigment of human red hair with those of red horse hair obtained by Zwicky and Almasy (1935) • He found that the red pigments of the two species showed similar absorption curves when dissolved in acid as well as when dissolved in alkali*

Fleseh and Rothman (1945) using a different ex­

traction technique found an iron containing red pigment in human rTitian red* hair.

They called this compound trichosiderin.

This

substance possessed different light absorption properties than the pigment extracted by Arnow.

Fleseh and Rothman compared acid extracts

of red hair samples from the horse, dog, cat and rabbit, and found that of the species studied, only red human hair contained trichosiderin. Results from the present investigation confirm the report of Nickerson (1946) that red and buff colored chicken feathers contain a reddishpurple pigment which appears to be identical with the iron containing pigment of human red hair.

Furthermore, it has been shown that the

same two brown pigments are probably present in all the species, breeds and strains studied.

Since the species studied were quite diverse, it

is logical to suggest that the two brown fractions are common components of all avian and mammalian red or red-brown epidermal pigment complexes. The presence of the purple group was less consistent.

At the present

time, the dark purple fraction has been found only in the domestic fowl and the Gallus bankiva, a closely related species.

The latter

a

is generally conceded to have played some part as the sole or one of several progenitors of the domestic fowl.

The red-purple pigment was

found in red or buff feathers of all breeds of domestic fowl studied, the Gallus bankiva and turkey, as well as in human red hair.

It

appeared to be absent In the bobwhite quail and red dog hair. The results of this investigation addsnew evidence to the problem of the relationship between red and buff pigmented chicken feathers. Bohren, Conrad and Warren (1943) advanced the hypothesis that red fea­ thers are genetically the seme as buff with an acid insoluble brown pigment superimposed upon the basic pigment.

This hypothesis was

suggested by their observations on the pigment granule makeup of the two different colored feathers and the occurrence of an acid insoluble fraction in red but not in buff feathers.

Also each breed has been

shown to carry the same sex linked gold gene.

Furthermore, these in­

vestigators showed that the light absorption curves of alkaline ex­ tracts of red and buff feather pigments differ significantly. difference was confirmed by Smyth (1947).

Ibis

Buss (1949) has since shown,

however, that if the quantity of buff feathers is large an acid in­ soluble fraction is found. The results of the present study indicate that red and buff feathers contain the same pigment fractions.

Although quantitative

determinations were not made, observations indicated that, buff fea­ thers contain less of the red-purple, brown I and brown II fractions, but appear to contain more of the dark purple fraction than an equal quantity of red feathers.

The different phenotypic appearance of

buff and red feathers is probably due to these quantitative differ­ ences in pigment, differences in pigment granule size as shown by

42

Buss (1949) with the electron microscope, and to differences in the arrangement of the pigment granules within the keratinous feather structure.

Multiple modifying genes apparently act on the substrate

produced by the major gold sex-linked gene and in this way affect these other characteristics which determine whether the feather is red or buff or some intermediate shade such as is found in the New Hampshire breed. The fact that alkaline extracts of buff and red feathers do differ in their light absorption properties is difficult to explain.

A logical

explanation would appear to be that keratinous impurities produce a greater effect on the light absorption characteristics of the smaller quantity of pigment found in buff feathers than they do on the larger quantities of red feather pigments.

However, Smyth (1947) showed that

large excesses of keratinous impurities from white feather extracts have only a slight effect on the light absorption curve of buff feather extracts.

Perhaps differences in the proportions of pigments is re­

sponsible for differences in the light absorption characteristics of red end buff extracted and read in alkali.

Thus, when extractions of

red and buff feathers are made in 0.5 N sodium hydroxide for one hour, the pigments precipitated with acid and then boiled in 0.5 N hydro­ chloric acid, the pigmented acid solutions of red feathers contained a small amount of the red-purple pigment.

Buff feather extracts treat­

ed in this way contained none of the red-purple pigment.

Apparently,

the alkaline digestion destroyed some of the red-purple pigment present in red feathers and all of the red-purple pigment present in buff fea­ thers. The role played by the purple pigment group in the final color

A3

of red feathers or human red hair is not known»

Flesch and Eothman

(1945) noted that despite exhaustive extraction of red hair, the original tint of the hair was not markedly modified»

However, they observed that

the red hair lost its sheen and became considerably duller»

The red

feathers used in this study appeared to lose some of their gloss, but as was found for hair the change was very slight. make up the bulk of the total pigment material.

The brown components It is interesting to

note that male chicken feathers show the most gloss and also contain more red—purple pigment than do feathers from females.

However, it

should be pointed out that black feathers also show considerable gloss, but no red-purple pigment can be extracted from these feathers» The existence of both acid soluble and acid insoluble fractions in red and buff feathers is interesting.

More interesting is the fact

that differences in light absorption curves are not found for the brown I and II pigments of the two groups*

Serra (1943, 1946 and 1947)

and Greenstein (1947) suggested that melanin is a combination of a chromatic grouping and a protein»

If this Is true, perhaps the differ­

ence in solubility of the pigments in acid depends on the basic pro­ tein part of the molecule.

Identical chromatic portions would then

give the same light Absorption properties to the two molecules of different solubilities.

It is interesting to note that in spite of

the difference in solubility these pigments have the same adsorption properties on a chromatographic column*

Therefore, the groupings

which determine the solubility of the compounds have little effect on the adsorptive properties of the molecules»

44

SUMMARY AND CONCLUSIONS Feather melanins from dark red Exhibition Rhode Island Red, medium, red production Rhode Island Red, light red New Hampshire, Buff Orpington, Dark Brown Leghorn and Dark Cornish breeds of domestic fowl. Callus bankiva, bobwhite quail and from the Jersey Buff and Bourbon Red breeds of turkey were separated chromatographically and studied spectroscop­ ically,

The pigments from the hair of a Red Setter male dog and from

red human hair were also studied. The chromatographic technique involved adsorption of pigments from either a dilute hydrochloric acid solution or an ethylene chloro— hydrin solution onto talc and cellte (50$50 by volume).

The chromato­

graphic columns were then developed with a solution of sodium carbonate at a pH of 8 ,6 . After development of the chromatogram the pigments were eluted from the talc with cold 0.5 N sodium hydroxide. Four chemically different pigments were isolated from feathers of the Exhibition Rhode Island Red chicken by chromatography.

These

pigments could be divided into two general color groups, of two purple and two brown pigments, each on the basis of their chromatographic behavior, light absorption properties and color at different pH* s. For descriptive purposes the two members of the purple group were called the "dark purple" and "red-purple" pigments, and the two brown fractions were designated as "brown I* and "brown II". The purple group was separated by introducing 0.1 N hydrochloric acid feather or hair extracts onto chromatographic columns. developed chromatogram showed two bands of pigment.

The

The dark purple

pigment was adsorbed above the red-purple group and represented in all cases a much smaller quantity of pigment.

45

The brown pigments were separated by introducing 0.5 N hydrochloric acid extracts onto chromatographic columns.

Upon development of the

chromatogram, two bands of pigments separated.

The brown I fraction

represented the bulk of the brown group and was adsorbed at the top of the chromatogram.

The smaller brown II band moved slowly down the

column. The purple group showed a reversible color change with changes in pH.

The brown group produced brown colored solutions regardless of

the solvent used.

The red-purple pigment was yellow to brown in color

depending on the Quantity of pigment in sodium hydroxide or sodium carbonate solution, while the dark purple pigment was yellow or brown with a definite peach tint in these solvents.

The red-purple pigment

produced a reddish-purple colored acid or ethylene ehlorohydrin solu­ tion, while the dark purple pigment produced dark purple colored solutions in the same solvents.

Hence, these descriptive names were

used. Light absorption curves of the red-purple and dark purple pigments each showed a definite maximum, but the two brown pigments had essenti­ ally linear light absorption curves.

The greatest light absorption of

all the pigments was found at the shortest wavelengths.

The red-purple

pigment showed a maximum at 555 millimicrons when dissolved in ethylene ehlorohydrin, at 535 millimicrons in acidic solutions and at 450 milli­ microns in alkaline solutions.

The dark purple pigment showed a max­

imum at 575 millimicrons in ethylene ehlorohydrin, at 567 millimicrons in acid and at 475 millimicrons in alkali. A part of the brown pigments of feathers and hair can be extracted with acid, but a considerable portion is acid insoluble.

The acid

46

insoluble portion is extracted with alkali,

"When the acid soluble and

acid insoluble brown pigments were combined and dissolved in ethylene ehlorohydrin and then chromatographed on a talc—celite column only two brown bands appeared.

Hence the two acid insoluble brown pigments,

are apparently chromatographically identical with the corresponding two acid soluble brown pigments.

It is further suggested that the differ­

ences in solubility may be due to the presence of achromatic groups which contribute nothing to the adsorptive properties of the acid in­ soluble pigments on talc.

The chromatic groups of the corresponding

pairs of the two groups of pigments are identical insofar as can be ascertained by light absorption studies. Feathers of all of the breeds and strains of chickens studied contained the red-purple, brown I and brown II pigments.

The dark

purple pigment was found in all of the chicken feathers except those of the female Brown Leghorn,

The absence of pigment in this case may

have been due to the small amount of total pigment extracted from these feathers with acid.

Light absorption analyses indicated that correspond­

ing pigments present in the different chicken feather samples investi­ gated were chemically identical. The quantity of dark purple pigment found in a sample of chicken feathers appeared to be inversely proportional to the shade of red in the original feathers.

The quantity of the red-purple fraction in the

saddle and cushion feathers of Exhibition Rhode Island Red chickens differed with the sex.

Feathers from males contained a significantly

greater amount of this pigment. Feathers from the Gallus bankiva and the Bourbon Red and Jersey Buff breeds of turkeys were found to contain the red—purple, brown I

A7

and brown II pigments.

The dark purple pigment was found only in the

feathers of the male Gallus bankiva, but the absence of this fraction in the female was again, as in the Brown Leghorn, probably due to the smal.3 quantity of total pigment extracted from these feathers.

The

feathers of the bobwhite quail contained only the two brown pigments. The pigmented extracts from red human hair and red dog hair each contained the two brown fractions.

Human red hair also contained the

red—purple pigment present in the feathers of birds. The brown and purple pigments of each species appeared to be identical chromatographically and spectroscopically with the corres­ ponding pigments of the other species studied.

However, observations

indicate that quantitative differences in total pigment and the rela­ tive amounts of avian pigments within and between species may account for differences in observed color of feathers and hair. The brown I pigment fraction represented the bulk of the pigment in all breeds and species studied*

The other three pigments were

found in comparatively small amounts whenever they were present.

48

BIBLIOGRAPHY Amow, L. Earle, 1938. The acid-soluble pigment of red human hair. Biochem. Jour. 32$ 1281—1284» Baker, M. R., and A. G. Andrews, 1944. The melanins. I. Studies of the hair pigments of the guinea pig. Genetics 29: 104-112. Bohren, B. B., R. M. Conrad, and D. C. Warren, 1943* A chemical and histological study of the feather pigments of the domestic fowl* Amer. Nat. 77$ 481—518. Buss, E. G., 1949. An electron microscopic study of feather pigments. Thesis, Purdue University. Danforth, G. H., 1937. Pigment cells in heterogenous feathers. Rec* 68 : 461-468.

Anat.

Daniel, Janet, 1938. Studies of multiple allelomorphic series in the house-mouse. III. A spectrophotometric study of mouse melanins. Jour. Genetics 36 % 139-143* Edwards, E. A., and S. Q. Duntley, 1939* The pigment and color of living human skin. Amer» Jour. Anat. 65 $ 1-33 * Einsele, 1937. Studies of multiple allelomorphic series in the house-mouse. II* Methods for the quantitative estimation of melanin. Jour. Genetics 34$ 1—18* Figge, F. H. J., 1947. Factors regulating the formation and the physical and chemical properties of melanin. Spec. Publ. N. Y* Acad. Sci. 4$ 405. Fischer, H., and J. Hilger, 1923. Zur kenntnis der naturlichen porphyrine. II. Uber das turacin. Z. physiol. Ghem. 128$

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Flesch, P., 1949. The role of copper in mammalian pigmentation* Proc. Soc. Exp. Biol, and Med. 70: 79-83* Flesch, P., and S. Eothman, 1945. Isolation of an iron pigment from human red hair. Jour. Invest. Dermatol. 6 : 257—270. Ginsburg, Benson, 1944* The effects of the major genes controlling coat color in the guinea pig on the dopa oxidase activity of skin extracts* Genetics 29$ 176-198* Goraits, K., 1923. Ueber die wirkung klimatischer faktoren auf die pigmentfarben der vogelfedem* Jour, fur ornithologie. 71$ 456-510. Gortner, R. A., 1911. Studieson melanin.

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Gortner, R. A., 19X2• On two different types of melanins. Exp. Biol. 9* 120-121. Graùbard, H., and G. Pincus, 1942. Steroid metabolism: phenolases. Endocrinology 30: 265—269• Gremmel, F., 1939•

Coat color in horses.

Greenstein, J. P., 1947. Acad. Sci. 4$ 433*

estrogens and

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Chemistry of melanomas.

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Greenstein, J. P., F. C. Turner, and W. V. Jenrette, 1940. Cancer Inst. 1: 377.

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Hamilton, H. L., 1940. A study of the physiological properties of melanophore s with special reference to their role in feather coloration. Anat. Rec. 73 : 525-547. Hamilton, H. L., 1940. Influence of sex hormones and desoxycortlcosterone on melanophore differentiation in birds. Proc. Soc. Exp. Biol, and Ned. 45$ 571-573. Hamilton, H. L., 1941* Influence of adrenal and sex hormones on the differentiation of melanophores In the chick. Jour. Exp. Zool. 83$ 275-305. Hawkins, L. E., 1930. Studies on inheritance in pigeons. X. Re­ lation of chocolate to black and dominant red* Genetics. 16: 547-573* Kritzler, H., 1943* Carotenoids in the display and eclipse plumage of Bishop birds. Physiol. Zool. 16: 241-255* L&debeck, E., 1921. Die farben einiger huhnerro seen. Induktive Abstenu u. Vererbungslehre. 30: 1-62. Lea, A. J., 1945*

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Lee, G. D., and L. S. Penrose, 1946. A contribution to the genetics of hair colour in man. Annals of Eugenics 13: 182-183. Lloyd-Jones, Orren, 1915* Studies on inheritance in the pigeon. II. A microscopic and chemical study of feather pigments. Jour. Exp. Zool. 18: 453-509. Mason, H. S., 1947. A classification of melanins. Acad. Sci. 4$ 399-404* Mayer, R. L., 1928. 7: 2471-2472.

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Metzelaar, J., 1926. Color breeding in pigeon plumage. Pigeon Keeper, Geneva, Illinois: 45*

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Neumann, Paul, 1937+ Haarpigmentuntersuchunger an verschiedenen farbrassen des kanlnchencu Mol, Zentr&lbl. 57$ 522-550, Nickerson, Mark, 194-6, Relation between black and red melanin pig­ ments in feathers. Physiol, Zool, 19$ 66-77, Randall, L. 0,, and G» H, Hitchings, 1944* Effect of tyrosinase on phenethylamine derivitives. Jour* Pharmacol, and Exp. Therap. 31$ 77-83. Paper, R, S., 1932.

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Riley, V, T., M. L, Hesselbach, Silvio Mala, M. ¥. Woods, and Dean Burk, 1949, Application of chromatography to the separation of subcellular, enzymatically active granules. Science 109$ 361—364,

Eothman, S., and P, Flesch, 1943. Isolation of an iron pigment from human red hair. Proc. Soc. Exp. Biol, and Med, 53: 134-135. Russell, W. L., E. S. Russell, and L, R. Branch, 1948. Problems in the biochemistry and physiological genetics of pigmentation in mammals. Spec. Publ. N. Y. Acad. Sci. 4s 447-453. Serra, J, A., 1943. Sur la nature des mélanines et la melanogenese. Genetica 23$ 300. Serra, J. A., 1946, 771-772.

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VITA John Robert Smyth, Jr. was b o m at Lexington, Kentucky, February 9, 1924.

He attended elementary school and high school in Orono, Maine,

graduating from the latter in 1941*

The next four years were spent

attending the University of Maine where he received a B. S. degree in Poultry Husbandry in June, 1945*

On July 1, 1945, he entered the

graduate school at Purdue University where he was an XE Fellow for two years.

An M. S. degree was received in August of 1947.

Graduate work

was continued at Purdue University, where he has held the position of Assistant in Poultry Husbandry since 1947.