...

Structural aspects of keratin fibres J. Soc. Cosmet. Chem.

by user

on
Category:

disorders

4

views

Report

Comments

Transcript

Structural aspects of keratin fibres J. Soc. Cosmet. Chem.
J. Soc.Cosmet.
Chem.23 427-445(1972)¸ 1972 Societyof Cosmetic
Chemists
of GreatBritain
Structuralaspectsof keratin fibres
N.H.
LEON*
Synopsis--Rapid progress has been made in keratin research since the availability of
ELECTRON MICROSCOPY and modern biochemistry. This REVIEW presentsa general
description of the structure of MAMMALIAN
KERATIN FIBRES with special reference to
HUMAN
HAIR. It is intended primarily for cosmeticchemistswho desirea brief surveyof the
subject.
INTRODUCTION
Hair and wool, in their natural (unstretched)state, belong to a
group of proteinscalled a-keratins.The designation'alpha' was usedby
Astbury and Woods (1) to indicatethat the crystallineportions of these
proteinshavea particularX-ray diffractionpatternin commonwith various
other fibrousproteins.This pattern was later shownto be associatedwith
the a-helical structure for proteins proposedby Pauling, Corey and
Branson(2). Keratins are definedby Lundgrenand Ward (3) as 'natural,
cellular systemsof fibrousproteinscross-linkedby cystinesulphur.They
have evolvedprimarily as a barrier to the environment,servingto protect
the higher vertebrates--amphibians,reptiles, birds and mammals--from
the stresses
of life. Keratins occuras the principalconstituentsof the horny
layer of the epidermisand of relatedappendages,
suchas horns, hooves,
scales,hair and feathers, that are derived from the skin'.'•
AMINO
ACID COMPOSITION OF KERATIN FIBRES
Like other proteins,keratin fibres are polypeptidescomposedof some
18 different types of a-I•-amino acid residues of the general formula
* Unilever Research Laboratory, 455 London Road, Isleworth, Middlesex.
•' For a discussion
of a moreprecise
definition,
see(4).
427
428
JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS
NHo.CHRCOOH; in the amino acid proline, the amino group forms part
of a pyrrolidinering. The sidegroupsR vary markedlyin sizeandchemical
nature; they may be hydrophobic,acidic or basic. In the specialcaseof
cystine,the groupsR form a cross-linkjoining two chains.An important
steptowardsa closerunderstanding
of the constitutionof keratinwastaken
with the useof columnchromatography,
supplemented
in someinstances
by specialmethodsfor certainamino acids,to obtaincompleteamino acid
compositionsof keratin hydrolysates.These techniqueshave shownthat
eachkind of mammaliankeratin fibre variesslightlyin compositionas a
result of genetic, nutritional and environmental differences,the main
variations occurring in cystine, proline, and glycine. Typical analytical
Table I
Amino acid compositionof keratin fibres
Merino sheepwool*
Human hair•'
MohairS'
Component
g 100 g-X Ixmoleg-X
Ala
g 100 g-X ,tzmole
g-X
g 100 g-X [xmoleg-X
4.10
460
3.07
345
4.03
452
Arg
Asp
Cys
9.58
6.65
12.02
550
500
1000
8.29
5.52
17.08
476
425
1422
8.53
7.24
9.70
490
544
808
Glu
14.41
980
13.02
885
15.52
1055
Gly
5.25
700
3.84
512
4.84
645
His
1.02
66
0.96
62
1.09
70
Hyl
0.16
10
....
Ile
Leu
3.41
8.26
260
630
2.78
6.08
212
464
3.57
8.70
272
672
Lys
3.22
220
2.60
178
3.26
223
Met
0.52
39
Phe
3.80
230
2.36
143
4.04
245
Pro
6.79
590
8.67
753
6.41
557
Ser
9.66
920
8.94
851
7.83
745
542
5.74
482
....
Thr
6.54
550
6.45
Trp
Tyr
1.43
5.25
70
290
....
2.28
126
3.51
194
Val
Ammonia
5.38
1.20
460
750
5.73
1.28
490
797
7.76
1.27
663
793
N (5/o)
S (%)
16.35
3.655
16.50
16.60
5.1õ
3.0•[
*Compiled by Ward (5).
•From Crewther, Fraser, Lennox and Lindley (6).
$Mean value from three analyticalmethodsby Fletcher and Robson (7).
õAveragevalue from Ward and Lundgren (8).
•From Lundgrenand Ward (3).
STRUCTURAL
ASPECTS
OF
KERATIN
FIBRES
429
valuesof somekeratin fibresare givenin Table I. The structuresand pK
valuesof the naturally occurringamino acids in the fibres are shown in
Table II.
The ammoniaproducedin the hydrolysisof keratin fibresis presumed
to arisefrom amidegroups,whichare associated
mainly with dicarboxylic
acids.It hasbeenfound recently,by enzymatichydrolysis,that about 70•o
Table
II
Naturally occurringamino acidsin keratin fibres
Amino Acid
Alanine
Structure
pK a at 25ø (9)
CHaCH(NHOCOOH
2.35; 9.89
ArginineHN=•--NH(CH•)
aNHa
•HCOOH
1.82;
8.99;
NH2
12.48(guanido)
Asparticacid
HOOCCHaCH(NH2)COOH
1.99; 3.90; 9.90
Cysteine
HSCH2CH(NH2)COOH
1.92; 8.35 (SH); 10.46
Cystine
Glutamicacid
(--SCH2CH(NHOCOOH)•
HOOCCH2CH2CH,(?qHOCOOH
< 1; 2.1; 8.02; 8.71
2.10; 4.07; 9.47
Glycine
Histidine
H2NCH•COOH
N
2.35; 9.78
1.80; 6.04 (imidazole); 9.33
L •--CH2CH(NHOCOOH
N
Hydroxylysine
•HaCH(OH)CH2CH•HCOOH
2.13;
8.62
9.67 (e-NH2)
NH,.
NH,.
Isoleucine
CaHsCH(CH)aCH(NHOCOOH
Leucine
(CHa)2CHCH2CH(NH2)COOH
2.33; 9.74
Lysine
HaN(CH2)4CH(NH2)COOH
2.16; 9.18; 10.79(•-NHa)
Methionine
CHaSCH2CH2CH(NH2)COOH
2.13; 9.28
Phenylalanine
C6HsCH2CH(NH•)COOH
2.16; 9.18
Proline
2.32; 9.76
1.95; 10.64
Serine
HOCH•CH(NH2)COOH
2.19; 9.21
Threoriine
CHaCH(OH)CH(NH•)COOH
2.09; 9.10
Tryptophan
• •'• CH
•--CHCOOH
2.43;
9.44
NH
NH•
Tyrosine
4-HOCsH4CH2CH(NH2)COOH
2.20; 9.11; 10.13(OH)
Valine
(CHa)•CHCH(NH2)COOH
2.29; 9.74
430
JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS
of the asparticacid residuesin wool appearto be aminated(10, 11) and
that the carboxylgroupsare presentmainly as glutamicacid residues(11).
It can be seenthat all keratin fibrescontaina very high level of sulphur,
most of which occurs as cystinc. Human hair has less alaninc, leucine,
tyrosine,phenylalanine,glutamicacid, aspatticacid, lysine, and arginine
than other keratin fibres and is richer in cystinc and proline. These
differences
probablyindicatea higherproportionof high-sulphurproteins,
as it is known that human hair has a greaterextent of cross-linkingthan
most wool fibres.The heaviercross-linkingis reflectedin greaterresistance
to attackby hot acidsand in slowerreductionby thioglycolateor sulphite
solution(12). The ready uptake of dyesby mohair is probablydue to the
presenceof a high contentof ionizableside-chaingroups(13). Human
hair, on the other hand, has a lower content of the amino acidswith ionic
side-chainsthan mohair or wool; this differenceis reflectedin its dyeing
behaviour.Thus, humanhair hasa lower capacityfor acids(14) as well as
for acid dyes,e.g. Orange II (15), than wool. On the basisof dyeingand
swellingexperiments,someinvestigatorshave suggested
that human hair
is predominantly'para' (videinfra)in its properties(16).
KERATIN
FIBRE HISTOLOGY
Keratin fibresare very complexboth at the histologicalleveland at the
chemicallevel owing to the multiplicity of protein moleculeswhich are
effectivelycross-linkedto form an integralstructure.Histologically,keratin
fibresconsistof three main components:(a) the cuticlecells(about 10•
of the fibre), whichenvelopthe fibre and overlaprather like fileson a roof;
(b) the corticalcells(about88• of the fibre),whichare longspindle-shaped
cells aligned parallel to the fibre direction; and (c) the cell membrane
complex(about 2• of the fibre), which separateseach cuticleor cortical
cell from its neighbour.The medulla is presentin sometypesof fibres;it
consists of a core of air-filled cells which runs down the middle of the fibre.
Fig. 1 is a schematicdiagramof humanhair structureand Fig. 2 is a scanning
electronmicrographof a human hair.
The cuticle
The outermostlayer of the cuticlewas observedby Allwbrden (17) as
a membranewhich was raisedfrom the surfaceof wool by treatmentwith
chlorinewater. It surroundseach cuticlecell individually,forming part of
Figure 2.
Scanningelectron micrograph of a human hair.
Facing page 430
STRUCTURAL ASPECTS OF KERATIN FIBRES
431
Microfil
Cuticle
--
Cell membranes
Nuclear
remnants
Figure 1.
Schematicdiagram of human hair structure.
the cell membranecomplex(18), and has been shownto reducethe rate of
penetrationof dyes(19) and acids(20) into the fibre. It wasfirst isolatedby
Lindberg,Philip and Gral•n (21), who calledit 'epicuticle',and later shown
by other workers (22) to be almost entirely proteinaceousand to have a
high content of cystine.However, it has now been demonstratedin this
laboratory that the 'epicuticle'of human hair is probably a portion of a
unit cell membraneand adheringmaterial (videinfra).
The exocuticleand the underlyingendocuticle,which comprisethe
main bulk of the cuticle,are differentiatedfrom one another on the basisof
differences
in their intensityof stainingwith a varietyof electronstains,e.g.
osmiumtetroxide,silver nitrate, and dodecatungstophosphoric
acid, and
also on the ground of chemicalreactivity.There is someevidencethat the
exocuticleitself is complex with an outer cystine-richlayer termed exocuticle 'a' (23). The endocuticleis digestedby enzymes(24) and hence is
432
JOURNALOF TIlE SOCIETYOF COSMETICCHEMISTS
consideredto have a low content of cross-linkingcystine. The cuticle is
more resistantto diffusionof reagentsthan the cortex (25) and the shape
of cuticlecellsvarieswith the sourceof fibres. It is possibleto separate
cuticlecells,corticalcells,and the cell membranecomplexby shakingwool
fibresin formic acid (26). The acid rapidly disruptsand dissolves
at least
part of the cellmembranecomplex,settingfreethe individualcells.Amino
acid analysesof the separated,whole cuticle show that it containsconsiderablylargeramountsof cystine,proline,serine,valine,and glycinethan
the fibre as a whole(27-29). Cuticularproteinis relativelyamorphousand
showsneitherorientationnor crystallineformation.The schematicdiagram
of Fig. 3 indicatesthe generallyacceptednomenclaturefor the various
submicroscopic
componentsof humanhair cuticlecells.
Hair
surface
'a'LAYER
--
Exocuticle
Endocuticle
Membrane
(cell
I)
Cement or 8-band
Membrane (cell 2)
Portion of adjacenf
cuticle
cell
Figure3. Schematicdiagramof the variouscomponents
of humanhair cuticlecells.
The cell membranecomplex
The cell membranecomplex,whichoriginates
from the fusionof two
unitcellmembranes,
onefromeachof the adjacentcuticleor corticalcells,
hasbeenobserved
by electronmicroscopy
of transverse
sections
by Rogers
(30, 31) and otherworkers.The wholestructure
is about30 nm thickand
consists
of a unit cell membrane,whichprobablycontainsproteinand a
bimolecular
lipid layer,thena fairly thicklayerof densematerialcalled
'intercellular cement' or fi-band and a second unit cell membrane. The
intercellular
cementis easilydigestible
by trypsin(32) andits composition
hasbeenthesubject
of muchspeculation
(33).Thecellmembrane
complex
maybe extracted
at leastpartiallyandpossibly
entirelyby formicacidand
certainmilderreagents.
Aminoacidanalyses
of the extractshowthatthis
STRUCTURAL
ASPECTS
OF KERATIN
FIBRES
433
protein is also very differentfrom that of whole wool in that it contains
smalleramountsof cystine,proline,threonineand serineand largeramounts
of lysine,histidineand the aromaticamino acids(26).
The cortex
Proceedinginwardsfrom the cuticle,the major structuralfeatureof the
fibre is reached,namely,the cortex. Horio and Kondo (34) noted that the
cortex of fine wool fibres has a bilateral structure,in the sensethat about
one-halfof the cortexalwaysabsorbsa given type of dye more intensely
than the other half.
Numerous
reactions indicate that the more reactive
sidealwayslies on the convexsideof the crimpwave. The sidewith greater
reactivity is called the orthocortex and that with lesserthe paracortex.
This asymmetrybetweenthe orthocortexand paracortexhasbeenshownin
differencesin morphologicalappearance(30, 35), stainingbehaviour (36)
and certainchemicaland physicalproperties(37). In bilateral wool fibres,
the sulphurcontentof the paracortexis appreciablyhigherthan that of the
orthocortex(38). There are more cystine,proline, and glutamicacidsbut
lessglycine,phenylalanine,and tyrosinein the formerthan in the latter (28).
The presenceof a highercontentof the cross-linkingcystinewould account
for the lower degree of swelling of the paracortex, its greater resistance
to acid hydrolysisand the slowerrate of reductionof its cystine(39).
Within eachcorticalcell of wool, which is about 80 •mlong and 5 •m in
diameter,there is great complexityof structure.Electron micrographsof
stainedtransversesectionsof wool showthat the cortexconsistsof approximately circular macrofibrils (also referredto as 'tertiary aggregates'of the
a-helices)havingthe appearanceof whorlsor spirals.Within the macrofibril
are microfibrils(also referredto as 'secondaryaggregates'of the a-helices)
about 7.5 nm in diameter arranged in pseudo-hexagonalpacking and
embeddedin a more heavily stainedamorphousmatrix.* The microfibrils
themselves
consistof a numberof protofibrils(alsoreferredto as 'primary
aggregates'
of the a-helices)about 2 nm in diameterarrangedin a regular
mannerand packedwithin the microfibril and embeddedin intramicrofibrillar matrix protein (40, 41). Independentsupport for the conceptof
*The electron-opaqueappearanceof structuralunits after fixation with metal compoundshas
been interpretedas an intensereactionof--SH or --S--S-- bondswith the metal, whereasan
electron-translucentappearanceis consideredto be a weak reaction. Accordingly, the dense
region seenin the cortex is thought to indicate the presenceof amorphousprotein(s) stabilized
by numerous --S--S-- bonds, while the less dense region reveals the presenceof a fibrous
protein stabilized by lessnumerous--S--S-- bonds.
434
JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS
a protofibrillarsubstructure
hasbeen obtainedby severalinvestigators
(42)
who succeededin isolating fine filaments, about 2 nm in diameter,from
e-keratinpreparationsfragmentedby ultrasonicirradiation.However,it is
contendedthat the filamentsobservedin thesepreparationsalmostcertainly resultfrom cellulosiccontaminants(41, 43). Moreover, experiments
by Sikorskiand associates
(44) on the interpretationof high magnification
electronmicrographsof keratin fibre sectionsindicatethat the distinctions
betweenthe microfibrillarpatternsof variouscorticalcellsmay not be as
simpleashasbeenindicated.Theyregardthe 'microfibril'asa manifestation
of an averagemode of aggregationin situ of protofibrils.
The medulla
The medullais a histologicalcomponentsituatednearthe centreof the
fibre in many keratin fibressuchas human hair, but it doesnot occurin
finewool fibres(45). It is formedfrom an axial streamof cells,the contents
of which shrivelup duringdehydrationleavinga seriesof vacuolesalong
the fibre axis.Many variationsoccurin the shapeand sizeof this part of the
fibre. It has been usual, however, to concentrate studies on wool fibres in
which medulla is either absentor presentin only small amounts. In any
case,the medullais believedto make little contributionto the chemicaland
mechanicalpropertiesof the fibre. That the materialof the medullarycells
differs from that of the surroundingcortical cells is indicated by the
differencesin stainingcharacteristics
of the two types of cells. Recently,
aminoacid analysesof the separatedmedullarycellsfrom rabbit,kangaroo,
andplatypushair showthat theycontainabout1 residuein 4-5 of glutamic
acid, 1 in 9 of citrulline, 1 in 12 of leucine,1 in 14 of glycineand only 1 in
35 of cystine (46). The citrulline is covalently bound in the peptide
linkagein the proteins(47). It has been shownthat the presenceof N6-q,
glutamyllysine
cross-linkin hair and quill medulla protein of mammalian
speciesis a generalphenomenon(48).
The structureof humanhair
An insightinto the histologicalstructureof humanhair hasbeengained
from the electronmicroscopicobservationsin this laboratory of sections
of ether-degreased
fibresstainedwith variousheavymetal compounds(49).
Human hair, like most other keratin fibres,consistsof a central core of
long spindle-shaped
interdigitatingkeratin-filledcorticalcells;this coreis
boundedby a sheathof overlappingleaf-likecuticlecells.Perhapsthe most
Figure4. Transversesectionof human hair stainedwith dodecatungstophosphoric
acid
showingsevenlayersof cuticlecells,cell membranes
(CM) and paracorticalcells(Para).
Pigment granules(PG) in the cortex are black.
Figure5. Transversesectionof human hair stainedwith silvernitrate to reveal the cxocuticle (Exo), the 'a' layer (A), the endocuticle(Endo), cell membranes(CM), the cortex,
macrofibrils(MF), microfibrils(MiF) and intermacrofibrillarmaterial (IMM).
Facing page 435
STRUCTURAL
ASPECTS
OF
KERATIN
FIBRES
435
notabledifferencebetweenhuman hair and wool, apart from the diameter
of the fibre, is that humanhair (Caucasian)is boundedby a layer of usually
six to eight cuticlecells(Figs. 4 and 5), whereasin wool the cuticleis only
one to two cells thick. The greater resistanceof human hair to chemical
attackby many reagentsis at leastpartly due to the thicknessof the cuticular layer. Caucasianhair consistsof a core of cortical cells mainly of the
'para-type', sometimessurroundedby one to two layers of 'ortho-type'
cells(49). However,humanhair doesnot showthe samedegreeof bilateral
segmentationas wool. Like wool, human hair is occasionallymedullated.
Infra-red spectroscopic
studiesof humanhair suggestthat the cuticleis
madeup of the (zand • structuresof polypeptidematerial,while the cortex
is composed
of boththeseconfigurations
plusthe randomcoil or amorphous
form (50).
It is interestingthat Negro hair containsa higherproportionof 'orthotype' cortical cells than Caucasianhair. Also, whereasthe cuticle of
Caucasianhair is usuallysix to eight layersthick and coversthe whole of
eachfibre, the cuticleof Negro hair is of variablethicknesswith six to eight
layersat the endsof the major axis of the fibresand diminishingto one to
two layersat the endsof the minor axis.Thus, the Caucasianhair approximates to a cylinder, the Negro hair to a twisted oval rod. Negro hair is
typicallyheavilypigmented.By meansof the electronmicroscope,
Swift(51)
has measuredthe size of isolatedmelanin granulesand found those from
Negro hair to be larger than those from Caucasianand Chinese hair.
However,no significantdifferences
in the chemicalbehaviourand physical
propertiescan be observedbetweenNegro and Caucasianhair (12, 52).
Human hair samplesgive detectableelectron spin resonance(ESR)
signalsat a g value (spectroscopic
splittingfactor) of 2.003 owingto the
free radical property of the melanin granules.The free radical content
variesfrom 4.7 x 10•6 free sping-• hair for black hair to 4.6 x 10x5for
medium brown hair and to 4.3 x 10•4 for blonde hair (53). During depigmentationof black hair with hydrogenperoxide,the intensityof the
ESR signaldeclinesexponentiallyin relationto the treatmenttime (54).
The highestconcentrationof cystincin human hair has been found to
occur in the 'a' layer and in the exocuficleby an electronhistochemical
technique(55). It appearsthat the highestconcentrationof tyrosineis also
in theseregions(49).
A cell membrane material has been isolated from the ethanol extract of
humanhair andidentifiedasa lipoprotein(56, 57). Ethanolextractioncompletelyremovesthismaterial,sinceethanolextracted
fibresnolongerundergo
436
JOURNAL OF THE SOCIETY OF COSMETIC CI-IEMISTS
the characteristicAllw6rden reaction. Furthermore, it has been found that
removalof this materialby ethanolextractionincreases
the rate of papain/
bisulphitedisintegrationof the fibre (58). Examination of sectionsof
metal-coatedfibresdemonstratedthat the cellmembranematerialis a layer
of electron-transparent,
non-stainablematerial approximately2.5 nm thick
on the surfaceof the fibre. This layer, whichis probablythe lipid portion
of a unit cellmembrane,is completelyremovableby ethanolextractionand
may be regardedas originatingfrom the componentsof the 'epicuticle'
described
by previousworkers.In fact, it hasrecentlybeenshown(60) that
therate of sorptionof n-propanolby woolisincreasedgreatlyby preliminary
extractionwith ethanol, which removeslipid and someprotein from the
cell membranecomplex.This hasled to the suggestion
that the bimolecular
lipid layerof the cellmembranecomplex,whichsurrounds
eachcellwithin
the fibre, presentsthe major barrier to the diffusionof moleculesinto the
intracellular
keratin.
END GROUPS OF KERATIN FIBRES
In view of the complexityof the histologyof wool and hair it is not
surprisingthat the fibres contain a number of both N- and C-terminal
amino acids. Methods for quantitative determinationof the former are
now well definedalthoughtechniquesfor determiningthe latter are still not
as precise.As suchgroups,as well as the side-chainamino and carboxyl
groups, can be involved in reactionswith many reagents,they are of
considerable
importance.
Sanger'sprocedure(61) hasbeenusedto identifythe N-terminalamino
adds. After treatment with 1-fluoro-2,4-dinitrobenzene
(FDNB) the fibres
are hydrolysed,and chromatographic
separationof the substitutedamino
acidsfrom the hydrolysateshowsthat, with fibresas dissimilaras human
hair and Lincoln,New ZealandRomney,and AustralianMerino wools,the
terminal groups are always provided by the same seven amino adds,
namely, glycine, threonine, valine, alanine, serine, glutamic add and
asparticacid (Table III).
Kerr and Godin (65) showedsimilarend groupsto be presentin human
and horsehair, and H•/hnel(66) foundthe sameend groupsin hair, callus,
nails, and psoriasisscales.Woodin (67) investigatedfeather keratin and
found the samesevenamino acidsto be N-terminal and in amountsroughly
similar to those of wool.
STRUCTURAL
ASPECTS
OF
KERATIN
437
FIBRES
Table Ill.
N-terminal
Amino
amino acids of wools and hair*
Lincoln
wool'•
acid
Merino
wools
Romney
woolõ
Human
hairõ
Glycine
5.2
7.8
4.5
3.9
Threonine
4.8
5.6
4.9
4.0
4.0
Valine
2.4
1.7
2.4
Alanine
1.2
1.5
1.2
1.0
Serine
1.2
1.7
1.2
1.0
acid
1.2
1.1
1.2
1.0
Aspartic acid
Glutamic
0.6
0.5
0'6
0.5
16.6
19.9
16.0
15'4
Total
*Value givenas/2moleg-X of dry keratin.
•'From Middlebrook (62).
:•From Thompson(63).
õFrom Tibbs and Speakman(64).
The total amounts of N-terminal residuesindicated that the average
molecularweight of the polypeptidechainsis approximatelythe same
(60 000) for all typesof fibres.Later, it wasfound that half-cystineis also
an N-terminal residueand is the secondmost abundant(68); the average
chain weight is then reduced to about 36 000. However, this kind of
calculationcannot be regardedas satisfactory.The presenceof N-acetyl
groupsin wool in comparatively
highamountshasbeendemonstrated
(69).
It wasassumed
that the e-aminogroupof lysinereactedquantitativelywith
FDNB (62), which would mean that a considerablenumber of the Nterminalamino groupsare maskedby acetylgroups.However,Siepmann
and Zahn (70) showedthat about 20• of the e-aminogroupsof lysineare
inertto FDNB andlaterwork of Asquith,ChanandOtterburn(71) supports
this finding.Hence,until the reasonsfor the lack of activity of this part of
the lysine residue have been elucidated,it cannot be assumedthat the
acetylgroupsare confinedto the terminalaminogroup.
A method(72) for determiningthe C-terminalamino acids,involving
reductionof carboxylgroupsto hydroxylgroups,hasbeenappliedto wool
(73), and end groupsof glycine,serine,alaninc,and threoninehave been
found. Blackburnand Lee (74) showedqualitativelythat the sameamino
acidswerepresentasendgroupsusingthe hydrazinolysis
method(75), and
Kerr and Godin (65) obtainedsimilarresultsfrom humanand horsehair.
A morethoroughstudyby Bradbury(76) addedasparticand glutamicacids
as C-terminalfor wool and gavea total amount of the C-terminal amino
acid of about 10 vmoleg4 of wool.
438
JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS
ISOLATION AND CHARACTERIZATION OF PROTEIN FRACTIONS FROM
KERATIN
FIBRES
In the last 15 years,numerousattemptshave beenmade to fractionate
wool keratin into homogeneousprotein fractions without main chain
hydrolysis.In general, two main kinds of protein fractions could be
obtained,i.e. the so-calledlow-sulphurand high-sulphurproteinfractions.
Oxidationof the disulphidebondsof keratinswith peraceticacid (77)
or performic acid (78) and extractionwith ammonia solutionleavesan
insolubleresiduetermed [•-keratose.
The low-sulphurfraction, a-keratose,
is precipitatedby acidificationof the filteredextractand the high-sulphur
fraction, T-keratose,remainsin solution.By keratoseestimationson wool,
human hair, and someisolatedcell components,Asquith and Parkinson
(79) have providedchemicalconfirmationthat a-, [•-, and T-keratoses
are
to be identified respectivelywith fibrillar, cell membrane, and matrix
componentsof the fibre. Similarly, reducedand subsequently
carboxymethylatedwool givesthe S-carboxymethylkerateines
(SCMK), which can
be fractionatedto give SCMKA low-sulphurfraction and SCMKB highsulphurfraction (80). a-Keratoseand SCMKA are heterogeneous
lowsulphurproteinfractionsof almostidenticalaminoacid composition(81),
while T-keratoseand SCMKB are extremelyheterogeneous
high-sulphur
proteinfractionswith similarbut not identicalam/noacidcomposition
(82).
The low-sulphurfractionsare relativelyrich in asparticand glutamicacids,
lysine,tyrosine,leucine,and alanine,while the high-sulphurfractionsare
poor in theseamino acidsbut contain relativelylarge amountsof cysteic
acid or S-carboxymethylcysteine,
proline, serine,and threonine. Studies
on these protein fractions are yielding useful information about the
molecular
structure on keratin fibres.
In [•-keratose,a substantialamount of the lysyl amino group has been
found to exist naturally as Nø-T-glutamyllysinecross-link (83) and in
T-keratose,83•o of the glutamicor asparticacidsare presentastheir amides
(84). Seasonalfactors have a marked influence on the proportion of
keratosesin wool keratin; duringmaintenanceof sheepon pasture,the
percentageof T-keratoseincreaseswhile in the wool grown during the
winter stall-maintenance,a-keratose increases(85). In the SCMKA
fraction the carboxyl groupsare presentmainly as glutamic acid rather
than aspartic acid residues,whereasin the SCMKB fraction there is a
relatively high contentof glutamine(11).
It has been shownthat low-sulphurSCMKA kerateinescontainabout
STRUCTURAL
ASPECTS
OF
KERATIN
FIBRES
439
50•o helix while high-sulphurSCMKB kerateinesare almost completely
devoidof helix (86). Partial digestionof the low-sulphurSCMKA fraction
from wool givesriseto helix-richmaterial(87, 88). UsingDavies'graphical
method (89), Asquith and Shaw (90) have calculatedthat low-sulphur
e-keratosecontains29•o (Val + Ile + Ser + Thr + Cys) and hence is
about 40•o e-helix, and that high-sulphur I•-keratosecontains 53}/0
(Val + Ile + Ser + Thr + Cys) and hence is non-e-helix. Although the
helicalregionsof the low-sulphurSCMKA fractioncontainmost of the
lysineresiduesof the wool fibre, they are highlyanionicbecausethey also
containmost of the free carboxylgroups.The non-helicalsectionsin the
SCMKA fraction are cationic(88).
From the amino acid compositionand physicalmeasurementdata, it
would appearthat the low-sulphurfractionsare derivedfrom the protofibrils and the high-sulphurfractionsfrom the intermacrofibrillar
matrix.
It is generallyagreedthat the protofibrilsconsistof severale-helicalpolypeptidechainstwistedaroundoneanotherin a rope-likemanner,andit is
essentiallythis orderedstructurewhich givesrise to the X-ray diffraction
patternof e-keratins.On thisbasis,the matrixproteinis considered
to be
disordered,at leastin sofar as there is insufficientlong-rangeorder to give
rise to an X-ray diffractionpattern.The matrix is more heavilystainedby
metalsthan the protofibrilsand thiswouldseemto indicatethat the former
containsmore cystine.However,it is not yet possibleto separateunequivocally the matrix proteinfrom the protofibrillarprotein,whichis not surprisingin viewof the obviousdifficulties
involved.Thus,all evidence
with
regardto the identityof the proteinsof the matrix and the protofibrilsis of
necessityindirect (6, 91).
The fractionationand separationof a singleproteinmolecularspecies
from sucha mixturehasbeenthe subjectof extensiveresearch.The work of
Lindley, Gillespieand Haylett (92) on a protein from SCMKB-2 highsulphurprotein fraction showsthe occurrenceof a high frequencyof
homodipeptides
suchasPro-Pro,Val-Val, Glu-Glu, andCys(CH•CO•H)Cys(CH•CO2H).By partialacidhydrolysis
of [asS]-cystine
labelledwool,the
Cys-Cys sequenceis shown to occur frequently(93). Asquith and coworkers(94) havealsofoundthat Cys(SOaH)-Cys(SOaH)
is a veryimportant sequence
in T-keratose,
over 30•o of the cysteicacidoccurring
in this
sequence,and thus have postulatedthat a large proportion of the
lanthionine formed in wool under suitable conditions is intramolecular.
Recently,a polypeptide
containing97 aminoacidresidues,
of which26 are
aromatic(tyrosineand phenylalanine)
and 24 are glycine,hasalsobeen
440
JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS
isolatedfrom reducedwool and partially characterized
by Zahn and Biela
(95). For the firsttime the completeaminoacid sequence
of threehomogeneousproteins, isolated from SCMKB fraction of Merino wool, has been
established
by Haylett, Swartandco-workers(96). The S-carboxymethylated
proteinshavea molecular weight of 11409 4- 150. They consistof single
chainscontaining97 or 98 residues.Their N-terminal sequence
is Ac-Ala-
Cys(CH•.CO•.H)
andtheyhaveS-carboxymethylcysteine
asC-terminus.
The
moleculescan be dividedarbitrarily at position49 into high- and lowsulphurproteinsbut the amino acidsthat preventhelix formation,such
as proline, serine,and threonine,are fairly evenlydistributedthroughout
the molecules.Thesehigh-sulphurproteinsthereforeappearto have no
helicalcontentand are ideallysuitedfor the function of matrix protein
in wool.
The work of Corfield,Fletcherand Robson(97) on a proteinfraction,
isolatedin 365/oyieldfrom oxidizedwool, hasled theseauthorsto suggest
that wool consistsof only one main proteincomposedof relativelyshort
helicalregionsinterspersed
with sequences
rich in cystinc,proline,serine,
and threonineresidues.It is suggested
that this fraction is derivedfrom a
protein that constitutesthe major part of the cortex. Further evidencein
this field may thereforeprovide direct chemicalevidenceregardingthe
validityof the conceptof the two-phasetwo-proteintheory.
ACKNOWLEDGEMENTS
The author is gratefulto ProfessorR. S. Asquithand Dr. J. A. Swift
for helpfulsuggestions
and comments.
Figs.1-5 werekindly suppliedby
Dr. Swift.
(Received:$rd January1972)
REFERENCES
(1) Astbury, W. T. and Woods,H. J. X-ray studiesof the structureof hair, wool and related
fibres. II. The molecularstructureand elasticpropertiesof hair keratin. Phil. Trans.Roy.
Soc. London.A232 333 (1933).
(2) Pauling, L., Corey, R. B. and Branson,H. R. The structureof proteins:two hydrogenbondedhelical configurationsof the polypeptidechain. Proc. Nat. Acad. Sci. U.S. 37 205
(1951).
(3) Lundgren,H. P. and Ward, W. H. in Borasky,R. Ultrastructure
of ProteinFibres39 (1963)
(AcademicPress,New York).
(4) Mercer,E. H. and Matoltsy,A. G. Keratin, in Montagna,W. and Dobson,R. L. Advances
in Biologyof Skin, Vol. 9, Hair Growth(1969) (Pergamon,Oxford).
(5) Ward, W. H. in Alexander, P. and Lundgren, H. P. Analytical Methods of Protein
Chemistry,Vol. 4, 185 (1966) (Pergamon,Oxford).
(6) Crewther,W. G., Fraser, R. D. B., Lennox, F. G. and Lindley, H. The chemistryof
keratins.Advan.Protein Chem.20 191 (1965).
STRUCTURAL
ASPECTS
OF
KERATIN
FIBRES
441
(7) Fletcher,J. C. and Robson,A. The occurrenceof bis-(2-amino-2-carboxyethyl)trisulphide
in hydrolysatesof wool and other proteins.Biochem.J. 87 553 (1963).
(8) Ward, W. H. and Lundgren, H. P. The formation, composition,and propertiesof the
keratins. Gdvan._ProteinChem. 9 39 (1954).
(9) Perrin,D. D. Dissociation
Constants
of OrganicBasesin AqueousSolution(1965) (Butterworth, London).
(10) Cole, M., Fletcher,J. C., Gardner,K. L. and Corfidd, M. C. A studyof enzymatichydrolysis applicableto the examination of processedwools. J. Appl. Polym. Sci., Appl. Polym.
Symp. 18 147 (1971).
(11) Holt, L. A., Milligan, B. and Roxburgh,C. M. Asparticacid, asparagine,glutamicacid,
(12)
(13)
(14)
(15)
(16)
(17)
(18)
(19)
(20)
(21)
(22)
(23)
(24)
(25)
(26)
(27)
(28)
(29)
(30)
(31)
(32)
and glutaminecontentsof wool and two derivedprotein fractions.Aust. J. Biol. $ci. 24 509
(1971).
Menkart, J., Wolfram, L. J. and Mao, I. Caucasianhair, Negro hair, and wool: similarities
and differences.J. Soc. Cosmet.Chem.17 769 (1966).
Simmonds,D. H. The amino acid compositionof keratins.V. Comparisonof the chemical
compositionof Merino woolsof differingcrimp with that of otheranimal fibres. Text. Res.J.
28 314 (1958).
Speakman, J. B. and Elliott, G. H. The combination of wool with acids and acid dyes.
Symposiumon Fibrous_Proteins,
Societyof Dyers and Colourists,Leeds116 (1946).
Robbins, C. R., Scott, C. V. and Burnhurst,J. D. A study of the causesof variation in the
acid dye-combiningcapacityof humanhair. Text. Res. J. 38 1130 (1968).
Menkart, J. and Coe, A. B. Microscopic studies on the structure and composition of
keratinfibres. Text. Res.J. 28 218 (1958).
yon Allw/Srden, K. Properties of wool--detection of damaged wool by chemical means.
Z. Gngew.Chem.29 77 (1916).
Leeder,J. D. and Bradbury,J. H. The discontinuous
nature of epicuticleon the surfaceof
keratinfibres.Text. Res.J. 41 563 (1971).
Whewell, C. S. and Woods, H. J. Measurement of damage in wool materials. II. A new
test for estimating small amounts of mechanicalmodification in wool materials. J. Soc.
Dyers Colour.60 148 (1944); Millson, H. E. and Turl, L. H. Influenceof the cuticlein the
dyeingof wool fibre. Gin. Dyest. Rep. 39 No. 20 647, _Proc.Amer. Gss. Tex. Chem. Color.
(1950).
Lindberg, J. Rate of acid sorptionby wool fibres. Text. Res. J. 20 381 (1950).
Lindberg, J., Philip, B. and Gral6n, N. Occurrenceof thin membranesin the structureof
wool. Nature (London)162 458 (1948).
King, N. L. R. and Bradbury, J. H. The chemicalcompositionof wool. V. The epicuticle.
Gust. J. Biol. Sci. 21 375 (1968); Lofts, P. F. and Truter, E. V. The constitutionof the
epicuticleof wool. J. Text. Inst. 60 46 (1969).
Sikorski, J. and Simpson,W. S. Electron microscopestudiesof the chemicalreactivity in
keratin cuticle.Nature (London)182 1235 (1958).
Mercer, E. H. _Proc.Int. Wool Text. Res. Conf., GustraliaF 210 (1955).
Speakman,J. B. The structureof animal fibres in relation to acid dyeing. J. Soc. Dyers
Colour.52 121 (1936).
Bradbury, J. H., Chapman, G. V. and King, N. L. R. The chemicalcompositionof wool
III. Analysis of cuticle, skin flakes and cell membrane material. 3rd lnt. Wool Text. Res.
Conf., _Paris1 359 (1965).
Bradbury, J. H., Chapman, G. V. and King, N. L. R. The chemicalcompositionof wool.
II. Analysisof the major histologicalcomponentsproducedby ultrasonicdisintegration.
Gust. J. Biol. $ci. 18 353 (1965).
Parisot, A. and Derminot, J. The amino acid compositionof various morphologicalwool
fractionsof wool isolatedduring progressiveacid hydrolysis.J. Gppl. Polym. Sci., Gppl.
Polym.Symp. 18 45 (1971).
Mazingue, G., Ponchel,P. and Lubrez, J.P. The compositionand propertiesof the wool
cuticle.J. Gppl. _Polym.Sci., Gppl. Polym. Symp. 18 209 (1971).
Rogers,G. E. Electronmicroscopestudiesof hair and wool. Ann. N.Y. Acad. $ci. 83 378
(1959).
Rogers,G. E. Electronmicroscopyof wool. J. Ultrastruct.Res.2 309 (1959).
Mercer, E.H. and Rees, H. L. G. An electron-microscope
investigationof the cuticleof
wool. Gust. J. Expt. Biol. Meal. Sci. 24 147 (1946).
Mercer,E. H. KeratinandKeratinization262 (1961) (Pergamon,Oxford).
(33)
(34) Horio, M. and Kondo, T. Crimpingof wool fibres.Text. Res.J. 23 373 (1953).
(35) Kassenbeck,P. and Leveau, M. New methodsfor examiningsectionsof fibres with the
electronmicroscope:applicationto the studyof wool structure.Bull. Inst. Text. Fr. 67 7
442
(36)
(37)
(38)
(39)
(40)
(41)
(42)
(43)
JOURNAL
OF THE SOCIETY OF COSMETIC CHEMISTS
(1957); Birbeck, M. S.C. and Mercer, E. H. The electron microscopyof the human hair
follicle. I. Introduction and the hair cortex. J. Biophys.Blochem.Cytol. 3 203 (1957).
Fraser, R. D. B. and Rogers,G. E. Microscopicobservationsof the alkaline-thioglycolate
extractionof wool. Blochim.Biophys.Acta 12 484 (1953); Fraser, R. D. B. and Rogers,
G. E. Origin of segmentation of wool cortex. Blochim. Biophys. Acta. 13 297 (1954);
Dusenbury,J. H. and Coe, A. B. Differential dyeingas an indicationof bilateral structure
in wool: new findings.Text. Res.J. 25 354 (1955); Dusenbury,J. H. and Menkart, J. The
presentstate of the ortho and paraconcept.Proc. Int. Wool Text. Res. Conf., Australia F
142 (1955); Ahmad, N. and Lang, W. R. Ortho-para cortical differentiationin 'anomalous'
Merino wool. Aust. J. Biol. Sci. 10 118 (1957); Thorsen, W. J. Estimation of cortical componentsin variouswools. Text. Res.J. 28 185(1958); Kassenbeck,P., Jacquemart,J. and
Monrocq, R. Mbthodesde caractbrisationdesfibres kbratiniquesen microscopicoptique.
3rdlnt. Wool Text. Res. Conf., Paris 1 209 (1956); Brown, T. D. and Onions,W. J. Anomaiiesin the microscopicstructureof somewools.Nature (London)186 93 (1960); Bon•s, R. M.
and Sikorski, J. The histologicalstructureof wool fibres and their plasticity.J. Text. Inst.
58 521 (1967).
Leveau, M., Cebe, N. and Parisot, A. Heterogeneityof the wool fibre and the Allwbrden
reaction. Bull. Inst. Text. Fr. 42 7 (1953); Dusenbury,J. H., Mercer, E. H. and Wakelin,
J. H. The influenceof chemicaltreatmenton the propertiesof wool. I. Alkali solubilityas a
measureof sulfuric acid degradation.Text. Res. J. 24 890 (1954); Dusenbury, J. H.
Characterization of the cortical structures of keratin fibres by urea-bisulphite solubility.
J. Text. Inst. 51 T756 (1960); Leach, S. J., Rogers, G. E. and Filshie, B. K. Selective
extraction of wool keratin with dilute acid. I. Chemical and morphological changes.
Arch. Blochem.Biophys.105 270 (1964); Mercer, E. H. A note on the digestionof wool by
clothes-mothlarvae. Blochim.Biophys.Acta 15 293 (1954); Dobb, M. G. Electron-diffraction studiesof keratin cells.J. Text. Inst. 61 232 (1970); Mirb, P. and Erra, P. Action of
sodium hydroxide on the morphologicalstructureof wool. Bull. Inst. Text. Fr. 23 841
(1969).
Kassenbeck,P. Considbrationssur la localisation des liaisons cystiniquesinter- et intrachaineset sur les teneurs en soufre compar&s des fractions corticales 'ortho' et 'para'.
3rd Int. Wool Text. Res. Conf., Paris 1 367 (1965).
Kulkarni, V. G., Robson, R. M. and Robin, A. Studieson the orthocortexand paracortex
of Merino wool. J. AppLPolym. Sci., Appl. Polym. Symp.18 127 (1971).
Filshie, B. K. and Rogers,G. E. The fine structureof •-keratin. or.Mol. Biol. 3 784 (1961);
Fraser, R. D. B., MacRae, T. P. and Rogers, G. E. Molecular organization in alphakeratin. Nature (London)193 1052 (1962).
Fraser, R. D. B., MacRae, T. P. and Millward, G. R. The structure of •-kerafin microfibrils. J. Text. Inst. 60 343 (1969).
Dobb, M. G. Protofibrils in ct-keratin.or. Mol. Biol. 10 156 (1964); Rogers, G. E. and
Clarke, R. M. Keratin protofilamentsand ribosomesfrom hair follicles.Nature (London)
205 77 (1965); Dobb, M. G. and Rogers, G. E. Electron microscopyof fibrous keratins.
Symposiumon FibrousProteins,Australia 267 (1968) (Butterworths,Sydney).
Millward, G. R. Cellulosecontamination:a possiblesourceof error in the interpretation
of previousexperimental
evidence
for thea-keratinprotofibril.J. Cell.Biol.42 317 (1969);
Fraser, R. D. B., MacRae, T. P. and Millward, G. R. Fact and artefact. J. Text. Inst. 60
498 (1969).
(44) Johnson,D. J. and Sikorski, J. Molecular and fine structureof alpha-keratin(I). Nature
(London)194 31 (1962); Dobb, M. G., Johnson,D. J. and Sikorski, J. Molecular and fine
structureof alpha-keratin(II). 5th Int. Conf. ElectronMicro;c., Philadelphia2 T4 (1962);
Johnson,D. J. and Sikorski,J. Molecular and fine structureof alpha-keratin(III). Proc. 3rd
Eur. Reg. Conf. Electron Microsc., Prague B63 (1964); Johnson,D. T. and Sikorski, J.
Molecularand fine structureof a-keratin (IV). Nature (London)205 266 (1965); Johnson,
D. J. and Sikorski, J. Fine and ultrafine structureof keratin (V). 3rd Int. Wool Text. Res.
Conf., Paris 1 147 (1965); Dobb, M.G. and Sikorski, J. Fine and ultrafine structure of
mammaliankeratin. J. Text. Inst. 60 497 (1969).
(45) Ryder, M. L. in Hearle, J. W. S. and Peters,R. H. Fibre Structure547(1963) (Butterworths,
London).
(46) Bradbury,J. H. and O'Shea,J. M. Keratin fibres.II. Separationand analysisof medullary
cells.Aust. J. Biol. Sci. 22 1205 (1969).
(47) Steinert,P.M., Harding, H. W. J. and Rogers,G. E. The characterizationof protein-bond
citrulline.Biochim.Biophys.Acta 175 1 (1969).
STRUCTURAL
ASPECTS
OF
KERATIN
FIBRES
443
(48) Harding, H. W. J. and Rogers, R. E. g-(¾-Glutamyl)lysinecross-linkagein citrullinecontainingprotein fractionsfrom hair. Biochemistry10 624 (1971); Harding, H. W. J. and
Rogers,R. E. The occurrenceof the ½-(•-glutamyl)lysine
cross-linkin the medulla of hair
andquill.Biochim.Biophys.
Acta257 37 (1972).
(49) Swift, J. A. Personalcommunication.
(50) Baddid, C. B. Structure and reactions of human hair keratin: an analysis by infrared
spectroscopy.J. Mol. Biol. 38 181 (1968).
(51) Swift, J. A., Ph.D. Thesis,Universityof Leeds(1963).
(52) Breuer, M. M. Personalcommunication.
(53) Mason, H. S., Ingram, D. J. E. and Allen, B. Free radical property of melanins.Arch.
Blochem.Biophys.86 225 (1960).
(54) Sacchi, S., Lanzi, G. and Zanotti, L. Electron spin resonanceresearchon human hairs
under varying physiologicaland experimentalconditions,in Montagna, W. and Dobson,
R.L. Advancesin Biologyof Skin, Vol. 9, Hair Growth(1969). (Pergamon,Oxford).
(55) Swift, J. A. The electronhistochemistryof cystinc-containingproteinsin thin transverse
sectionof human hair. J. Roy. Microsc. $oc. 88 449 (1968).
(56) Holmes,A. W. Degradationof humanhair by papain. II. Experimentsin the isolationand
identificationof the protectivesubstance.Text. Res. J. 34 777 (1964).
(57) Leon, N.H. Unpublisheddata.
(58) Holmes, A. W. Degradation of human hair by papain. I. The pattern of degradation.
Text. Res. J. 34 706 (1964).
(59) Swift, J. A. and Holmes, A. W. Degradationof human hair by papain. III. Someelectron
microscopeobservations.Text. Res. J. 35 1014 (1965).
(60) Bradbury,J. H., Leeder,J. D. and Watt. I. C. The cellmembranesof wool. J. Appl.Polym.
$ci., Appl. Polym. $ymp. 18 227 (1971).
61) Sanger,F. The free amino groupsof insulin. Blochem.J. 39 507 (1954).
62) Middlebrook,
W. R. Thechainweight
ofwoolkeratin.
Blochim.
Biophys.
Acta.4 547(1951).
(63) Thompson, E. O. P. Terminal amino groups in wool and S-carboxymethylkerateine 2.
Aust. J. Biol. $ci. 10 225 (1957).
(64) Tibbs, J. Ph.D. Thesis, University of Leeds(1951); Speakman,J. B. Neue Forschungsarbeiten fiber die Chemic der Wolle. Melliand Textilber. 33 823 (1952).
(65) Kerr, M. F. and Godin, C. N- and C-terminal end groupsof hair keratin. Can. J. Chem.
37 11 (1959).
(66) H'mreel,R. Comparative chemicalstudiesof physiologicaland pathologicalkeratins. I.
Quantitative determination of N-terminal amino acids in calluses,psoriasisscales,nails and
hair. Arch. Klin. Exp. Dermatol. 209 97 (1959).
(67) Woodin, A.M. Structure and compositionof solublefeather keratin. Blochem.J. 63 576
(1956).
(68) Thompson,E. O. P. A method for the isolation of DNP-cysteic acid and its application to
the determination of N-terminal half-cystineresiduesin wool. Aust. J. Biol. $ci. 12 303
(1959).
(69) O'Donnell, I. J. and Thompson,E. O. P. Oxidized wool. VI. Interactions betweenhigh- and
low-sulphur proteins and their significancein the purification of extractedwool proteins.
Aust. J. Biol. $ci. 15 740 (1962).
(70) Siepmann,E. and Zahn, H. Automatic analysisof water-soluble2,4-dinitrophenylamino
acids by chromatographyon nylon powder. 3rd Int. Wool Text. Res. Conf., Paris 1 303
(1965).
(71) Asquith, R. S., Chan, D. and Otterburn, M. S. An electrophoreticmethod for the determination of 'bound' e-aminolysinein proteins.J. Chromatogr.43 382 (1969).
(72) Fromageot, C., Jutisz, M., Meyer, D. M. and P6nasse,L. The characterizationof terminal
carboxylgroupsof proteins.Application to insulin. Blochim.Biophys.Acta 6 283 (1950).
(73) Speakman,J. B. Proc. Int. Wool Text. Res. Con(, AustraliaC 474 (1955).
(74) Blackburn, S. and Lee, G. R. Terminal carboxyl groups of wool keratin. J. Text. Inst. 45
T487 (1954).
(75) Akabori, S., Ohno, K. and Narita, K. HydrazJnolysis
of proteinsand peptides:methodfor
the characterization of carboxyl-terminal amino acids in proteins. Bull. Chem. Soc. Japan
25 214 (1952); Braun, V. and Schroeder,W. A. A reinvestigationof the hydrazinolytic
procedurefor the determinationof C-terminal amino acids. Arch. Blochem.Biophys.118
241 (1967).
(76) Bradbury, J. H. The hydrazinolysisof insulin, lysozyme,wool proteinsand wool. Blochem.
J. 68 482 (1958).
(77) Alexander,P. and Earland, C.F. Structureof wool fibres.Isolation of an a- and 13-protein
in wool. Nature (London)166 396 (1950).
444
JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS
(78) Blackburn, S. and Lowther, A. G. The action of organic acidson somefibrous proteins:
the oxidationof wool keratin.Blochem.J. 49 554 (1951).
(79) Asquith, R. S. and Parkinson,D.C. The morphologicalorigin and reactionsof some
keratin fractions. Text. Res. J. 34 1064 (1966).
(80) Crewther, W. G., Gillespie, J. M., Hatrap, B. S., O'Dormell, I. J. and Thompson,E. O. P.
S-Carboxymethylkerateines.
3rd lnt. Wool Text. Res. Conf., Paris 1 475 (1965) and the
references there cited.
(81) Corfield, M. C., Robson, A. and Skinner, B. The amino acid compositionsof three
fractionsfrom oxidizedwool. Biochem.J. 68 348 (1958); Crewther,W. G. and Dowling,
L. M. Effectsof chemicalmodificationson the physicalpropertiesof wool: a model of the
wool fibre. 2nd QuinquennialWool Text. Res. Conf., Harrogate (1960) (J. Text. Inst. 51
T775 (1960)).
(82) Gillespie, J. M. in Benesch,R., Benesch,R. E., Boyer, P. D., Klotz, I. M., Middlebrook,
W. R. and Szent-Gy6rgyi,A. G. Sulphurin Proteins51 (1959) (AcademicPress,New
York); Gillespie, J. M. and Simonds, D. H. Amino acid composition of a sulfur-rich
proteinfrom wool. Biochim.Biophys.Acta 39 538 (1960); Joubert,F. J., de Jager,P. J. and
Swart, L. S. Studieson the high-sulphurproteins of reduced Merino wool. Symposiumon
FibrousProteins,Australia 343 (1968) (Butterworths,Sydney);Darskus, R. L., Gillespie,
J. M. and Lindley, H. The possibilityof common amino acid sequences
in high-sulphur
proteinfractionsfrom wool. Aust. J. Biol. Sci. 22 1197 (1969).
(83) Asquith, R. S., Otterburn, M. S., Buchanan,J. H., Cole, M., Fletcher, J. C. and Gardner,
K. L. The identification of ½-N-(¾-glutamyl)-•.-lysine
crosslinksin native wool keratins.
Biochim.Biophys.Acta 221 342 (1970).
(84) Asquith,R. S. and Watson,P. A. Estimationsof amidenitrogenin ¾-keratose.
Text. Res.J.
35 381 (1965).
(85) Lobur, V. V., Makar, I. A. and Gzhitokii, S. 2;. Keratose contentin the wool of Merino
sheepunder different maintenanceconditions.Dopov. Akad. Nauk Ukr. RSR. Ser. B. 32
71 (1970).
(86) Harrap, B. S. The molecularweightof a purifiedhigh-sulphurproteinderivedfrom wool.
Aust.J. Biol. Sci. 15 596(1962); Harrap, B. S. The conformationof a solublewool keratin
derivative.Aust. J. BioL Sci. 16 231 (1963); Gillespie,J. M. The isolationand properties
of somesolubleproteinsfrom wool. V. The isolationof the high-sulphurprotein SCMKB2.
Aust. J. Biol. Sci. 16 241 (1963); Gillespie, J. M. and Harrap, B. S. The isolationand
propertiesof some soluble proteins from wool. VI. The physicochemicalpropertiesof
SCMKB2. Aust.J. Biol. Sci. 16 252 (1963).
(87) Crewther,W. G. and Harrap, B. S. Helix-rich fraction from the low-sulphurproteinsof
wool. Nature(London)207 295 (1965); Crewther,W. G. and Harrap, B. S. The preparation
and properties of a helix-rich fraction obtained by partial proteolysisof low sulphur Scarboxymethylkerateine
from wool. J. Biol. Chem.242 4310 (1967); Crewther, W. G. and
Dowling, L. M. The preparationand propertiesof large peptidesfrom the helical regions
of the low-sulphurproteinsof wool. J. Appl. Polym. Sci., Appl. t'olym. Syrup.18 1 (1971).
(88) Crewther, W. G., Dobb, M. G., Dowling, L. M. and Harrap, B. S. The structureand
aggregationof low-sulphurproteins derived from alpha keratins. Symposiumon Fibrous
Proteins,Australia329 (1968) (Butterworths,Sydney).
(89) Davies, D. R. A correlation between amino acid compositionand protein structure.
J. Mol. Biol. 9 605 (1964).
(90) Asquith,R. S. and Shaw,T. A noteon the compositionof the fibrillar and matrix proteins
of wool. Text. Res. J. 35 670 (1965).
(91) Bradbury,J. H. The structureof wool. Proc.Roy. Aust.Chem.Inst. 35 98 (1968); Robson,
A. Die Struktur der Wolle. Textil-Praxis 23 106 (1968); Fraser, R. D. B., MacRae, T. P.,
Millward, G. R., Parry, D. A.D., Suzuki, E. and Tulloch, P. A. The molecularstructureof
keratins.J. Appl. Polym. Sci., Appl. Polym. Syrup.18 65 (1971).
(92) Lindley, H., Gillespie, J. M. and Haylett, T. Recent studieson the high-sulphurproteins
of alpha keratins. Symposiumon Fibrous Proteins, Australia 353 (1968) (Butterworths,
Sydney);Lindley, H. and Haylett, T. Occurrenceof the half-cystine-half-cystine
sequence
in keratins. J. Mol. Biol. 30 (1) 63 (1967).
(93) Buchanan,J. H. and Corfield, M. C. Isolation of cystine-containing
peptidesfrom wool.
J. Appl. Polym. Sci., Appl. Polym.Syrup.18 101 (1971).
(94) Asquith,R. S. and Shaw,T. A semi-quantitative
investigationof the cysteicacid-containing
peptidesin a partial acid hydrolysateof ¾-keratose.
Makrotool. Chem.115 198 (1968);
Asquith, R. S. and Parkinson,D.C. Semi-quantitativedeterminationof some peptide
sequences
in wool keratin. Proc. Soc.Analyt. Chem.5 206 (1968).
STRUCTURAL
ASPECTS
OF
KERATIN
FIBRES
445
(95) Zalm,H. andBiela,M. OberdieIsolierung
tyrosinreicher
ProteineausWolle.TextilePraxis 23 103 (1968).
(96) Haylett, T., Swart, L. $. and Joubert, F. J. Studieson the high-sulphurproteins of reduced
Merino wool. II. The isolation of a homogeneous
protein. Text. Res. J. 39 912 (1969);
Haylett, T. and Swart, L. S. Studieson the high-sulphurproteins of reducedMerino wool.
III. The amino-acid sequenceof protein SCMKB-IIIB2. Text. Res. J. 39 917 (1969);
Haylett, T., Swart, L. S. and Parris, D. Studies on the high-sulphurproteins of reduced
Merino wool. Amino acid sequence
of proteinSCMKB-IIIB3. Blochem.J. 123 191 (1971);
Swart, L. S. and Haylett, T. Studiesof the high-sulphurproteinsof reducedMerino wool.
Amino acid sequenceof protein SCMKB-IIIB4. Biochem.J. 123 201 (1971).
(97) Coffield, M. C., Fletcher, J. C. and Robson, A. Recent work on the chemicalstructureof
wool proteins.Symposium
onFibrousProteins,Australia289 (1968) (Butterworths,Sydney);
Corfield, M. C., Fletcher, J. C. and Robson, A. Wool proteinsin relation to wool structure.
Symposiumon FibrousProteins,Australia (1968) (Butterworths,Sydney); Cotfield, M. C.
and Fletcher,J. C. Amino acid sequences
of peptidesfrom a chymotrypticdigestof a ureasolubleproteinfraction(U.S. 3) from oxidizedwool. Blochem.J. 115 323 (1969).
Fly UP