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. 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