J. Soc. Cosmet.Chem.

25 355-366 (1974) 1974 Societyof CosmeticChetnistsof Great Britaht

The chemistry humanhair cuticleof

II: The isolation and amino acid

of analysis A-layer

the cell membranes

and

J. A. SWIFT

and B. BEWS*

Synopsis--Mixturesof papain and dithiothreitol have been used to effect the separation of the A-LAYER and CELL MEMBRANE COMPLEX and the cell membrane complex alone from
human HAIR CUTICLE. The course of these ENZYMATIC DIGESTIONS has been followed

gravimetrically and by the ELECTRON MICROSCOPE examinationof digested hair sections, and it is evidentthat the componentsmentionedare isolatedcleanly. The AMINO ACID compositionsof the cell membranecomplex and of the A-layer (obtained by difference)are quite differentfrom the compositionof the whole cuticle.The significance the analyses discussed. of is

INTRODUCTION

The previous paper of this series(1) describeda method for the physical isolationof cuticlefrom human hair and emphasized lamellar the
substructure of each cuticle cell sheet. Our interest is now in the further

separation theselaminae for chemicalanalysis.Various chemicalproof cedureshave been described isolatingdifferent fractionsfrom keratin for fibres(cf. reference Many of theselead to uncertainties 2). about the significanceof the subsequent chemicalanalysisand most do not allow the
* Unilever ResearchIsleworth Laboratory, 455 London Road, Isleworth, Middlesex.
355

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accurate specification the morphological of origin of the isolated fractions. Enzymic digestionprocedures, virtue of their high chemicalspecificity, by are likely to lead to much cleanerfractionationand indeedBradbury and Ley (3) have recently usedpronaseto dissolvewool endocuticularcomponentsselectively. Using mixturesof papain and dithiothreitolwe have shown(4), by the electron microscope examination digested of hair sections, that a distinctlayer remainsundissolved the vicinity of the cuticle cell in membranes. The presentpaper is concerned with the further study of this digestion procedure whichhasenabledus to separate from isolated human hair cuticlethe cell membranecomplex and A-layer.
METHODS AND MATERIALS

The isolationof humanhair cuticle

This material was prepared by shaking root-end pieces of brown Caucasian hair in water according procedures to described the previous in paperof this series Cuticlefragments (1). obtainedafter 2 h shaking (i.e. 5 by weight of the original hair) were separatedand dried in vacuoover phosphorus pentoxide.

Digestion cuticle of fragments withpapain/dithiothreitol

Preliminary gravimetric experiments,in which isolated cuticle was digestedby dispersionin a solution containing I mg/ml papain (crude powdertype 2, Sigma)and 2 mg/ml dithiothreitol(Calbiochem) 0.1M in phosphate buffer at pH 6.7, showedthat 92o of the cuticlewas dissolved after 18 h at 65C. In addition, from observations the transmission in electron microscopeof thin transversehair sectionsdigestedunder similar conditions,it was evident that somecomponents were almost completely dissolved as little as 15 min at 65C. The rate of digestioncould be in reducedby working at 50C and this lower temperature was used in all subsequent experiments. The courseof the digestion was studiedby using the followingprocedure. Weighedsamples cuticle(30 mg) weretriturated of with phosphatebuffer (0.1M, pH 6.7) to form a uniform suspension. A solutionof papain(2.5 mg) and dithiothreitol(10 mg) in buffer(2 ml) was then addedand the suspensions incubatedin a water bath at 50C.After appropriate time intervals, samples were withdrawnand chilledto quench the enzymereaction. The undigested cuticle residues were recoveredby

CHEMISTRY OF HUMAN HAIR CUTICLE

357

centrifugation,washed twice with distilled water, dried in vacuo over phosphorus pentoxideand weighed.
Examinationof morphological sitesof digestion humanhair cuticleby of papain/dithiothreitol
For this work, root-ends of brown Caucasianhair were embeddedwith

Spurr'sresin (5) in Beemcapsules (LKB Produkter). It is noteworthythat theseresinsare unlikely to diffuseinto the fibres,but nevertheless bonding of the resin with the fibre surfaceis sufficiently strongto provide the rigid supportof the hair for subsequent thin sectioning. Transverse sections of the hair approximately60 nm thick were cut on a diamondknife (Du Pont de Nemours)at a Porter-Blum MT-2 ultramicrotome (Sorvall)and collected on 100 mesh gold electronmicroscope grids(Polaron) previously covered with a thin collodion/carbon support film. Papain/dithiothreitol reagent,of the samecompositionas that usedin the gravimetricexperiments, was prepared and filteredimmediately beforeusethrough0.45 tm Millipore discs. The gridswere immersedin the reagentat 50C, and after varioustimes of digestion,rinsedbriefly in a solutioncontainingdithiothreitoland buffer, then in two changes distilledwater eachfor 5 min and dried. Some of of the grids were 'shadowed'by the oblique vacuum evaporation of carbon/ platinum on to the upper sideof the grids(subtending angleof 30with an the grid surfaces). The shadowing techniquedid not permit the discrimination of structure within the cellmembrane complexon subsequent examination in the electronmicroscope. The remainderof the gridswere therefore stainedwith various heavy metal compoundsaccordingto the following scheme,designedto yield maximum structural information: grids were immersed at room temperature successively 2o aqueous osmium in tetroxidefor 2 h, distilledwater for 1 h, saturatedaqueousuranyl acetate for 2 h, distilledwater for 1 h, Reynold'slead citrate (6) for 10 min, 0.02 sodiumhydroxidefor 10 s, distilledwater for i h and then dried. The variousdigested hair sections, shadowed stained,were examined or in a JEM 7 transmission electronmicroscope 80 kV and usinga 20 gm at diameter objective aperture. In the case of the shadowed sectionsit is alreadyknown (7) that the surfaces undigested of hair sections not flat are and that the irregularitiesare related to the underlying morphological structure the sections. the other hand the presentdigestions cleanly of On so
removed some of the structures of the hair cuticle that no confusion arose

with the originalirregularities the hair sectionsurfaces. of

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Amino acid analyses isolated of fractions

Insolublematerialremaining after digestion hair cuticlewith papain/ of dithiothreitol reagent for 45 and 90 min was centrifugedat 30 000 g, the supernatant removedand the residuewashedwith three changes disof tilled water, centrifugingafter each wash. Material obtainedby exhaustive digestionfor a total of 3 days with two changesof reagent,was treated similarly.The residues were dried in vacuoover phosphorus pentoxideand their amino acid compositions determined conventional by autoanalysis.
RESULTS AND DISCUSSION

Course digestion isolated of of cuticlewithpapain/dithiothreitol A plot of the amount of residueremainingagainsttime of digestion of isolated cuticle shown Fig. 1 (A). From thiscurveit appears is in that at least two components are dissolving different rates. That this is the caseis at revealedby the logarithmicplot of Fig. 1 (B). The undissolved residue at long timesof digestion approximately is 7 by weightof the originalcuticle and by extrapolationof the log. plot to zero time it would appearthat the fast dissolving components represent about 82o and the slow dissolving component about 11o by weightof the cuticle.
oo

(B)

5o

3o

8
? 6

5
A

e,
1
I

e----.// e-7 8 18
Digest,on tme (h)

e.,
24 72

1
2

I
3

I
4

I
5

Figure 1. Graph showing the gravimetric course of digestionof human hair

cuticlewith papainand dithiothreitol. CurveA, axisA showthe linearplot and curveB, axisB the corresponding logarithmic plot. Each point shown represents
the mean of three separatedeterminations.

CHEMISTRY OF HUMAN

HAIR CUTICLE

359

The morphological progress digestion the hair cuticle of in

Examination of digested hair sections in the transmission electron microscope revealedsignificant progression the dissolution the various in of structuralcomponents the cuticle.In addition, sincewe believethat the of accessibilityto enzyme attack of the sectionedhair cuticle and of the physicallyisolatedfragmentsare probably similar, somecorrelation could be made between the removal of cuticle components observed in the electron microscopeand the various stagesof digestionrevealed by the foregoinggravimetricresults. After digestionof the sections only 15 min therewasalmostcomplete for
removal of the endocuticle and there was some loss of material from the

exocuticle(Fig. 2). (It is pertinent to note that Figs 2 and 3, which are electron micrographsof 'shadowedsections',have been prepared from intermediatenegatives that shadowregionsare dark. In achievingthis so normal consistency shadows, for regionsof increasing electronopacity are depictedby increasing brightness the photographs.)Even at 15 min in digestionit is striking that a distinct layer approximately 100 nm wide associated with the cuticle cell boundariesexhibits an electron opacity similar to that of the sectioned epoxyresin,indicatingthat this component is unaffected the digestion. by After digestion 45 min virtually all the for exocuticle was removedleavingthe cell boundarymaterial still apparently unaffected (Fig. 3). At this stage wasnecessary examinemetal-stained it to
rather than shadowed sections to reveal the detailed substructure of the

cellboundarylayer.It wasfound that this consisted the A-layer to which of was attached,on the outer-facingaspectof the hair, the completecuticle cell membranecomplex(Fig. 4). With further increasing time of digestion the width andgeneral opacityof the A-layerslowlydiminished. periods For of digestion exceeding h the A-layer had more or lessdisappeared 24 still leavinga residue associated the cellboundaries. with Closeexamination of this revealedthat the completecell membranecomplexwas still present and boundedon either sideby a layer approximately nm thick of stain80 able material(Fig. 5). It is noteworthy that at this stageit was difficultto discernthe laminatedsubstructure the membranecomplexbut this was of probablybecause disorientation the residueon the electronmicroscope of grid occurs that the membrane so laminaeare no longerparallel to the electronbeam of the microscope. On the other hand, by examiningthe point whereadjacentcuticlecellsoverlapto give a type of T-junction of cell boundaries, initial orientationof the membranes the was maintained

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JOURNAL THE SOCIETY COSMETIC OF OF CHEMISTS

and the laminated substructure the cell membranecomplex could be of seen(Fig. 5). From the presentresultsit is clear that the initial fast-dissolving component observed the gravimetricstudiesconsists endocuticle in of and exocuticle, that the slowly-dissolving componentconsists A-layer and of that the final insoluble residue consistsmainly of cuticle cell membrane material.Indeedthereis goodcorrespondence between percentage the areas occupied the endocuticle exocuticle inner layer, the A-layer and cell by + + membrane complexin hair cuticlesections (82-85o, 10-12o and 5-6 respectively) and the foregoinggravimetricdeterminations what are for apparently samecomponents the (82o, 11o and 7o respectively). Little mention has beenmade of the fate of the cuticleinner-layerin the present work but it is assumed becauseof its similarity in structureand cystine

content that it behaves a manneranalogous that of the exocuticle. (1) in to The progress digestion of leadingto the isolation onecaseof A-layer in and cellmembrane complex and in the otherof the cellmembrane complex
alone is summarizedin the schematicdiagram of Fig. 6.
Cell membrane

corn plex
90 rnin

A-layer .....

Exocuhcle

Inner layer
Endocuticle

72 h

Figure 6. Schematicdiagram illustrating the courseof digestionof human hair cuticlewith papain and dithiothreitoland leadingto the separationof two major morphologicalcomponentsof the cuticle.

Amino acid analysis

The first columnof Table I containsthe amino acid analysis isolated for cuticleand columns2, 3 and 4, the analyses the papain/dithiothreitolfor
insolublefractionsof the cuticleobtainedafter digestion 45 min, 90 min for and 3 daysrespectively. The analysis column5 was obtainedby calculatin ing the differences amino acid composition in between '90-minute'and the '3-day' residues,

JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS

Figure Transverse 2. section humanhair cuticle of digested 15 min and then for shadowed. Co, cortex; En, endocuticle;Ex, exocuticle,A+CM, A-layer-t-cell
membrane complex.

Figure3. As Fig. 2 but digestion 45 min. for

(Facingp. 360)

JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS

'

Figure4. Transverse sectionof human hair cuticle digestedfor 45 min and

stainedwith heavy metals. A, A-layer; CM, cell membranecomplex.

Figure5.

As Fig. 4 but digestion 24 h. for

CHEMISTRY

OF HUMAN

HAIR

CUTICLE

361

362
Membranes

JOURNALOF THE SOCIETY COSMETIC OF CHEMISTS

As discussed previously,the insolublematerial remainingafter digestion for 3 daysconsists mainly of cell membranecomplexand contains approximately 39o lipid material.The amino acid analysis this fractionprobfor ably represents for the membrane-associated that proteins and it is interesting in that it differsconsiderably from that for intactcuticle.The lastthree columnsof Table I show the amino acid compositions membranefracof tions obtainedfrom wool by Bradbury and co-workers(8, 9) while column 6 showsthe compositionof wool cuticle (3). In each casethe membrane fractionsshow substantially higher concentrations asparticacid, glycine of and lysine,and lower concentrations serine,proline and cystinc of than the corresponding cuticle samples.The cuticular membrane fraction from humanhair alsocontainshigherproportions isoleucine, of leucineand the aromatic amino acids than does intact cuticle. Closer comparisonof the human hair and wool membranefractionsis not possiblesincethosefrom wool are preparedfrom whole wool rather than isolatedcuticle. The presence high concentrations basicamino acidsin the cuticle of of membranecomplex is consistent with the intense stainingwhich is seen under the transmission electron microscope, in the intermembranous cement(b-band)and the thin layersbounding complex hair sections the in stainedwith dodecatungstphosphoric (1). This heteropolyacid acid exists in solutionas a trivalent anion and is generallybelievedto bind readily to the basicgroupsof proteins(10). Electronhistochemical stainingof hair sections cystinc(7, 11) indicates for that this amino acid is absent from the cuticlecell membranecomplex. Sincesomecystincis presentin our membrane fraction, at least part of the fraction is probably derived from the protein on either side of the lamellatedmembranecomplexproper and indeedthis is consistent with our presentelectronmicroscope observations (rig. 5). The main advantageof the presentmethod for preparingcell membrane fractionsfrom human hair compared with the previously usedoxidafire procedures is that the destruction sensitive (8) of amino acids such as tyrosine and methionineis minimized. Furthermore the lipid components of the membranesare well preservedso that further study of thesematerials is possible.
The A-layer

The fractions obtained after digestionfor 45 and 90 min evidently

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363

contain mainly A-layer and cell membrane complex, with the 45-min fraction containing a small amount of undissolvedexocuticle and the 90-min fraction having had some A-layer removed (cf. Fig. 1). As in the caseof the membranefraction, the accuracyof the amino acid analyses is limited by the presence non-proteinaceous of components.A more serious limiting factor is the difficultyof obtaininga well-characterized sampleof the A-layer and cell membranecomplexfree from other contaminantssuch as exocuticlebut not having lost any of the A-layer. Sincedigestionof the A-layer is essentially completein lessthan 7 h, small errors in sampling time or inefficiency quenchingthe enzymereaction result in substantial of variationsin composition.These difficultiesare reflectedin the coefficients of variation shownin the third column of Table I. In spite of theselimitations the calculateddifferences amino acid compositionbetween the in '90-minute' and '3-day' fractions shown in the fifth column of Table I, approximateto the composition the A-layer, and showmajor differences of from the compositionof the intact cuticle.The A-layer thuscontainshigher concentrations aspartic acid, basic and aromatic amino acids and lower of concentrations serine,proline and cystinethan the whole cuticle.The of low content of sulphur-containingmaterial in the A-layer/membrane residues wasconfirmed semiquantitatively X-ray microanalysis by wherethe sulphurpeak/background ratio for the '90 minute residue'was substantially lower than that obtainedfrom intact cuticleor from exocuticleisolatedby pronase digestion according the methodsof Bradburyand Ley (3). to The A-layeris probablya complex mixtureof differenttypesof proteins. Analysis of residuesremaining after various times of digestionbetween
45 min and 3 h showed that the relative concentration of aromatic amino

acidsincreased digestionproceeded, indicatedin Table II. No such as as trends were shownby the other amino acids.
Table II

Residue as o by weight
of cuticle 27 14.3 13.3 12.4 9.8

Moles/1000 tyrosine Moles/1000 phenylalanine

17.2 4.25

18.4 11.6

33.7 23.5

41.9 30.9

48.9 40.7

These resultsindicate the presence a componentrich in aromatic of amino acidsand more resistantto proteolyticenzymes than the rest of the A-layer, though whether or not this componentforms a discretelamella closeto the membraneis not yet clear.

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JOURNALOF THE SOCIETYOF COSMETIC CHEMISTS

The low cystinecontentof the A-layer is surprising and contrary to electronhistochemical evidence 11). It is possible (7, that a cystine-rich component has been removedspecifically from the A-layer during the enzyme digestion this is considered but unlikelysinceour electron microscope observations indicate that the overallstructural integrity andelectron opacityof this layer is maintained the analysed for specimens. further A possibility that the electron is histochemical methods not as specific are in their staining cystine cysteine of or residues hasbeenclaimed.Using an as organomercurial method,Dobb, Murray and Sikorski(11) have showna near-stoichiometric uptake of mercuryby cysteine reducedwool. On in the otherhandthe uptakeof mercury such in minorcomponents the Aas layer may not be necessarily relatedto the presence cysteine, other of for aminoacids non-proteinaceous or materialcouldbeinvolved the reaction. in In this respect is interesting note that Levy (12) has demonstrated it to reaction between an organomercurial halide and the amino groups of insulin. Indeed this reactionmay explain the existence the thin layer of stainedby mercuryadjacentto the cuticlecell membranes wool and of described Dobb et al. (11). Sucha reaction by wouldbe consistent with our observations high lysine concentrations the membrane-associated of in fractionsand the staining a thin layer eithersideof the cuticlecell memof branes complexby dodecatungstophosphoric With respectto the acid.
silver-methenemine method for the electron histochemical demonstration

of cystine,anomalousstainingof the A-layer of human hair cuticle has beendescribed already(7). The silverglobules usedin the latter methodas a criterionfor the presence cystine(7, 13) have a physicalappearance of whichis quitedifferentin the A-layer than in the othercomponents the of hair, perhapsindicatingthat the histochemical reactionis modified by groupsotherthan cystine. the light of thesediscrepancies the electron In in histochemical demonstrations cystine the A-layer, we believethat our of in analyses showing low cystinecontentof this component realistic. the are
The chemistry papain/dithiothreitol of digestion

One strikingfeatureof the present work is the rapiditywith whichhuman hair cuticle dissolves mixtures of papain and dithiothreitol compared in with papain alone (usuallynegligible)or mixturesof papain and sodium bisulphite(55o dissolves after 8 h rising to 70o after 28 h). One of the main reasons this is undoubtedly high percentage for the reductionof the keratin cystineby dithiothreitol(14, 15). Whereascompletereductionof

CHEMISTRYOF HUMAN HAIR CUTICLE

365

cystine keratinsby thioIsis difficultto achieve in because reactionis the determined the law of mass by action,dithiothreitol reduction readilysplits 85 of the disulphide bonds wool undermild conditions in with only a small excess dithiothreitol(14, 15) owing to the formation of a stable of cyclic disulphide (4,5-dihydroxy-l,2-dithiane). reduction the disulAfter of phidebondsthe proteins, previously resistant attack by proteolytic to enzymes, rapidlydigested are under mild conditions. Borenfreund, and Fitt Berdich (17) have used combinations trypsin and reducingagents of including 2-mercaptoethanol and 2,3-dimercaptopropanol degrade to 'proteolytic enzyme resistant, keratin-like' components sperm of cellsand similarly PfauandMcCrea(18) haveused pronase 2-mercaptoethanol and to release DNA from vacciniavirus. Preliminaryexperiments us have by shownthat a combination pronaseand dithiothreitolrapidly digests of humanhair cuticle (80o beingdissolved 1-}h at 37CpH 8.0 and over in 91 within 16 h) indicating that pronase may prove to be a valuable
alternativeto the use of papain at 50C.

The activationof papainis dependent upon the existence cysteinyl of residues the prosthetic (16) sothat the presence dithiothreitol at site of will give maximumyield of the groupsand therebymaximumproteolytic
activity.
ACKNOWLEDGMENTS

We are gratefulto Mr F. J. Bailey of Unilever Research Laboratory, ColworthHousefor undertaking amino acid analyses us. Thanks are for
also due to Mrs S. J. Smith for her valuable assistance with the electron

microscope work.

(Received: 28th February1974)

REFERENCES

(1) Swift,J. A. and Bews,B. The chemistry humanhair cuticle. of Part 1. A newmethodfor the physical isolationof cuticle.J. $oc. Cosmet. Chem.25 13 (1974). (2) Fraser,R. D. B., Macrae, T. P. and Rogers,G. E. Keratins: Their Composition, Structure andBiosynthesis (1972) (CharlesC. Thomas, Springfield, Illinois). (3) Bradbury, H. and Ley, K. F. Separation analysis exocuticle endocuticle. J. and of and
Aust. J. Biol. $ci. 25 1235 (1972).

(4) Swift,J. A. andBews, The isolation membranes B. of from keratinfibres with papainand
dithiothreitol. J. Text. Inst. 65 222 (1974).

(5) Spurr, A. R. A low-viscosity epoxyresin embedding mediumfor electronmicroscopy. J. Ultrastruct,Res. 26 31 (1969).

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JOURNALOF THE SOCIETY COSMETIC OF CHEMISTS

(6) Reynolds,E. S. The useof lead citrate at high pH as an electron-opaque stainin electron microscopy.J. Cell Biol. 17 208 (1963). (7) Swift, J. A. The electronhistochemistry cystine-containing of proteinsin thin transverse sectionsof human hair. J. Roy. Microsc. Soc. 88 449 (1967). (8) Bradbury, J. H. and Leeder,J. D. The cell membranecomplexof wool. Appl. Polymer Syrup.18 227 (1971). (9) King, N. L. R. and Bradbury, J. H. The chemicalcompositionof wool V. The epicuticle. Aust. J. Biol. Sci. 21 375 (1968). (10) Kiihn, K., Grossman,W. and Hofman, U. Die elektronenmikroskopische anffirbung des kollagens und die ausbildungeiner hochunterteilen querstreifung. Z. Naturforsch. 136 154 (1958). (11) Dobb, M. G., Murray, R. and Sikorski,J. Specific labellingof thiol groupsin mammalian keratin suitablefor electronmicroscope studies.J. Microsc. 96 285 (1972). (12) Levy, D. The selective chloromercuration insulin.Biochim.Biophys. of Acta 317 473 (1973). (13) Swift, J. A. The electroncytochemical demonstrationof cystinedisulphidebonds using silver-methenemine reagent. Histochemie35 307 (1973). (14) Weigmann, H-D. and Rebenfeld,L. Reduction of wool with dithiothreitol.Text. Res. J. 36 202 (1966). (15) Weigman, H-D. Reduction of disulphidebonds in keratin with 1,4-dithiothreitol.Part 1. Kinetic investigation. Polym. $ci. 6 (AI) 2237 (1968). J. (16) Glazer, A. N. and Smith, E. L. Papain and other plant sulphydrylproteolyticenzymes.In: The EnzymesPart 3, ed. P. D. Boyer, 501 (1971) (Academic Press,New York). (17) Borenfreund,E., Fitt, E. and Berdich,A. Isolation and properties deoxyribonucleic of acid from mammalian sperm. Nature, 191 375 (1961). (18) Pfau, C. J. and McCrea, J. F. Releaseof deoxyribonucleic acid from vacciniavirus by 2-mercaptoethanoland pronase. Nature, 194 894 (1962).