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J. $oc. Cosmet.Chem., 21, 875-900 (Dec. 9, 1970)
The Mechanismof Hair Bleaching
LESZEK J. WOLFRAM, Ph.D., K. HALL, B.Sc.,and I. HUI, M.Sc.*
PresentedDecember2, 1969,New York City
Synopsis--Thecolor of mammalian HAIRS is due mainly to the inclusion of discrete,darkly
colored MELANIN granules in the keratinized cytoplasmicprotein of the fiber-forming
cells. During BLEACHING the melanin pigment undergoes irreversible physicochemical
changeswhich result either in the toning down or complete elimination of the original
fiber color. The modificationof the fiber protein (KERATIN) attendant upon bleaching is
largely confined to the oxidation of combined CYSTINE. The cysteicacid residuesformed
in this reaction causea significantchangein the distribution of electrostaticcrosslinks.
INTRODUCTION
Peroxide bleachingof pigmented keratin fibers has been practiced for many years,yet no published account of any comprehensive
studyof the kineticsor mechanismof the processis available. Literature concerningthe physicochemical
changesin the pigment is practically nonexistent; that dealing with keratin modification attendant
upon bleachingis relativelyrich (1-15), but mainly devotedto generalities, and often contradictory. This is not surprisingin view of the fact
that the various authors who have studied the bleaching processemployedwidely differingconditionsof treatment.
As the aim of bleaching is to eliminate or tone down the natural
hair color, the processis directly related to the structure and reactivity
of the hair pigment. Two principal approachesto understandingthe
structure of melanin have been tried. The analytical approach has
* Gillette CompanyResearchInstitute, 1413 ResearchBlvd., Rockville, Md. 20850.
875
876
JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS
been employed with greatest successby Nicolaus and his coworkers
(16, 17). It hasled to a conceptof melanin structureas a polymerof
multiple subunitsjoined by multiple typesof bonding,i.e., a poikilopolymer. The syntheticapproachwasinitiated by Raper (18). It has
led to the secondconceptof melanin structureas a regular polymer
involvinga singletype ot5monomerjoined by a singletype of linkage,
i.e., a homopolymer. Unfortunately,little effort wasmade to interpret
the postulatedstructuresin terms of the melanin reactivity which remains an enigma.
The pigment granulesare distributed within the cortex of the fiber
and it is thusnot surprisingthat during the bleachingprocess
someoxidation of the keratin matrix
occurs.
This is often referred
to as "oxidative"
or "bleaching"damage. With regard to the specificityof this oxidative
attack,the lossof cystinehasbeen ascertained(5, 7) and the modification
of other amino acid side chains (tyrosine, tryptophane, lysine, and
arginine)hasbeen postulated(10, 13, 15). However, the majority of
the publisheddata refers to the bleachingof wool, which is usually
carriedout at elevatedtemperaturesin neutral or slightlyalkaline media.
In the bleachingof hair, the useof ambient temperatureis compensated
for by a higher pH value of the system. The publishedinformationon
the physicochemical
changesin hair keratin taking placeunder suchconditions is almost exclusivelyqualitative, and it is quite inadequateto
serveas a firm guide for improving existing bleaching systems. This
communicationis an accountof an investigationaimed at obtaining
a better understanding of the complex processesassociatedwith the
reaction of hydrogen peroxide with both the melanin pigment and
hair proteins.
MATERIALS
AND METHODS
The Caucasianhair,* brown and white, used in this investigation
wasshampooed,
rinsed,and conditionedat 65% RH and 70øF.
The black poodle hair was obtained from random samplesof hair
clippings. The hair was purified by Soxhlet extraction for 4 hours
each with methylene chloride followed by absolute ethanol. It was
then rinsed well with
deionized
water and conditioned
as above.
Commercially available, reagent grade solvents and chemical reagentsutilized in this studywere not further purified, unlessotherwise
specified.
* Suppliedby De Moo Brothers,New York.
HAIR
BLEACHING
877
The melanin wasisolatedfrom the hair by acid hydrolysisaccording
to the method of Green and Happey (19). Purified black poodle hair
was placed in a round-bottom flask equipped with a reflux condenser
and hydrolyzedwith 6N HC1 for 4 hoursunder reflux. A liquor-to-hair
ratio
of 40:1
was used.
The
mixture
was cooled
and
the melanin
was
separatedby centrifugingfor 30 tnin at approximately 1000 g's. The
sedimented
melanin
was washed
with
deionized
water
until
the solu-
tion in equilibrium with melanin had a pH value of 5.2. The melanin
was then rinsed
several times with
acetone
and dried
in vacuo at 60øC.
Microscopicinspectionof the product indicated the absenceof any
fibrouscontamination. The high purity of the isolated pigment was
confirmed by examination of the melanin granules with the electron
microscope. Altogether, 50 g of poodle hair was hydrolyzed,yielding
3.75 g of melanin.
A nonhydrolyticmethod (20) of melanin extraction was employed
mainly for a comparative examination of chemical properties. The
hair samplewasmaintained at reflux for 24 hours in a phenol hydratethioglycolic acid mixture (PHT). The filtration step was omitted,
due to a previousunsuccessful
attempt in the isolationof the pigment.
After separation of the brown pigment by centrifuging, the melanin
was washed two times with fresh portions of PHT.
The isolated
melanin was further washed with deionized water as described above,
followed by severalacetonerinsesand drying. The purified melanin
contained
a considerable
amount
of fibrous material,
which
was re-
moved manually.
Visible and UV absorptionspectraof melanin were obtained on a
Perkin-Elmer Model 202 Spectrophotometer.
Mechanical propertiesof hair were determined on the table model
Instron. The fiberswere mounted on plastictabsat 2-in. gaugelength,
equilibratedunder the desiredconditions,and stretchedto break at required ratesof extension. The broken endswere then cut off the tabs,
conditioned,weighed,and the denier of the testedfiberswascalculated.
Amino acid analysesof untreated and bleached hair were carried
out on a Phoenix Model M-7800 Micro Analyzer.*
The swellingof hair was determined by the liquid retention tech-
niquedescribed
by Valkoand Barnett(21). When specified,
a reduction-
* PhoenixPrecisionInstrument Co., 3803-05North Fifth Street,Philadelphia,Penna. 19140.
878
JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS
oxidationcyclewasusedjust beforethe determination. The purpose
was to intensify the damageand thus allow a differentiation between
sampleswhich are difficult to resolveotherwise. The hair was treated
with 0.2M ammonium thioglycolate(pH 9.6, 35øC) for 10 min at a
20:1 liquor ratio, followed by a brief rinse and treatment with 0.2M
H•O2 (pH 3.4, 35øC) for 5 min. The hair was then rinsed free of
peroxidewith deionizedwater.
Thin-layer chromatography
wasemployedfor separatingthe melanin
oxidation products. The adsorbentlayer wassilicagel (Merck), having
a thicknessof 250 v. Plate size was 5 X 20 or 20 X 20 cm. In caseswhere
the subsequent
elution of components
wasto be performed,the thin-layer
plateswere freed of possibleimpurities by their immersionin spectral
grademethanol [or at least30 min. The plateswere then ready for use
without any prior activation.
Sampleapplicationwasusually made by streakingthe aqueoussolutions onto the plate with the aid of a Brinkmann streaking piper.
Wheneverlimited quantitiesof samplewere available,the sampleswere
spottedonto the plateswith microliter pipers.
Developmentof the plateswasperformedin a rectangularchamber
(11 X 11.5 X 4 in.) containing300 ml of solvent,namely, ethanol:ammonia:water in the ratio of 80:4:16. Overnight equilibration of the
solvent,in a closedtank, lined with solvent-soaked
paper, was necessary
before its usage. A solventmigration of 17 cm required development
timesof 3-3.5 hoursat ambient temperature.
After the plateswere developed,they were dried for about 20 min
at 105øC. The resolvedcomponents
were locatedby exposingthe plate
to ultraviolet irradiation from a 4-watt lamp with a spectraldensity of
3600fk.* The spotsor bandswerelocatedby their fluorescence.
Other methods of identification included the following spray reagents,preparedaccordingto Stahl's (22) procedure:
(a) Bromocresol
green(0.04%) in ethyl alcohol
(b) AgNO:,-NH.•: equalpartsof 0.1NAgNO:,and5N NH•
(C) 50% H2SO4
(d) Ninhydrin, 0.3% in ethanol.*
* Ultra-Violet
Products, Inc., San Gabriel, Calif. 91778.
?As suppliedby SigmaChemicalCo.,P.O. Box 14508,St. Louis,Mo. 63178.
HAIR
BLEACHING
879
RESULTS AND DISCUSSION
Preliminary 0 bservations
Stabilizedaqueoussolutionsof H202 undergolittle decomposition
at ambient temperatureseven at high pH values. However, the introduction of a solid into a system brings about an increase in the
decompositionrate which is roughly proportional to the surfacearea
of the solid. An additional incrementin the rate of decompositionis
observedwhenever the introduced solid undergoeschemical reaction
with
H202.
Table
I shows the rate differences obtained
under
such
conditions.
Table
I
Decomposition of Hydrogen Peroxide in the Presenceand Absence of Hair a
H202 Decomposition (%)
Time of Reaction,
rain
No fibers
present
White hair
Brown hair
5
0
O8
0.8
10
0
21
2.5
20
0.3
42
6.5
30
0.5
61
60
0.9
94
14.2
90
1.4
13 5
21.1
8.0
a Bath ratio, 33:1; Initial [H20•] = 35 gl-•; pH 10.0; 35 ø C.
Initially, with the reaction confinedto the surfaceand to the cuticular
region of the fiber, the rate of the peroxide decmnpositionis ahnost
identical for both brown and white hair. This is not surprisingin
view of the similarityof the dimneterand of the che•nicalcronposition
of both smnplesof hair. It is likely that the divergence in the decompositionratesobservedin the later stagesof the reactionis associated
with the responseof the piginent,the granulesof which are locatedwithin
the cortexof hair and thereforenot soreadilyaccessible
to the peroxide.
Bearing in mind the low inelanin contentof the studiedhair (,-•2%),
these observationsconnote a much higher reactivity of melanin than
that of keratin.
Galculations
based on the data of Table
I show that
the overall rate of peroxide decompositionin the presenceof brown
hair is 9.0 X 10-2 •nM •nin-• g-•, while the correspondingvalue for
white hair is 6.4 X 10-2mM min -• g-•. If the difference in H202
880
JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS
decompositionwas to be accountedfor by the melanin alone, then the
latter would yield a value of 130 X 10-2 mM min -• g-•, a difference
factor of over 20. A rate of this magnitude (95 X 10-2 mM min -• g-•)
was actually observedin the study of the oxidation of melanin with
H202 at pH 10 and 35øC. Assumingthat the rate of peroxidedecmnposition is related to that of oxidative reactionstaking place within the
fiber, then this large difference in reactivity is obviously a desirable
feature from the point of the bleaching process. A further increase
in the reactivity ratio should lead to faster and less damaging bleaching. Such an approachhas been utilized in the bleaching of wool by
using the iron mordanting technique (23). The preferential binding of
iron by the pigment sensitizesthe latter to the peroxide attack and results in significantaccelerationof bleaching.
Although qualitative observationsof colorchangeswhich hair undergoesduring bleaching,combined with quantitative evaluation of peroxide consumption,provide somemeasureof melanin reactivity, they
add little to our understanding of the mechanismof the process. In
addition, the intimate associationof the pigment with the hair fiber is
likely to interfere with many physicochemical
aspectsof the process
and to obscuretheir relative importance. To obviate these difficulties
eachof the components(melaninand keratin)wasexaminedseparately.
It was assumedthat isolation of the pigment from its keratin environment would not significantlyaffectits chemicalbehavior.
Reaction of Melanin Pigment with Hydrogen Peroxide
The
melanin
was isolated
from
the hair
in the form
of discrete
granules,approximately0.8-1.2 • long and 0.3-0.4 u thick (Fig. 1).
Examination of the pigment with the electron microscopeat several
magnificationlevels (5,000-50,000) did not reveal any structural organizationof the g•:anules.
This wastrue for both the PHT- and HC1isolated
melanin.
The densityof melanin was determinedby the flotation technique
(benzene/bromobenzene/3-bromochlorobenzene
system)and wasfound
to be 1.53 g/cmg. This is much lower than the value of 1.71 reported
by Swift (24).
The pigment •-anules are hygxoscopic,
attaining the equilibrium
regain of 16.4% at 65% relative humidity. Although no attemptwas
made to identify the water binding sites,the acid and basecombining
capacitiesof the pigmentwere determined(0.32 and 2.5 meq/g, re-
HAIR
BLEACHING
881
.'
.
Figure 1. Electron micrograph of melanin granules extracted by hydrolytic method
spectively). It is very-likely that thesepolar residuesact as the primary
centersof water sorption.
Solubilizationof Melanin Pigment
The cross-linked,polymeric structure of melanin manifestsitself in
its high resistanceto numerous organic and inorganic solvents. Some
dissolution of melanin was detected in DMSO,
concentrated H.,SO4, and
1N NaOH, but only at elevatedtemperatures (100øC and above). Yet,
evenprolongeddigestionwith thesesolventsleft the bulk of the pigment
insoluble.
Extensive treatments of melanin (up to 48 hours) with reducing
agents such as thioglycohcacid, borohydride, sulfide, and sulfite produced no apparent physicalchangein the pigment. Neither did oxidation with persulfate, perchlorate, iodate, and permanganateperformed
over a wide range of pH (1-10). A different behavior wasdisplayedby
hydrogen peroxide. Dilute aqueoussolutions of this reagent caused
disintegTationof the pigment gTanules,which slowly dissolvedin the
reaction system. The solution becameintenselycolored, the dark color
persistingfor a considerablelength of time, after which some fading
was evident. This observation, obviously relevant to our bleaching
882
JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS
studies,was also very significantin the senseof a general method for
melanin
solubilization
and its usefulness toward
better
characteriza-
tion of this highly resistantpolymer.
The reactionappearedamenableto spectroscopic
techniques,and
both the visible and the UV spectraof the melanin solutionswere examined. The visible region proved uninformative;a monotonicrise
in optical densitywith increasingtime of reactionwas observed. The
rise was very gradual and did not appear to reflect the qualitative
changes
takingplacein the system
under investigation.Initial attempts
to utilize the UV region were also of little avail becauseof the high
absorptionintensity of H•O2, which overshadowed
any absorption
changescausedby the solubilization of the melanin. However, by
resortingto the techniqueof differentialspectroscopy,
the peroxide
absorbancewas suppressed
and the spectroscopic
changesdue to the
reactioncouldbe readilyfollowed. A typicalrecordingis reproduced
in Fig. 2. Within a few momentsof the contact of the reagents,a
well-definedabsorbance
peak wasdeveloped. The peak intensityincreased with
the time of the reaction
and reached a maximum
at the
time of completedissolutionof the melanin. Then a slowdecreasein
absorbance
wasobservedasthe bleachingof melaninby H202 continued.
mm.•..• --
õmirl•_
15mi•----•"'•
\
-0.4
•4
mirt
0.5
8 mi
o7•
•N
0m.i,•
0.s•z
8mln
0,9
•
1.0
1,1
1.2
1,3
1.4
1,5
3 0
350
3
WAVELENGTH (millimicrons)
Figure 2. Solubilization of intact melanin in 1% H202 at pH 11.5
HAIR
BLEACHING
883
The time required for dissolutionof melanin in aqueousH:O2 can
be readily determined from the absorbancepeak and then used as a
convenient parameter in further investigationsof the reaction. The
generalexperimentalprocedureemployedin the solubilizationstudies
was as follows: 1 mg of melanin was introduced into a volumetric
flask containing 10 ml of aqueousH20.,. The reaction mixture was
stirred magneticallyat 25øC; at a given time, aliquots were withdrawn,
transferredinto a 5-ram quartz cell, and their spectrarecorded. The
reference cell contained a solution of H:O2 at a concentration identical
to that in the sample. Concomitant with the recording'of spectra a
visual observationwasmade of the state of the melanin dispersion,and
the time of its completedissolutionwas noted. Usually the reaction
was followed
for at least 60 min
after
the dissolution
time.
Effect o[ pH on the Rate of Solubilization--The pigment was treated
with 1% aqueoussolutionsof HeO2 adjustedto different pH values by
means of sodium hydroxide. Both the dissolution times (tD) and the
absorbancesat tD were recorded,and are given in Table II. The reaction appearsto have a maximum rate in the region of pH just below the
pK value of H20,(11.75), indicating that although the peroxide anion
is definitely involved in the solubilization process,it may not be the
soleattackingspecies.
Table
II
Effect of pH on the Dissolutionof Melanin in 1% H202 at 25øC
pH
Time for Complete Dissolution (t•)
(Min)
Absorbance
10.45
30
10.80
14
1.29
1.27
11.25
9
1.15
11.55
10
1.21
12.20
14
1.21
12.70
13
1.02
Duke and Haas, who studied the homogeneousbase-catalyzeddecompositionof H== (25), noted a similar pH dependenceand accountedfor it by postulatinga reactive,cyclicintermediateformed from
the neutral H=•
molecule and its anion:
[ .4' decomposition
__ _ H•o/OxH'
ßH20
+OH+0,2
H,,O,,
+ HO,- •
884
JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS
The rate of decompositionwasaccordinglyexpressedby'
Rate = K[H.,O2][HO2-]
the productreachinga maximumat pH = pK•2o2. Our resultsare
not entirely consistentwith the aboveequation as the rate of solubilization appearsto be more affectedby the decreasein concentrationof
the ionized speciesthan that of the neutral molecule. However, it
shouldbe borne in mind that ionization of the acidic side chainspresent
in the melanin is likely to lead to a buildup of negativechargeson the
pigment. This may form an effectiveelectrostaticbarrier to the penetration of the peroxideanion and thusbe an important factorin affecting
the rate of oxidation.
Effect of Hydrogen Peroxide Concentrationon the Rate of Melanin
Solubilization--The experimentswere run at the optimal pH of 11.5.
An accelerationin the dissolutionrate wasobservedwith increasingconcentrationof the peroxide. Although a plot (Fig. 3) of the reciprocal
I
I
I]
1
2
3
24.(
20.,
x 16.•
i
r.
12.0
.--
.--
:3
'•
8.0
©
©
._•
4.0
Figure 3. Effectof H,20.o
on rate of melanin dissolution
HAIR
BLEACHING
885
of the dissolutiontimes (tD) againstH202 concentrationyields a straight
line indicative of ordinary kinetics for the bimolecular reaction, this
simple dependenceis probably fortuitous in view of the heterogeneity
of the solubi!i?ationprocessA scrutiny of the changesin absorbanceat to for various concentrations of H202 reveals a possibleclue to the physical mechanismof
bleaching. If one assumesthat the bleaching of melanin by peroxide
is an inherent part of the solubilization of the pigment granule, then
the absorbancesof melanin solutionsat to should be independent of
H202 concentration. This is not the case (Table III). Not only are
the absorbanceintensities of dilute Heemelanin systemsvery much
higher than thosewith prolongedtime, but they remain virtually unchangedfor a prolongedtime. The "lack" of bleachingis 'not caused
by depletion of the reagent. Indeed, even in the most dilute solutions
studied(0.01% H.oO2)the molar ratio of H202 to the melanin (indole
residue)at to is at leastof the order of 5:1.
Table
III
Maximum Absorbancesat tofor Various Concentrationsof H202 at pH 11.5
[H2021, %
Absorbance
[H202], %
Absorbance
0.1
2.95
1.0
1.24
0.4
1.41
2.0
1.14
0.6
1.36
3.0
1.06
The resultscanbe plausiblyexplainedin termsof a two-stepprocess:
solubilization of the granule followed by decolorization of the dissolved melanin. The data imply that the bleaching processis relatively slow when compared to the solubilization of the pigment and
thus controls
the overall
rate.
This hypothesiswas supportedby electromicrographicexamination
of the melanin which had been subjectedto H202 treatment for various
lengthsof time. At the end of the reaction time, excessperoxidewas
decomposedby platinum black, the solution was filtered, and the undissolvedpigment was examined. There was little apparent change
in the sizeof the granulesas a function of time. Yet, only 5 min of
treatment was required to dissolveas much as half of the original
weight of the melanin. Evidently the disintegrationof the pigment
granuleswasvery fastonce it commenced.
886
JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS
Physicochemical
Propertiesof SolubilizedMelanin
Solubilizationof melanin by dilute solutionsof H•O,., presented
itself as a potentially very useful tool for better characterizationof this
intractable polymer, provided that the modificationbrought about by
the peroxideattackwasnot too great. It wasthoughtlikely that under
mild conditions of treatment the primary reaction would be the
elimination of the solubility-restrainingcross links and the overall
chemicalnature of the pigment would be retained. Solubilizedmelanin
wasthereforeprepared,and someof its propertieswere examinedand
comparedto thoseof intact melanin,where possible.
Solubilization of the granules was effected at low concentrationsof
H..,O2(1%) in 0.5:1I ammonia at pH 10 and 200:1 liquor ratio.
Complete dissolutionof melanin under these conditionstook place
within 60 min. At this point the peroxidewasdestroyedby platinum
black, the water wasremovedby evaporationon a steambath, and the
product was isolatedas a water-soluble,highly lustrousmaterial. On
acidificationto pH 2, the solubilizedmelanin precipitated. This product wasdenotedas melanin free acid (MFA). Its solubility behavior is
typical of a polymer containingfew ionizablegroups,dissociationof
which forcesthe polymeric chain into solution. Thus MFA remains insolubleat low pH and dissolves
rapidly when the pH of the systemis
raised above 4.
The solubilizationprocessincreasedthe baseuptake capacityof the
melanin from 2.7 to 3.8 meq/g. This correspondsto a neutralization
equivalent of 262, and indicatesvery slight oxidative breakdown of
the melanin
structure.
SpectroscopicSt•tdies--The potential usefulnessof infrared spectroscopyis limited when chemicallyill-defined polymersare examined;
this certainly appearsto be the casefor melanin. There waspractically
no changein the spectrumfollowing the solubilization. The UV region
proved to be more informative. While the intact melanin showsno
absorbance maxima,
and a monotonic
rise of absorbance with the de-
creasein wavelengthbeing observed,the solubilized melanin exhibits a
well-definedmaximum at 222 mv (Fig. 4). Although no positiveidentification of the absorbing sites can be made, a tentative assignmentof
this maximum to a peroxide-typestructureis postulated.
It is perhapsappropriateat this stageto discussin more detail the
spectroscopic
changesoccurringin the UV region during the solubilization of melanin. As reported earlier in this investigation, dissolution
HAIR
BLEACHING
887
0.4
0.5
0.6
•>
I).7•
11.8
•
z
0.9m
ø
1.0
1.2
1.3
1.4
200
f
•
•
•
•i
2 0
•
•
I
I
I
300
•
f, 1.5
WAVELENGTH (millimicrons)
Figure 4.
Absorbance of solubilized melanin in water
of the pigment in aqueousH.,O2is characterizedby an increasein the
absorbance
and a formationof a well-pronounced
peak. The position
of this peak is congruentwith the absorbancemaximum displayedby
H.,O2 itself, and varies with the changein the peroxide concentration
in an identical manner. Upon destructionof the peroxidewith platinum black, the peak shiftsto a new positionillustrated in Fig. 4. The
shift is accompaniedby an increasein the absorbanceintensity. The
positionof the peakduring the solubilizationprocessis suggestive
either
of a peroxide-typecompoundexhibiting an absorptionpattern identical
to that of H202 or of the generationof H202 during the reaction. The
latter alternative, however, would not satisfactorilyaccount for the existenceof an absorbance
maximum after the decompositionof hydrogen
peroxidewith platinum black.
Molecular Weight--The extent of the degradation suffered by
melanin during its dissolutioncould be assessed
by the determination
of molecularweightchangesbroughtabout by the solubilizationprocess.
Unfortunately, no data on the molecular weight of the intact melanin
are available and our attemptsto determine it with a vapor pressure
osmometerwere unsuccessful.In fact, it is the solubilizationprocess
itself that presentedthe opportunity to assess
the molecular weight of
the pigment. The apparatuschosenfor this study was devisedby Bull
(26) for osmoticpressuremeasurements. Measurementsfor this type
usually require some form of extrapolation to infinite dilution. The
advantageof Bull'sosmometeris that it eliminatesthe needfor extrapola-
888
JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS
tion by allowing measurementsto be made at sufficientlylow dilutions
for Van't
Hoff's
law to be valid.
Melanin solubilizedby ammoniacalhydrogenperoxide wasdialyzed
for 24 hours prior to the measurements. The molecular weight was
calculatedfrom Burk and Greenberg'sequation (27):
M = CdRT/lOOP
where C is the concentrationof polymer in gramsper 100 ml of solvent,
d is the densityof the solvent,R is the gasconstant,T is the temperature,
and P is the osmoticpressure. The equation reducesto
C
M = 2.527X 10.5dff
after insertingthe numericalvaluesfor the constants. A melanin concentrationof 0.310 g per 100ml of saltsolutiongaverise to an osmotic
pressureof 6.86 cm of water. The molecularweight calculatedfrom
thesedata yielded the value of 11,400.
A value of the same order, viz., M z
15,000, was also obtained from
the molecular weight determination using the thin-layer gel-filtration
technique.
Free Radical Content--Samplesof melanin were examined in a
Varian X-band esrspectrometer.Both the intact and solubilizedmelanin gave rise to virtually identical structureless
absorption,with line
widths o1:the order of 6 gaussand g valuesof 2.003. The spin density
wasdeterminedfor both of the samplesby comparisonwith a known
DPPH (o6cr-diphenyl-/:t-picryl-hydrazyl)
standardand gave a value of
10•9 spinsper gram.
The most important point emergingfrom this brief study is that
the free radicalcharacterof the melanin is not affectedby the solubilization process.This meansthat theseradicalsare extremelystableand
do not rely for their existenceand stability on a physicaltrapping
mechanism.
Decolorization--The solubilizationof melanin by H.,O• is only the
first step in the reactionsequence.Prolongedtreatmentresultsin
bleachingor decolorizationof the intenselydark solution. Although
thehighefficacy
of hydrogenperoxide(ascomparedwith otheroxidants)
for the solubilizationof melanin wasclearly establishedin this investigation,it did not connoteits superiorityin the bleachingstep. Consequently,
theeffectof a numberof oxidizingagentson thecolorchange
of theaqueous
solutions
of solubilized
melaninwasassessed.
The reac-
HAIR
BLEACHING
Table
889
IV
Effect of Oxidizing Agent on the Bleaching of Solubilized Melanin
Oxidizing Agent
Bleaching Ability
Conditions, pH
1-10
(NH4)2S.•O8
None
KI O 3
None
1-7
K2Cr•O7
None
1-7
NaC104
12
None
None
5.2
q+ qq- q- qq- q-
10
7
<3
7-8
H202
NaOCI
KMnO4
CHaCOOOH
1-7
tion was carried out at room temperature. In each case,excessof the
oxidant waspresentin the system. The resultsare given in Table IV.
The most surprisingfinding was the high decolorizationefficacy
of the permanganate,particularly in view of its inability to react with
the intact melanin. Are the crosslinks which are broken during the
solubilization important to the preservationof color? Or does the
solubilizingaction of peroxidesensitizethe melanin polymer, e.g., by
generationof labile, peroxide-typestructuralelements?Approximately
0.03 meq of KMnO4 was required to bleach 1 mg of soluble melanin
to a pale yellowcolor. Assumingan averageunit weight of the melanin
as 145, 1 mole equivalentof KMnO4 is utilized for 2 melanin units.
The contribution of peroxy anion speciesto the bleaching process
canagainbe readily seenin the caseof peraceticacid. In slightlyacidic
media thisreagentis specificfor oxidativecleavageof the disulfidebonds
in hair but has little effect on the melanin.
The
latter is, however,
readily attackedunder alkaline conditionsand a maximum decolorization effectwas observedin the pH range closeto the pK value (8.2) of
the peracid (28).
It is worth pointingout that while the disintegration
of the pigment
granulesand their solubilizationare the necessaryprerequisitesfor
bleaching,theseprocesses,
by themselves,
are not likely to affect the
color of hair significantly. At best,the conversionof pigment particles
into solublemelanin dye might bring about a slight changein hue.
The decolorizationstep,on the other hand, althoughcontingentupon
the former, is more readily perceivedand thus may be consideredot•
greaterpracticalimportance. From the experimentalevidenceobtained so far, it is impossibleto elucidatethe precisechemicalnature
890
JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS
Table
V
Ri Values of the Componentsof the Decolorized Melanin
Rf
Relative Intensity of Fluorescence
0.00
0.02
0.02
Strong
Medium, very narrow band
Strong
0.04
Medium
0.08
Strong
0.16
Weak
0.23
Medium
0.35
Weak-medium
0.41
Weak-medium
0.43
Medium
0.51
Weak
of the reactionsoccurringduring the solubilizationof the pigment and
its subsequent
decolorization.In the former process,
the solubilityrestraining crosslinks are eliminated and the chromophoricgroups appear to be left virtually intact. There is a goodcaseto argue that these
cross links
are much
more
labile
and thus different
from
the residues
which undergo much slower reaction in the decolorizationstep. The
bleachingproper, on the other hand, reliesupon oxidative destruction
of the highly conjugatedsystemof the indole residues. It is likely that
the oxidation is centered initially on the benzenoid portion of the
ring. The acceleration of the decolorization processobservedwith
both permanganate and peracids is in accordancewith such a view
(29). Some additional support for the postulatedpath of oxidative
breakdowncan also be derived from the resultsof qualitative chromatographic analysisof the productsof the decolorizationreaction. All
the resolvedcomponents (Table V) were identified as acids but no
aromaticderivativeswere present. The test for pyrrolic acidswas also
negative. The latter were detectedby Piattelli (16) in the permanganateoxidized melanin. It appears,therefore, that under ordinary bleaching
conditions,which we have employedfor the preparation of decolorized
melanin, even the indole nucleusundergoescompletedegradationupon
fissionof the benzenoidportion of the ring to yield smaller fragments
with acidic functions, such as oxalic acid which was identified as one of
the decolorization
products. We were unable,however,to identify positively the remaining components.
The oxidation of melanin by peroxideis accompaniedby development of fluorescence
which increasesin intensity with the progressof
HAIR
BLEACHING
891
the reaction. All the decolorizationproductsare stronglyfluorescent
and indeed it was this property which greatly assistedtheir chromatographicseparation. To our knowledgethis hasbeen the first report of
the phenomenon. The only other relevant report was the observation
by Binnsand Swan(30) of the purple fluorescence
from the synthetic
melanins.
Reactionof Hair Keratin with Hydrogen Peroxide
The fact that the reactivityof melanin with regardto hydrogenperoxide happensto be so much higher than that of keratin almost automatically connotesthe bleachingprocessas a commercialsuccess.However, the melanin pigment representsonly a very small fraction of the
fiber weight (usuallyabout 2%) and thus it is reasonableto expect that
some oxidative
modification
of the fiber
matrix
will
occur.
Conven-
tional bleachingprocesses
utilize hydrogenperoxide in alkaline media
at pH 10 and above;the perhydroxyanion (HO2-) is the predominant
reactivespecies.The abundanceof sitesin keratin which might yield to
a nucleophilicattack by this ion precludesany firm prior assignment
of a specificinteraction. In addition, the presenceof some radicals
derived from H202, the reactivity of which is not particularly sensitive
to pH changes,addsto the uncertainty concerningthe type of reactions
involved. The physicalmethodsusedto detectdamageassociated
with
bleachingare satisfactoryfor measuringthe extent of deterioration,but
are of little value for elucidating the chemical character of the damage.
The latter can best be ascertainedby chemical analysis,and such a
methodwasusedasa startingpoint of this investigation.
ChemicalCompositionof BleachedHair
Tressesof brown hair were bleachedwith 3% Haaat pH 10 (0.5M
NH3) and 35øC for 4 hours. The bleachedtresseswere sampled,the
sampleswere hydrolyzed,and the hydrolyzateswere analyzedon the
Phoenix M-7800 Micro Analyzer. The resultspresentedin Table V1
showconvincinglythat, under the conditionsstudied,the reactionbetweenkeratin and H202 is confinedmainly to the cystineresidues. The
decrease
in cystineis almostquantitativelymatchedby a corresponding
increasein cysteicacid. The amino acid analysisdoesnot reveal any
intermediateoxidation productsof cystinewhich might be formed
during the bleachingprocess. These compoundsare, however,very
unstable under alkaline condiitons, and any remaining would dis-
proportionate
to cystineandcysteic
acidduringthe hydrolysis.
892
JOURNAL OF THE SOCIETY OF COSMETICCHEMISTS
Table
VI
Amino Acid Compositions
of Untreated and BleachedCaucasianHair
Amino Acid Content in/•mol/g
Amino Acid
Untreated
Bleached
Cysteicacid
Asparticacid
55
455
289
447
Threonine
Serine
Glutamic acid
Proline
653
870
871
672
642
820
868
700
Glycine
539
525
Alanine
471
460
1380
1130
Valine
Isoleucine
Leucine
538
250
554
542
247
530
Tyrosine
Phenylalanine
Lysine
132
130
213
120
119
225
Histidine
Ammonia
63
780
69
870
Arginine
512
540
• cystine
During bleachingsomeof the keratin dissolves
in the reaction
medium. The weight lossesare, however,very small. After 4 hours'
treatmentof brown hair with 3% H22 at pH 10 and 35øC, the amount
of extractedprotein did not exceed1% of the fiber weight. Even
smallerproteinextracts
wererecordedin the caseof whitehair treated
under similar conditions.
Althoughwe could not yet assess
the averagemolecularweight of
the dissolved
protein,its aminoacid contenthasbeendeterminedand
the resultsare givenin Table VIII.
The origin of the dissolvedfraction is uncertain,as the relatively
largedifferences
in aminoacidcomposition
betweenthe extractand the
untreatedhair argueagainsthomogeneous
solubilizaton. Someof the
solubleproteincouldconceivably
resultfrom the destruction
and elution of the melanin-keratincomplex.Sucha view is stronglysupported
by the fact that the extracted
proteincontains
two aminoacidsnot
[ound either in the bulk of hair or in the bleachingextractof the white
hair. These are taurine and /•-alanine. Nevertheless,the major portion
of the extractmostprobablyrepresents
peptidesassociated
directlywith
the oxidizedcystine. The cysteicacid residueis knownto facilitate
HAIR
BLEACHING
Table
893
VII
Amino Acid Compositionof Keratin Material DissolvedDuring Bleachingof
Brown
Caucasian
Hair
Amino Acid Content, %
Amino
Acid
Extract
Cysteic acid
14.1
Untreated
Whole
0.9
Taurine
0.2
Aspartic acid
9.2
6.0
Threonine
5.3
7.8
Serine
...
11.8
9.1
17.6
12.8
Proline
0.6
7.7
Glycine
7.6
4.0
Alanine
4.1
4.2
•
3.7
16.8
Valine
4.9
6.3
Isoleucine
1.7
3.3
Leucine
3.9
7.1
Tyrosine
Phenylalanine
•5-Alanine
Lysine
2.8
2.1
0.3
3.9
2.4
2.1
Histidine
0.4
1.1
Arginine
7.8
8.9
Glutamic
acid
cystine
Hair
...
3.1
greatly the hydrolysisof the adjacentpeptide bond and thus create
favorable
conditions
for destructive
solubilization.
Swellingof BleachedHair
Of the manywaysin whichtheoxidativedamageof keratinattendant
upon bleachingmanifestsitself (deteriorationof tactile properties,mechanicalweakeningof the fiber, increasein alkali solubility,etc.), the increasein swelling representsa convenientmeansfor the assessment
of
the extent of damage. This increasein swelling is brought about by
changesin the bulk of the fiber, and thus is directly related to the overall chemicalmodificationof keratin by H,•O_o.
Evidencehasbeen presentedhere that of all the amino acidspresent
in keratin, only the cystineundergoesa measurableextent of reaction
with the peroxide;possiblythe oxidativebreakdownof disulfidebonds
alone would satisfactorilyaccountfor the increasedswelling. However,
the principallocusof the bleachingreaction,aswe havelearnedearlier,
is the melanin granule. It is conceivablethat the oxidative destruction
of the •nelanin granulesmight result in formation of discretevoidswith-
894
JOURNAL
OF THE SOCIETY OF COSMETIC
CHEMISTS
in the fiber structure. It was thought that the contribution of this factor to the total swelling characteristicsof the bleached hair could be ascertainedby specificoxidation of cystincin brown hair with peracetic
acid (without attacking the melanin) or by control bleachingruns on
white hair.
Table VIII correlatesthe swellingdata with the extent of disulfide
bondbreakdown. The swellingwasmeasuredat pH 7 usingthe liquid
retentionmethodwith the reduction-oxidationcycle.
Table
VIII
Swelling of Oxidized Hair
Liquid Retention (%)
Cystinc
Oxidized,
White Hair,
Oxidized with
Brown Hair,
Oxidized with
Brown Hair,
Oxidized with
% of Original
H202
H20•
PeraceticAcid
0
54.5
50.2
50.2
10
63.2
63.6
64.0
13
68.8
70.5
67.0
15
78.5
97.8
77.2
At the same levels of disulfide bond oxidation
the bleached brown hair
appearsto be more damagedthan its white counterpart. This difference
could be due to the breakdownof melanin, a contentionsupportedby
the fact that the damagein brown hair can be lowered when the disintegrationof pigmentis prevented(peraceticacid oxidation).
It is, however, obvious that the main source of damage residesin
the destructionof disulfidebondswhich not only opensthe structureof
hair but providesadditional hydrophilic centersin the form of cysteic
acid residues. The introductionof theseresiduessignificantlyaltersthe
swellingcharacteristicof hair as illustrated in Fig. 5. A novel feature
of the swellingbehavioris its unusualdependenceupon pH. A precipitousincreasein swellingof bleachedhair occursin the pH region
5-7.5, while no suchchangeis observedin caseof untreated hair. This
phenomenoncan be bestexplainedby acceptingthe chargerearrangement mechanismpostulated for oxidized wool by Thompson and
O'Donnell (31). The stronglyacidic c¾steicacid residuesare, in the
courseof their formation, being cmnpensatedfor by the ionized basic
groupsof the keratin. As a result, the carboxylicgroupsof the acidic
side chains are being expelled from their salt links and remain essentially un-ionized at pH 4 and below. This decreasein their acidity
HAIR
BLEACHING
895
.• 46
Figure 5. Effect of pH on swellingof bleachedhair
(in the intact fiber their pK = 2) is broughtabout by the growth in
negativechargedensityfollowingthe formationof the cysteicacid residues. AbovepH 5 the displaced
carboxylicgroupsbeginto titrate; this
is reflectedin an increasein swellingwhich reachesa maximum value as
complete ionization is approached. The available evidence thus
stronglysuggests
that the ionizationof the carboxylicgroupsleadingto
increasedswelling contributes to the resultant fiber damage. The
ionizationeffectcan be convincinglydemonstratedon the behaviorof a
hair tresswhichwasexposedfor a shorttime (10 min) to the oxidizing
actionof a dilute solutionof peraceticacid (pH 3.5). When the treated
tresswasevaluatedfor feel and combing,it wasindistinguishablefrom an
intact tress. However, when the oxidized tress was subsequentlyim-
mersedfor a few minutesin pH 9 buffer, rinsed, and rated again, its
tactile and combingpropertieswere similar to thoseof bleachedtresses.
Apparently,sufficientetchingof the keratin took placein the buffer to
changethe surfacecharacteristic
of the oxidizedfibers.
896
JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS
Table
IX
Mechanical Properties of Oxidized Fibers
Yield
Stress,g/den
Breaking
Stress,g/den
Breaking
Extension,
Min
Dry
Wet
Dry
Wet
Dry
Wet
...
51.4
Time,
Fiber Treatment
Untrcated
control
3% H20•, pH 10, 35øC
3% H•O•, pH 10, 35øC
3% H•O•, pH 10, 35øC
0.5% CHaCOOOH, 25øC
(then 0.1M NH4OH,
•nin)
1.10
0.41
1.92
1.68
40.1
30
1.08
0.36
1.84
1.61
41.6
56.5
90
1.07
0.28
1.79
1.05
44.2
58.1
180
1.07
0.20
1.43
0.75
42.7
57.6
30
10
1.08
0.36
1.86
1.58
41.0
53.5
20
1.02
0.30
1.78
1.30
43.5
56.5
30
1.02
0.27
1.74
1.20
41.8
58.2
60
0.99
0.20
1.72
1.10
46.1
59.1
MechanicalPropertiesof BleachedHair
The disulfidebondscontribute greatly to the wet strengthof keratin
fibers,which decreases
almostlinearly with the cystinecontent. On the
other hand, the strengthof the dry fibersis not appreciablyaffectedby
the breakdown of covalentcrosslinks, being dependentlargely on the
main chain length and interchain hydrogenbonding.
When viewed from this standpoint,the changesin mechanicalproperties of hair keratin brought about by bleachingcan be satisfactorilyinterpreted in terms of the oxidative attack on the disulfide bondsalone.
Thus, we observe(Table IX) a steadydecreasein wet moduluswith increasedtime of bleachingand virtually no changein either the modulus
or ultimate strengthof dry fibers. The latter observationsupportsthe
view that the extent of the main chain scissionduring the bleaching
processis negligible;it wasshownby Elod (32) that the breakdownof
1% of the peptidebondsin keratin bringsabout an almost20% lossin
the dry strengthof the fiber.
The dry breaking extensionis slightly affectedby bleaching. Certainly, no evidenceof the brittlenessoften referred to is found. This is
not changedby varyingthe rate of strainingfrom 1 to 50 in./min.
The apparentretentionof dry strengthby the bleachedfibersdoesnot
result from a decreasein regain. On the contrary, the regain of the
fiber increaseswith th? extent of bleaching(Table X) over a wide range
of tested humidities. Bearing in mind the charge rearrangement involving the cysteicand carboxylicacid residues,it would appear that
this increasein regain may be directly related to the ionization of the
HAIR
BLEACHING
Table
897
X
Effect of Bleaching on Moisture Absorption by Hair
Regain (%) at Relative Humidity of:
Sample
17%
41%
61%
72%
84%
94%
23.9
Intact
5.4
8.7
13.5
15.9
19.2
Bleached 30 rain
5.6
9.3
13.9
16.1
19.5
25.0
Bleached
120 rain
6.0
9.8
14.4
16.6
20.1
25.9
Bleached 240 rain
6.5
10.1
14.8
17.5
20.5
28.7
carboxylicgroupsand by depressingsuch an ionization a return to regain valuescloseto thoseof intact fiber could be attained. This indeed
is the case. When bleachedhair is soakedbriefly in acid, then rinsed
in deionizedwater to removeany bound acid and its regain is redetermined, the value obtained is almost identical with that of untreated hair.
The wet mechanicalpropertieswere measuredwith fibers immersed
in pH 7 buffer (Fig. 6). However, unlike the untreatedhair, the hydration of bleachedhair is stronglypH dependentand thus should manifest itself accordinglyin the mechanicalperformanceof wet fibers. Let
1.0
'•]•C"•'• • •'•
reduced
0.9
0.8
•
0.7
oxidized
_:
o.6
F-0.5
I
0.4
0.3
0.0
•
2I
3I
4I
5I
6I
7I
8I
pH
Figure 6. ElFoctof pH on yield index of oxidized and reduced hair
898
JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS
us briefly considerwhat are, in terms of interchain bonding, the consequencesof charge rearrangementattendant upon bleaching. The
displacementof carboxylicgroupsfrom their salt linkageswith positively chargedaminogroupsby cysteicacidresiduesgreatlystabilizesthis
linkage. This is simplydue to the fact that cysteicacid residuesremain
ionized at low pH valuesat which the carboxylategroups,even those
in the intact fiber, would protonate,leading to destructionof the electrostatic bond.
Thus, breakdown
of the covalent disulfide bond is com-
pensatedfor somewhatby formation of a more stable electrostaticcross
link. In slightly bleachedfibers only a fraction of the cystineundergoesthe oxidative breakdown,and only a few of the carboxylic•oups
are displaced[TOmtheir salt links and just as many new, strongerbonds
of the sametype are formed. In an extensivelybleachedfiber, the displacementis virtually complete and theoretically,the strengthof the
fiber shouldbe only slightlyaffectedby increasingacidity. Someexperimental support[or this view is derived from a brief study of the effect
of pH on the mechanicalperformanceof bleachedand reduced fibers.
In both casesthe fraction of broken disulfide bonds was closeto 40%.
We
have chosen to use reduced
fibers rather
than
intact
fibers as our
controls becausethe importance of charge rearrangement has to be
viewed againstbackgroundsof similarly disorganizedstructures. The
following test procedure was employed: Both bleached and reduced
hair were soaked in 0.01N
HC1 for 6 hours at 25øC and then rinsed with
freshchangesof deionizedwater until no more acid wasreleasedby the
keratin.
The fibers were then dried, mounted on tabs, and stretched
5% in deionizedwater (calibration step). They were then released,
kept in deionized H20 for 12 hours, and transferred for an additional
12 hoursto buffer solutions,in which they were restretchedagain. The
force to attain the yield point wascalculatedin both casesand the ratio
Yield
Yield
force in buffer
force-calibration
denotedas the yield index (Fig. 6). The resultsconformsatisfactorily
to the pattern expectedon the basisof our theoretical considerations.
Thus, the bleached fibers exhibit a region of maximal mechanical stability between pH 3 and 5 where the displacedcarboxylic groupsare
undissociated
and those ionized
are bound
in the salt links.
An increase
in pH above5 leadsto the ionization of free carboxyls,the fiber hydration
increases
and
so does
the
ease of
its
deformation.
This
be-
havior is sharply contrastedby that of reduced fibers. With no free
HAIR
BLEACHING
809
carboxyl
sidechainsto ionize,the regionof theirmechanical
stabili'ty
staysalmostunchangedup to pH 8. This is not so under acidicconditionswherereducedfibersshowa precipitousfall in yield force.. It is
obvious that the combination
nation
of electrostatic
of the disulfide
bond breakdown
and elimi-
cross links have a disastrous effect on mechanical
performanceof the fiber. Althoughbleachedhair alsoshowssomeweakening (apparentlya sizeablefraction of acid-labile,salt links is still
present),
thestabilizing
effect
of newelectrostatic
bonds,
involving
•he
cysteicacidresiduesand the chargedbasicgroupsof arginine and lysine
is prominently evident.
ACKNOWLEDGEMENTS
The authorsare indebtedto the followingpeoplefor their assistance
in providing someof the data presentedin this paper. The electron
micrographicexaminationof melanin wascarried out by Mr. A.. Dano
of the Gillette Safety Razor Research Laboratories in Boston. Dr.
Kokoschka
of the NationalBureauof Standards
examined
samples
of
melanin in a Varian X-band esr spectrometerand Dr. R. K. Brown of
WayneStateUniversitydeterminedthe molecularweightof solubilized
melanin by thin-layer gel chromatography.
:
(ReceivedMarch 10',197,0)
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900
JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS
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