J. Biol. Macromol. , 13(3), 92-106, 2013

A novel purification procedure for keratin-associated proteins and keratin
from human hairToshihiro Fujii*，Shunsuke Takayama，Yumiko Ito
Faculty of Textile Science and Technology, Shinshu University, 3-15-1 Tokida, Ueda,
Nagano, 386-8567, Japan
Received November 22, 2013; Accepted December 16, 2013
The proteins in human hair consist primarily of microfibrillar keratins with amolecular mass of 40–65 kDa and keratin-associated proteins (KAPs) with amolecular mass of 6–30 kDa, according to the results obtained from sodium
dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Because aneffective purification procedure of KAPs has not been established, little is known
about the protein chemistry of KAPs as compared with that of keratin. When hairsamples were incubated in the Shindai solution containing alcohols such as
methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, and 2-methyl-1-propanol,
the extraction of KAPs was enhanced, while extraction of keratin was suppressed.
Using ethanol, we established a selective purification procedure for KAPs andkeratin. According to Tricine/SDS-PAGE, the KAPs fraction contained six
polypeptides with molecular masses of 3.5, 4.4, 5.2, 7.8, 15, and 28 kDa. The
keratin fraction contained two polypeptides with molecular masses of 45 and 67
kDa and was free of low-molecular-weight components. The amino acidcompositions of the KAPs and keratin fractions were mostly in agreement withthe values found in the literature. The recoveries of the KAPs and keratin
fractions from the hair samples were approximately 10 and 50%, respectively.
Scanning electron microscopy (SEM) showed that hair samples retained fine
fibrous structures in the cortex after extracting the KAPs and that the additional
extraction of keratin caused the fibrous structures to disappear. These results
indicated that KAPs may function by surrounding the fibrous structures andsupporting the keratin fibers in the cortex. In this study, we propose a novel andconvenient isolation procedure for KAPs and keratin from human hair.
Keywords: human hair, keratin-associated protein, keratin, purification, selective
solubilization*To whom correspondence should be addressed:
fujiit1@shinshu-u.ac.jp

93

Human hair keratin-associated proteins and keratin

fibers. Due to the damage that is caused

Introduction
The

proteins

hair

to hair by bleaching or permanent wave

comprise approximately 80% of theprocesses, KAPs are interesting for hair

total mass of the hair and consist

care research. Kanetaka et al. haveprimarily of keratins, with a molecularconfirmed

weightcomponents,

of

in

40-65

keratin-associated

human

kDa,

elutionincludingofproteinsulfurand(KAPs),

cysteic acid, when bleach-treated hairwith a molecular weight of 6-30 kDa,

is immersed in a solution of 6%

according to SDS-electrophoresis [1-4].

thioglycolic acid solution (TGA) as a

The keratin family can be further

reducing

resolved

substances

into

proteins

and

the

two

subfamilies

reagent [6]. Such elutedarepresumed

tobeconsisting of type I (acidic; 40-50 kDa)

associated with KAPs, whose sulfur

and type II (neutral/basic; 55-65 kDa)

content is approximately 2.6 times thatmembers. KAPs are classified based onof keratin. Inoue et al. reported that

their

when human hair was immersed in a

amino

acid

content

intohigh-sulfur proteins, ultra-high-sulfur

solution

proteins,

low-molecular-weightandhigh-glycine/tyrosine

containing

6%

TGA,

proteins(less

proteins. Rogers et al. have reported

than 15 kDa molecular weight) wereamino acid sequences obtained fromobserved in the solution [7].
We have developed the “Shindai

the genetic analysis of KAPs as well as
their detailed distribution within hairmethod”[5].

human hair easily and efficiently. ThisA number of investigations havemethodtoextractdeliversaproteins

high

from

yield

of

focused on the biochemical properties

solubilized proteins by using thioureaof the keratins because they form theand urea as denaturants; the recovery

fibrous structures that are found in thewas threefold higher than that of thehair cortex. On the other hand, there

conventional method using urea only

has been little investigation into the[4]. The solubilized proteins consisted

KAPs,

of

which

functionintheamorphous space between the keratin

keratin

Gillespie

and

KAPs.

reportedaPreviously,
methodof

T. Fujii et al.

94

separating KAPs and keratin from woolof proteins from human hair by ethanol[1]. When the alkylated wool protein

solutions were examined with respectsolution was mixed with zinc acetate attopH 5.8-6, the KAPs fraction wastemperature. By applying the findings,

recovered as the filtrate. However, weweare interested in assessing the KAPs

selectively isolate KAPs and keratin

and keratin fractions that did notwithout using 2-ME or SDS.

undergochemicalthe

reducing

proposeagents,

a

novel

pH,

methodMaterials and Methods

developed a selective isolation method

Effectforextraction from human hair(matrix),(microfibrils),

tomodification

occurring by this method. Kon et al.

KAPs

and

keratinhigh-molecular-weight

of

Human

alcohols

hairproteinsamples

obtained[8].

avolunteers and did not have any

characteristic of KAPs that, whenchemical treatments such as bleach,human hair was immersed in a 1% SDS

hair dyes, or perms. The hair fragments

solutionM

were cut with scissors, mixed at 50

2-mercaptoethanol (2-ME), the KAPs

mg/ml with the extraction solution

were specifically solubilized from theconsisting of 25 mM Tris-HCl (pH 8.5),

hair samples. However, it was unknown

2.6 M thiourea, and 5 M urea, 250 mM

why keratin did not elute despite thedithiothreitol (DTT), and diluted withpresence of the reducing agent.

distilled water or various alcohols (3methodcontaining

utilized2

numerous

wereproteins, and cuticles from human hair
This

from

on

Japanese

We focused on the role of theparts the extraction solution plus 1 parthydroxyl group of 2-ME and therefore

diluent) [4, 9]. After incubation for 24

added various low molecular weighth at 50℃, the samples were centrifugedalcohols to the extraction solution. We

at 12,000 g for 10 min at 25℃. The

found that elution of KAPs from hairsupernatants were recovered in test

increased with this treatment, whiletubes and used to measure proteinthat of keratin was suppressed. In thisconcentrations and for electrophoresis.

study, the details of the solubilization

Human hair keratin-associated proteins and keratin

95

Fractionation of KAPs and keratin(NeoscopeJCM-5000,

JEOL

Ltd.,

KAPs and keratin were separated

Tokyo, Japan). The samples wereby combining reagents including aplaced on specimen mounts using

denaturant, a reductant, and ethanol.

double-sided adhesive tape and wereFirst, the hair fragments were incubatedmade electrically conductive by coating

at 50 mg/ml with a “KAPs solution”

them with a thin layer of gold in aconsisting of 25 mM Tris-HCl (pH 9.5),

vacuum. The images were collected at

25% ethanol, 200 mM DTT, and 8 M

an excitation voltage of 10 kV andurea for 72 h at 50℃. The solution was

500-fold magnification [9, 10].

filtered and centrifuged at 12,000 g for
10 min at 25℃, and the supernatantProtein

was used as the KAPs fraction. The

electrophoresis

residue obtained from filtration wasconcentration

and

gel

The protein concentration waswashed with distilled water and dried atdetermined

room

was

Bradford method using a protein assay

extracted from the dried hair residue bykit (Bio-Rad) [11], using bovine serum

suspending it at 60 mg/ml in thealbuminas

Shindai solution containing 200 mM

dodecylsulfate-polyacrylamide

DTT and incubating the mixture for 24

electrophoresis

temperature.

Keratinh at 50℃. This suspension was alsofiltered and centrifuged at 12,000 g for
10 min at 25℃, and the supernatant

by

the

the

colorimetricstandard.

Sodium(SDS-PAGE)

Tricine/SDS-PAGE

were

gel
and

performed

according to the Laemmli method [12],using a 5-20% gradient polyacrylamidegel, and the Schagger and Jagow

was used as the keratin fraction.

method

[13],

using

an

18%

gel,

respectively. Gels were stained with
Scanning electron microscopy (SEM)The

morphology

of

the

hair

samples after treatment with the KAPs
and Shindai solutions was examined byascanning

electronmicroscope

0.1% Coomassie brilliant blue R-250,
10% acetic acid, and 40% ethanol for 2
h and destained in 10% acetic acid.

Amino acid analysis

T. Fujii et al.

The KAPs and keratin fractions
werecarboxymethylated

iodoacetic acid,usinghydrolyzed in 6 M

96(2-ME) and 1% SDS inhibited thedissociationofkeratin

from

hair

structures

[8].

Based

on

this

HCl for 24 h at 110℃ under a nitrogencharacteristic, they proposed a selective

atmosphere, and dried by a rotary

preparation procedure for keratin andevaporator. The samples were analyzed

KAPs

on an automated amino acid analyzer

indicating that the hydroxyl group of(JLC-500/V, JEOL Ltd., Tokyo, Japan).

2-ME was presumed to affect the

from

human

hair

samples

interactions between keratin and the
KAPs molecules. Thus, we prepared

Results
Effectsofalcoholonproteinsolutionscontaining(methanol,

extraction from human hair
In our research on the applications

25%

ethanol,

2-propanol,

alcohol

1-propanol,

1-butanol,

orof human hair, nail, and wool proteins,

2-methyl-1-propanol) and DTT as a

we

first

convenientdevelopedaprocedure,

rapid

and

reducing agent, and we mixed thiscalledthesolution

with

hair

samples.

After

Shindai method, for protein isolation

incubation for 24 h at 50ºC, the

from

hard

solution was centrifuged at 12,000 g for

keratin [4, 14]. Briefly, in this method,

10 min at 25ºC. The supernatants were

human hair was incubated with therecovered,

and

the

Shindai solution (25 mM Tris-HCl, (pHconcentrationwasmeasured.

8.5), 2.6 M thiourea, 5 M urea, and 250

protein concentrations of the samples

mM DTT) at 50ºC for 1–4 days. After

treated with the six types of alcohol

filtration

thewere considerably lower than that ofcomposed

the samples treated with distilled water

predominantly of keratin and KAPs,(Fig. 1A). Electrophoresis showed thatand no significant degradation of thethe alcohol-extracted solutions from theprotein components was observed.

ethanol,biomaterials

andcontaining

centrifugation,

supernatant

was

Kon et al. found that a solution
containing

2M

2-mercaptoethanol

methanol,proteinThe

1-propanol,

2-propanol, and 1-butanol treatments
consisted primarily of KAPs, whereas

97

Human hair keratin-associated proteins and keratinthese alcohols not only inhibited theFig. 1 Effects of various alcohols on
the solubilization of proteins fromhuman hair samples and on thesolubilized protein components.
Hair samples were incubatedwith the extraction solution (#1)
containing 25% distilled water (#2,
control), 25% ethanol (#3), 25%
methanol (#4), 25% 1-propanol (#5),
25% 2-propanol (#6), 25% 1-butanol(#7), and 25% 2-methyl-1-propanol(#8) at 50℃ for 24 h. The solution
was recovered and centrifuged at
12,000 g for 10 min at 25℃. The
supernatant thus obtained was usedtodeterminetheproteinconcentration (A) and was analyzed
by 5-20% SDS-PAGE (B).
the non-alcohol solutions consisted ofboth keratin and KAPs (Fig. 1B). These
results suggested that the presence ofFig. 2 Effects of the ethanol
and urea concentrations on
the solubilization of proteinsfrom the hair sample.
Hairproteins
wereextracted with the solution
containing 0-25% ethanol (Aand B) and 0-8 M urea (C) at
50 ℃
for 24 h. Aftercentrifugation at 12,000 g for
10 min, the supernatant wasused to determine the protein
concentration
andwasanalyzed
with
5-20%
SDS-PAGE.

T. Fujii et al.

dissociation

of

the

further experimentation because of its

hierarchical architectures of the hairhigh safety profile. The quantities ofproteins

but

dissociationofkeratin

also
KAPs

from

98

inducedthesolubilized protein upon changes in the

from

such

ethanol concentration in the extraction

macromolecules.

solution were examined (Fig. 2A), andthe keratin and KAPs contents were

Characterization of KAPs extraction

analyzed by SDS-PAGE (Fig 2B). The

Of the six types of alcohols that

total solubilized protein decreased with

were tested, we selected ethanol forincreasing ethanol concentration. The

Table 1 Effects of the solution parameters on the amount ofsolubilized protein obtained from hair samples.
The standard solution contained 25 mM Tris-HCl (pH8.5), 25% ethanol, 200 mM DTT, and 8 M urea and was
incubated for 50℃ for 24 h. Hair proteins were extracted
under varying conditions by changing the reducing agent(DTT and 2-ME), the pH (7.5, 8.0, 8.5, 9.0, and 9.5), andthe temperature (30, 40, 50, and 60℃). After centrifugation
at 12,000 g for 10 min, the supernatants were used todetermine the protein concentration.

99

Human hair keratin-associated proteins and keratin

KAPs content increased at greater than
10% ethanol, while the keratin content
decreased

steadilyandonly the KAPs solution.
Variousconditions

for

the

almostextraction of KAPs, including different

disappeared at 25% ethanol. Because

reducing agents, pH, and temperatures,

25% ethanol is calculated to be 4.3 M,

were examined and are presented inapproximately

theTable 1. In the solution consisting of 25

concentration of ethanol was requiredmM Tris-HCl (pH 8.5), 25% ethanol,

as for 2-ME [8].

and 8 M urea, the addition of DTT was

two

times

Figure 2C shows the effect of urea
concentration

proteinfold), indicating that DTT is a stronger

solubilization in the presence of 25 mM

reductant than 2-ME. The quantity of

Tris-HCl (pH 8.5), 25% ethanol, andrecovered protein was increased by

200

of

increasing the pH value of the solution

solubilized protein increased linearly(pH 7.5-9.5). When the incubation

with

The

temperature was changed over theprotein concentration (2 mg/ml) at 5 M

range from 30 to 60℃, the quantity of

urea was similar to that obtained by therecovered protein increased with theShindaithat

increase of temperature. Therefore, thethiourea (2.6 M) will not contribute tosolution consisting of 25 mM Tris-HCl

the extraction of KAPs from hair

(pH 9.5), 25% ethanol, 200 mM DTT,

samples.

and 8 M urea was identified as the

mM

the

on

DTT.

The

urea

concentration

quantity

concentration.

solution,

The

the

more effective than that of 2-ME (5-20

indicating

solubilized
obtained

proteinthe

KAPs extraction buffer and named thesolution containing 25 mM Tris-HCl

KAPs solution. When hair samples(pH 8.5), 25% ethanol, 200 mM DTT,

were incubated with the KAPs solution

and 8 M urea was 1.7 times higher thanat 50 mg/ml for 24, 48, or 72 h at 50℃,

that from the alcohol-diluted solution

the quantity of KAPs was 3.5, 5.6, andcontaining 25% ethanol and 200 mM

6.9 mg/ml, respectively. The protein

DTT (3.4 mg/ml versus 2 mg/ml,

concentration was almost saturated at

respectively).

fractions72 h (3 days). Taken together, werecovered at 1 to 8 M urea consisted of

considered this to be a selective method

Allproteinfrom

T. Fujii et al.

for extracting KAPs from human hair.

100

protein solubilization from human hair,
we attempted to establish a convenient

Selective fractionations of KAPs andmethod for the fractionation of KAPs

keratin from human hairand keratin from human hair (Fig. 3A).

Using the effects of ethanol onFirst, the hair samples (1 g) were cut

Fig. 3 Scheme for the fractionation of KAPs and keratin from human hair.
Human hair fragments (1 g) were cut with scissors and immersed in
20 ml of the KAPs solution. After incubation for 72 h at 50℃, the solution
was filtered and centrifuged at 12,000 g for 10 min, and the supernatant
was used as the KAPs fraction (Lane 1). The residue was washed with
distilled water and used as KAPs-free hair. The washed hair was further
incubated with the Shindai solution at 50℃ for 24 h, to extract the keratin(Lane 2) (A). As a control experiment, human hair was also incubated with
the Shindai solution at 50℃ for 24 h, and a mixture of keratin and KAPs
was extracted (Lane 3). The protein components were analyzed by
Tricine/SDS-PAGE (B).

101

Human hair keratin-associated proteins and keratinwith scissors and incubated with theused the Shindai solution (25 mM

KAPs solution (20 ml) at 50 ℃ to

Tris-HCl (pH 8.5), 2.6 M thiourea, andsolubilize the KAPs. After incubation

5 M urea containing 200 mM DTT) forfor 72 h with shaking, the hair fibers

the solubilization of keratin from thebecame an aggregate like muddy paste

KAPs-free

and were filtered. The filtrate wasincubation was performed at 50℃ for

centrifuged at 12,000 g for 10 min at

24 h, and then, the suspension wasroom temperature, and the supernatantfiltered [9, 10]. The filtrate was

was recovered and used as the KAPs

centrifuged at 12,000 g for 10 min, andfraction. The residue from the filtration

the supernatant was recovered and used

was thoroughly washed with distilled

as the keratin fraction.

water and dried at room temperature.

hairsamples.

The

The fractions thus obtained were

The aggregates thus obtained were used

analyzedbyTricine/SDS

gelas the KAPs-free hair sample. We thenelectrophoresis (Fig. 3B). Our KAPs

T. Fujii et al.

fraction

consisted

seven

Because both keratin and KAPs

polypeptides with molecular masses ofare considered to be multi-protein

3.5, 4.4, 5.2, 7.8, 15, and 28 kDa

polypeptides, we examined their amino

according to the electrophoresis. Thisacid compositions to identify them. The

fraction did not contain significant

half-cystine

amountsotherfraction was 2.5 times higher than that

proteins.

of the keratin fraction (Table 2). The

However, the keratin fraction, which

contents of aspartic acid, threonine,

did not contain significant amounts ofglutamic acid, proline, alanine, andKAPs, consisted primarily of keratin

leucine differed between the KAPs andtype I and II polypeptides. From 1 g ofkeratinhair samples, approximately 120 andcompositions of the KAPs and keratin

510 mg of protein were recovered in

fractions in this study were mostly intheagreement with those in the literature

of

of

102

keratinandhigh-molecular-weight

KAPs

and

keratinfractions,

respectively.

content

fractions.

of

The

the

amino

KAPs

acid

[8]. Based on these results, KAPs andAs a control, we also prepared akeratin could be fractionated by using ahair protein fraction (KAPs + keratin)

combination of different solutions, andbythe obtained samples were of adequate

using

the

Shindaisolutioncontaining 250 mM DTT as previously

purity.

described [9, 10]. The hair protein
solution contained both keratin type I

SEM observation of hair samples

and II polypeptides and low-molecularafter selective extraction

weight KAPs polypeptides (Fig. 3B).

Morphologicalchangeofhair

The recovery from the hair protein

samples was observed by scanning

fraction was 610 mg, which was mostly

electron microscopy. The surface ofin agreement with the summation of thehair samples after treatment of KAPs

KAPs and keratin fractions.

solution, that is, KAPs free hairs in thelateral direction was retained cuticle

Amino acid composition of the KAPs

structures and apparently unchanged as

and keratin fractions

untreated hairs. However, the sectional

103

Human hair keratin-associated proteins and keratin

Fig. 4 Morphological observation of the hair samples after protein
extraction.
(a) Untreated hair, (b) KAPs-free hair, and (c) KAPs-free hairresidue after extraction by the Shindai method.

view indicated a number of fibers withtechnique for KAPs and keratin was

several micrometers in diameter werereported that utilized their differential

projected from the cortex region (Fig.

solubilities in different concentrations

4a and 4b). It seemed that the glueof 2-ME [8]. In this conventional

substance

surrounding

microfibers

had

the

cortexmethod, human hair samples were

removedandincubated with the buffer (25 mM

disappeared. After further treatment

Tris-HCl (pH 8.3), 2 M 2-ME, and 1%

with the Shindai solution, the form ofSDS) for 72 h at 50℃, and the KAPs

the hair samples was irregular andwere selectively released and recovered

became flat and the sectional viewin the supernatant after centrifugation.

showed the fibrous structures

had

After the extraction of the KAPs, thedisappeared (Fig. 4c). On the otherhair sample was further incubated withhand, the cuticle was resistant to these

the buffer (25 mM Tris-HCl (pH 8.3),

treatments

0.4 M 2-ME, and 1% SDS) for 144 h at

and

the

structurewasmaintained [4, 8].

50℃, and the keratin was recovered.
Compared

Discussion

novel purification method for KAPs
keratinPreviously,

theconventional

method [8], there are three advantages

In this study, we established a

and

with

an

from

human

alternative

hair.

isolation

to our method. ① SDS, a commonlyused detergent, has been known tointerfere with chemical and biological

T. Fujii et al.

104

analyses, and its complete removal ishair alternative to accurately evaluatedifficult. No detergent was used in our

the effects of reductive damage from②

solution.

The

KAPs

solution

UV

irradiation,

perm,

and

heat

contained ethanol and DTT, while

treatments [9, 10, 17, 18]. As with hair

2-ME and SDS were used in thesamples, reductive treatment by TGA

conventional method. In Japan, 2-ME

caused the selective release of KAPs

has

toxic

from the keratin film, and the amount

substance since 2008. ③ Processingof protein that was eluted was two

by our method was complete within

thousand times greater than the amountfive days, while six to twelve days were

eluted from hair samples [17, 18].

required for all of the operations in theProcedures have been developed for theconventional method.

preparation of films and sponges frombeen

designatedasaSome types of hair damage arewools and human tissues containing

thought to arise from structural changes

keratins and their related proteins [19].

to KAPs. Kon et al. applied their

Cellular adhesion and proliferation of

method for the analysis of protein

mouse fibroblasts on keratin substrates

composition in hair samples and found

was comparable to those on collagenthat the keratin content had decreasedmaterials [20]. Keratin sponges werein the end and middle regions of hairalso used as scaffolds for long-term cellsamples after perm treatment [8]. Inoue

cultivation [21]. In mammalian hair,

et

that

KAPs are believed to control the steric

low-molecular-weight proteins such asconfiguration of the keratin filaments.

S100A3 and ubiquitin were eluted from

Recently, Fujikawa et al. reported that

perm-treatedThis

KAP2, one of the high-sulfur KAPs,

phenomenon will be a useful index of

was prepared by a gene expression

hair damage. We have developed asystem and induced self-aggregation

convenient procedure for preparing aand interacted with the head domain of

keratin film consisting primarily of

the keratin molecule [22]. Because thekeratin and KAPs from human hairs

molecular interactions of KAPs with[16]. The keratin film can be used as akeratin and other KAPs have not yetal.

reported

hair

[7,

15].

105

been

Human hair keratin-associated proteins and keratin

fully

studied,themethodpresented in this paper will be usefulfor analyzing protein architectures inhuman hair tissue in the future.

Acknowledgements
This research was supported by
Grant-in-Aid for Scientific Research(B) (24360375).

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2. Langbein, L., Rogers, M. A.,
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Communicated by Tate Shinichi