J. soc. cos. CHEM.
TWO-DIMENSIONAL
DESCENDING
CHROMATOGRAPHY
SEPARATION
OF
15, 459-4-63 (1964)
AS
THE
A METHOD
AMINO
ACIDS
PAPER
FOR
IN
THE
HAIR
By ALBERT SHANSKY,P•.D.*
ABSTRACT
Results of paper chromatographic analyses of the amino acid composition of normal, of reduced, and of reduced and oxidized human
hair are presented. These preliminary data suggest that reduction of
hair with alkaline thioglycolate not only splits disulfide linkages of
cystinc but causes chemical changes in other amino acids.
Human hair can be subjectedto many chemicaltreatments in order
to producevarious physical effects. It appearslikely that the changes
taking place at the various bondswhich structurally support the keratin
moleculecan be elucidatedthroughamino acid analyses. A particularly
promisinganalytical tool is two-dimensionalpaper chromatography(1)
which can be applied readily to the qualitative microanalysisof protein
hydrolysatesor other amino acid mixtures. In the studiesreportedhere,
the proteinhydrolysatewasfirst separatedinto fractionscomprisingacidic,
basicand neutral amino acidswith the aid of ion exchangeresins. These
threefractionswere then individuallysubjectedto paper chromatography.
MATERIALS AND EQUIPMENT
Whatman No. 1 filter paper was used (standardsheet, 18 X 22.5 in.).
The advancingfront of liquid is yellowish-brown,but this contaminant
of the paper movesso rapidly that it doesnot usually interfere. A commerciallyavailabletroughfor two-dimensional
paper chromatography
was
employed. The chamber,a glass-sided
lead box (about 75 X 75 X 12.5
cm.) wasmadeairtight with a leadcover.
The first solventusedwas a phenolmixture whichis preparedas follows:
To a mixture of 100mi. of water and 400 mi. of liquid phenol,madehomogeneousby warming, is added 20 mg. of 8-quinolinol. During chroma* Rilling DermeticsCo., Bridgeport7, Conn.
459
460
JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS
tographywith thissolvent,a beakercontaining30 mi. of 0.3% ammoniais
placedinto the chamber.
The secondsolventis preparedas follows: To 55 parts of 2,6-1utidine
are added20 partsof isopropanol
and 25 partsof water. To each500 mi.
of this mixture is added3.3 mi. of diethylamine. During chromatography
with thissolventa beakercontaining100mg. of sodiumcyanidein 4-6 mi.
of water is placedinto the chamber.
EXPERIMENTAL
Various hair swatcheswere exposedto the following treatments for
comparativestudy:
A--untreated
hair.
B--hair subjectedto threeminutesof total immersion
in a reducingsolution of pH = 9.3 (alkali = 0.67 N; thioglycolate= 6.62%).
C--hair subjectedto treatmentB followedby a four-minutetotal immersionin 1.5% H202 at pH = 4.8.
D--hair subjectedto 8 minutesof total immersionin solutionof B followedby a four-minutetotal immersionin 1.5% H20=at pH = 4.8.
The hair swatchesin all casesabovewere givenfinal water rinsesin order
to removeresidualmatter and thenweredriedwith a hair dryer.
Two hundredrag.hair charges
wereplacedin 5 mi. of 1:1 HCl in Pyrex
test tubes. The tubeswere sealed,and the hair was hydrolyzedin a parafIin ovenfor 6 hoursat 120øC. At the end of the hydrolysis,the tubeswere
allowedto cool,thencrackedopen. HCl removalwasaccomplished
by repeatedevaporationin a vacuumoven. The hydrolysates
werethenfiltered
and madeup to 5 mi. with deionizedwater.
The filtered hydrolysateswere separatedinto their acidic, basic and
neutral fractions by means of ion exchangecolumns. The hydrolysate
wasfirstpassed
through
a column
ofAmberlite
IR-4B©*;theacidic
amino
acidsare adsorbed,and the neutral and basicamino acidspassthrough.
The adsorbedacidic amino acidswere eluted with N/10 HC1 and collected
separately. (The columnis convenientlyregeneratedwith 4% NaOH.)
The basicand neutral hydrolysatefraction was then passedthrough a
column of Amberlite IR-50-C which was previouslybufferedat pH 4.0
with an acetate buffer. The basic amino acids are adsorbed,and the
neutralaminoacidspassthrough. The basicaminoacidswereelutedwith
N/10 HCl. (The columnis regeneratedwith 4% NaOH and bufferedat
pH 4.0 with acetate buffer.) By bufferingthe Amberlite IR-50-C at pH
4.0, cystineappearsin the neutral fractionof aminoacids. That cystine
* Amberliteis a tradenameof Rohm& Haas Co., Philadelphia,Pa.
METHOD
FOR
SEPARATION
OF AMINO
ACIDS
IN
HAIR
461
was found exclusivelyin the neutral fraction was demonstratedby the
platinic iodidetest (2).*
The solution(6 to 12 ml.), corresponding
to 200-400 mg. of protein hydrolysate,was placednear a cornerof the filter paper sheet,6 cm. from
either edge. The paper was held with one edgeslightly overlappingthe
openingof the troughand pressedinto it with a strip of sheetglasssomewhat longerthan the paper. This assemblywas then transferredto the
chamberwhichhadbeenpreparedasfollows:
A removablelead tray, the bottom of which was coveredwith a twophaselayerof water andthe firstsolvent,wasplacedon the floorof the boxin
orderto securea saturatedatmosphere. The chromatogramwas allowed
to developfor 24 to 72 hours. The paperwasdriedin a dryingcupboard,
turned througha right-angleand returnedto the trough in order to be developedby the secondsolvent. After drying, the paperwassprayedwith
0.1% ninhydrin in n-butanol, again dried, and then heated at 80ø for 5
minutes. The spotswereoutlinedwith pencilbecauseof eventualfading.
RESULTS
A--Untreated Hair. It was shownthat all the aminoacidsexpectedto
be presentin untreated hair could be accountedfor. There were twelve
spots on the chromatogramfor the neutral fraction, two spots on the
chromatogram
for the acidicfraction,and four spotson the chromatogram
for the basicfraction. The spotsfor methionine,leucine,phenylalanine
and proline in the neutral fraction were so closetogether that they could
be regardedas one spot. No specialeffort was made to separatethe individual membersmakingup this spot.
The amino acid compositionof each fraction is consideredto be as
follows:
•lcidic
Aspartic acid
Rs
0.14
Glutamic
0.24
acid
Basic
Lysine
0.50
Histidine
0.72
Hydroxylysine
Arginine
0.76
0.67
Neutral
Glycine
0.40
Alanine
0.57
* Another--lessconclusive--proof
of the presence
of cystinein the neutral fractionis the
formationof typicalcystinecrystalswhentheneutralfractionofuntreatedhair waspermitted
to remainover a weekendat refrigeratortemperature. This crystallineprecipitatemust be
cystinebecause
it is the only aminoacidin thisfractionwhichexhibitssuchhighinsolubility.
462
JOURNAL OF THE
SOCIETY
OF COSMETIC
CHEMISTS
Serine
Proline
Valine
Threonine
Leucine
Methionine
0.33
0.87
0.78
0.50
0.84
0.82
Phenylalamine
Tyrosine
Tryptophan--theoretical
Cystine
0.86
0.59
0.76
0.13
The acidicfractioncontaineda ninhydrinspot at the solvent"frontal."
This wasconsidered
to be due to inorganicsalt impuritiescollectedduring
the ion exchangeseparation. The spotwas not characteristicof a typical
aminoacid and was,therefore,discounted.
B--Hair TreatedThreeMinutes in ReducingSolution. Ninhydrin development of the chromatogramsof the three amino acid fractions of
treated hair disclosedthat a marked changehad taken place: The acidic
fraction was completelyvoid of amino acids. However, two new spots
appearedin the neutral fraction, and it is possiblethat they are due to the
presenceof somedegradationproduct of the native acidic amino acids.
The basicfraction,however,was completelyaccountedfor. This finding
was so unexpectedthat replicationof this seriesof experimentswas necessary to excludethe possibilityof experimentalerror. The resultsof the
secondexperimentalserieswere identical with the first one and suggest
that the acidicaminoacidswere alteredduringthe reducingtreatment.
It is believedthat this changemay be a resultof deaminationby the reducingsolutionto produceoxaloaceticacidfrom the aspatticacid:
--CO-•CH--
CH•--COO-
-------•
HOOC-
CH2--CO--COOH
I
- -NH
and a-keto glutaric acid from glutamic acid:
---CO•CH--CH•--CH•-
CO()- -- ....
HOOC--CH,,.---CH2-
CO---COOH
- NH
C & D--Hair Reducedby Method"B" for Four and Light Minutes RespectivelyThen Oxidizedin 1.5% H202 at pH ¾.8. Both methodsof treatment (C & D) yielded identical chromatograms. The chromatogramsof
the acidicfractionsfor both of thesetreatmentshad a newspotwhichwas
not encounteredin untreated hair. For reasonsnot clearly understood,
the spotsfor glutamicand aspatticacidsreappearedin the hydrolysates
from reduced-and-oxidized
hair.
The basic fractions of both treatments
revealedchromatograms
which could accountfor all amino acids. The
neutral fractionsof both treatmentscontainedtwo new spotswhich were
not encounteredbefore. A spot at an Rf value of 0.10 appearsto match
METHOD
FOR
SEPARATION
OI; AMINO
ACIDS
IN
HAIR
463
the positionof a known chromatogramof lanthionine. In addition, there
appeareda very largespotor groupingof spotsat an Rf valueof 0.50.
Sincethesenewspotsappearedonly asa resultof treatmentsC & D, it is
assumedthat their presenceis the resultof somechangeswhichtook place
during reductionand oxidation of the hair. These changesprobably occurred at the disulfide linkage since lanthionine was produced; other
changesprobablyoccurredat the polar linkageas well sincethe new spots
are some modification of the acidic areinc acids.
DISCUSSION AND CONCLUSION
In all treated hair swatches there seemed to be a distinct reduction
of
the total quantity of aspatticand glutamicacids. The appearanceof a
new spotin the neutral fraction as a resultof a typical coldwave procedure
(total immersionnotwithstanding)suggests
formationof lanthionine.
It is known that acid hydrolysisof proteinsdestroysonly tryptophan
by polymerizingits residuewith a prostheticcarbohydrateto form humin.
On the other hand, it is known that alkalinehydrolysisof proteinsdestroys
cystine and hydroxy amino acids,convertsarginine to ornithine, causes
lossof ammonia,and rapidly racemizesall the areinc acids. It is likely,
therefore,that the changesin areinc acid compositionof hair keratin reported here take place not during hydrolysisbut during (alkaline) cold
wave
treatment.
It is proposedthat, duringcoldwaving,the reducingsolutionnot only
convertsdisulfidelinkagesto sulfhydrylgroups,but alsocausesa variety
of reactionsdue to its high alkalinity. Thus, the conversionof cystine(to
lanthionine)and deaminationof acidicareinc acids(glutamicand aspattic
acids)and of basicareinc acids(arginine and lysine) might well account
for the unusualfindingsreportedhere. Evidently thesereactionsare not
quantitative, and considerablymore work will be required to elucidate
completelythe chemistryof the interactionbetweenkeratin and cold
wavingpreparations.
(ReceivedFebruary 7, 1964)
REFERENOES
(1) R. Consden,A. H. Gordon,and A. J.P. Martin, Biochem.
St.,38, 224 (1944).
(2) J. W. Sease,et aL, dnaL Chem.,20, 431 (1948).