Indian Journal of Textile Research

Vol. 9, June 1984, pp. 64-69

Isolation and Characterization of Two Main Sub-component Proteins of Wool

BEHZAD AHMADI

Department of Textile Industries, Tehran Polytechnic University, Tehran, Iran

Received 21 July 1983; accepted 13 December 1983

Treatment of wool with aqueous alkaline thioglycollic acid converts 65% of it into soluble fragments, which are insoluble atpH 4.6 and aggregate spontaneously and irreversibly on storage. Polyacrylamide gel electrophoresis in the presence of sodiumdodecyl sulphate shows that the soluble protein consists mainly of two components. The calibration curves for molecularweightsjelectrophoretic mobilities are presented. The main components from the plasmolysed wool have molecular weights44,000 and 58,000 daltons. Their amino acid compositions have been determined; electron microscopy shows that they formfibrous aggregates.

After the cystine residues in intact wool have beenreduced, the thiol groups may be alkylated to yield S­carboxymethylkeratin (SCMK). At pH 4.4, SCMKyields two fractions: (1) an insoluble, low-sulphurprotein fraction (SCMK-A) containing partly-helicalproteins originating in the microfibrils of wool cortex,and (2) a high-sulphur protein fraction (SCMK-B)containing non-helical proteins originating in theintermicrofibrillar matrix 1,2 .

The starch gel electrophoresis of low-sulphurfraction 3 shows that it contains at least eightcomponents, of which components 7 and 8, havingmolecular weights 51,000-58,000 and 40,000-46,000daltons respectively4, are the most abundant ones.Attempts to isolate these sub-fractions by acombination of chromatography on DEAE-celluloseand gel filtration on Sephadex G 200 (ref. 5) or byfractional precipitation from n-propanol-water andfrom 4,M LiBr (ref. 6) yielded mutually contaminatedfractions.

Another method for extracting low-sulphur proteinis plasQ:lolysis7 • In this method, wool is treated withalkali and a reducing agent, and is subsequentlydisrupted in distilled water and centrifuged. The low­sulphur protein is soluble.

This paper describes the isolation of the low-sulphurprotein fraction by the plasmolytic method, and itsanalysis by electrophoresis in sodium dodecylsulphate-polyacrylamide gels. The two most abundantcomponents, having molecular weights correspondingto those of sub-fraction 7 and 8 of SCMK-A, wereisolated and purified electrophoretically, and theiramino acid compositions were determined. The

protei~ obtained by plasmolysis was treated withtrypsin and the product was fractionated on Sepharose6B. Electrophoresis of the main fraction gave a patternsimilar to that obtained on tryptic digestion of reduced

64

wool (merokeratin)8. Electron microscopy ofplasmolyzed protein revealed that it aggregatesspontaneously into long threads with an average widthof 2 nm.

Materials

Stock solutions were prepared as reported by Weberand Osborn9 and Harrap and Gillespie 7 with slightmodifications.

Stock solution of monomers-A mixture containingacrylamide (222 g/litre, 3.1 M) and N ,N' -methylenebis­acrylamide (6 g/litre; 40 mM) and another mixturecontaining acrylamide (444 g/litre, 6.2 M) and N,N'­methylenebisacrylamide (12 g/litre, 80 mM) wereprepared and kept in dark at 4°C.

Solution A (for reduction)-A mixture containingthioglycollic acid (18.4 g/litre, 0.2 M) and disodiumhydrogen phosphate (14.2 g/litre, 0.1 M) and adjustedto pH II with 2 N sodium hydroxide.

Solution B-Disodium hydrogen phosphate (2.85g/litre, 20 mM) adjusted to pH 10.5 with 2 N sodiumhydroxide.

Solution C-A mixture containing urea (480 g/litre,8 M), 2-mercaptoethanol (20 g/litre, 0.26 M) andsodium dodecyl sulphate (20 g/litre, 70 mM) andadjusted to pH 8.5 with 2 N sodium hydroxide.

Solution D-A mixture containing disodiumhydrogen phosphate (19.9 g/litre, 0.14 M), sodiumdihydrogen phosphate (7.2 g/litre, 60 mM) and sodiumdodecyl sulphate (2 g/litre, 7 mM).

Solution E (for electrophoresis)-A mix.turecontaining disodium hydrogen phosphate (14.2 g/litre,0.1 M) and sodium dodecyl sulphate (10 g/litre, 35mM) and adjusted to pH 8.5 with Nhydrochloric acid.

Staining solution-Coomassie brilliant blue G 250(2.5 g/litre, 3 mM) dissolved in methanol (454 ml) andacetic acid (92 ml) and made up to I litre with water.

.. ~'T·

AHMADI: ISOLATION & CHARACTERIZATION OF TWO MAIN SUB-COMPONENT PROTEINS OF WOOL

Methods

Isolation of low-sulphur protein by plasmolysis­Lincoln wool (2 g) was soaked in solution A (llitre) atroom temperature for 18 hr and then filtered; 33%wool passed into the filtrate. The residual swollen andreduced wool was disrupted in distilled water (20 ml)with a Dounce hand homogenizer and the suspensionwas centrifuged (45,000 x g) for 30 min; 27% wool wasinsoluble and the supernatant contained 32% wool.

Limited proteolysis-A sample of the supernatantwas dialysed against four changes of sodiumphosphate (2 litre, 20 roM) at pH 8 and 6 hr and thesolution was treated with trypsin (40 mg). After 1hr atroom temperature, the trypsin was inhibited by theaddition of phenylmethylsulphonylfluoride (8 ml,0.25% in 50% vol/vol isopropanol-water). The pH ofthe solution was adjusted to 4.6 and the precipitate wascollected in a bench centrifuge. After the solids hadbeen redissolved in sodium phosphate (10 ml, 0.1 M) atpH 8 and the solution had been dialysed againstsodium phosphate (2 litre, 0.1 M) at pH 8 for 2 hr, itwas centrifuged at 25,000 x g for 30 min. The clarifiedliquor was fractionated on a column of Sepharose 6Busing sodium phosphate (0.1 M) at pH 8 as the eluant.

Column chromatography-After the supernatanthad been dialysed against solution B (2litres) for 24 hr,the concentration of protein was measured byinterferometrylo. A sample (5 ml) of the dialysedsolution containing 3mg/ml protein was fractionatedon a column (40 x 2.5 cm) of Sepharose CL-4B, whichhad been treated with solution B for 72 hr. The eluant(solution B)was collected in cuts of 50 drops (approx. 3ml), the absorption being measured continuously at254 nm.

Electrophoresis-Polyacrylamide gel elect'ro­phoresis was carried out by the method of Weber andOsborn 9 with some modifications.

The protein (0.5 mg) in solution C (1 ml) wasmaintained at 60°C for 5 hr and then glycerol (80 JlI)

and bromophenol blue (30 JlI, 1g/litre) were added. Analiquot (50 Jll) of the mixture was submitted toelectrophoresis in 5%gel, with solution E as the buffer,at a current of 9 mA per tube. Subsequently, the gelswere soaked in a solution of trichloroacetic acid (10ml,98 g/litre) for 5 hr, rinsed twice with water, andimmersed in the staining solution (l0 ml) at 35°C for 2hr. Free dye was removed electrophoretically (24 V, 2hr) in 10% acetic acid in the presence of ion-exchangeresin AG 501-X8 (Bio-Rad) in the Pharmacia geldestainer.

Preparative electrophoresis-Replicate samples ofprotein (100 JlI, 1 g/litre) were submitted toelectrophoresis in batches of 12on polyacrylamide gel(7.5%). One gel was stained in the normal way, whilethe others were immersed in the staining solution for

.•.\

.",' \.

10min and rinsed thoroughly with water. This processcaused ring-dyeing of the proteins. Correspondingbands from the ring-dyed samples were cut, combined,homogenized and soaked in aqueous sodium dodecylsulphate (5 ml, 10 gjlitre) at 35°C. After 12 hr, the gelwas spun down in a bench centrifuge. The precipitatewas submitted to the soaking process twice more, andthe combined blue supernatant liquors were dialysedagainst water (2 litre) for 24 hr and then freeze-dried.The freeze-dried material (l mg) was dissolved insolution C (l ml) and the aliquots of the solution (100Jll)were submitted to electrophoresis on 5% gels untilthe much more mobile dye had been separated fromthe protein. The protein was recovered from the geleither by a soaking process or electrophoretically usingan apparatus designed earlier11 • After the position ofthe protein had been determined by staining one of thegels, the corresponding segments (2-3 mm) of theunstained gels were cut and packed (5 or 6 segments)into one end of a glass tube (35 x 7 mm), which wasthen attached to a dialysis sack. The tube was invertedand the space was filled with new 7.5% gel. The proteinwas transferred directly into the dialysis sack byelectrophoresis (100 V, 2 hr) and after it had beendialysed against water (2 litre) for 12 hr, it was freeze­dried.

Electrophoretic mobility as a function of molecularweight-Aliquots of solutions of standard proteins(0.5 mg each) dissolved in solution C (l ml) weresubmitted to electrophoresis in both 5% and 10%gels.The standards were chymotrypsin,. haemoglobin,myoglobin, trypsin, chymotrypsinogen, pepsin,alcohol dehydrogenase, ovalbumin and bovine serumalbumin. The standards of higher moleCUlar weightswere prepared by crosslinking the subunits of alcoholdehydrogenase (yeast, 1 mg) with dimethylsuberi­midate (2 mg) in triethanolamine hydrochloride (l ml,0.2 M) at pH 8.5 and diluting the product with an equalvolume of solution C. The electrophoretic mobility asa function of molecular weight for various proteins isshown in Figs 1 and 2.

Hydrolysis-A solution of the freeze-dried protein(0.5 mg) in constant boiling point hydrochloric acid (1ml) was degassed under vacuum. After the tube hadbeen heated at 105°C for 24 hr, its contents wereevaporated to dryness under reduced pressure. Theresidue was dissolved in water (0.1 ml) and the solutionwas evaporated to dryness as above. This process wasrepeated twice more.

Electron microscopy-A sample of the freshsupernatant was dialysed against four changes of tris­(hydroxymethyl)aminomethane hydrochloride (2 litres,10 mM) at pH 8 for 24 hr. The concentration wasadjusted to I mg/ml and the solution was centrifugedat 25,000 x g for 30 min. One drop of the solution was

65

80

Fresh sam pit'

Aftt'r 3 daysAfter 6 days

After 10days

:II

Molt'cular weights

of compont'nts1-250002-300003-440004-580005-720006-1050007-1500008-2040009-3000006

i•.

I

10 50 60 70TUBE NUM B'ER

Fig. 4-Fractionation of protein in the supernatant on SepharoseCL-4B

50

Fig. 3-Pherogram of a sample of supernatant on 5%polyacrylamide gel

Ec 40"'"NI-et 30

Results and Discussion

Electrophoretic analysis of the freshly preparedsupernatant revealed the presence of at least ninecomponents, two of which were much more abundantthan the others (Fig. 3). Fractionation of the protein inthe supernatant on Sepharose CL-4B yielded twopoorly resolved fractions (Fig. 4), and although itbrought about concentration of the substances of highmolecular weight in fraction I, the components of lowmolecular weight were distributed through all the cuts.Fig. 5 shows the pherograms of the two cutscorresponding to the maxima in Fig. 4 on 10%polyacrylamide gel.

Storage of the dialysed supernatant, before it wasfractionated, caused a progressive increase in theproportion of fraction I at the expense of fraction II,and after 10 days, the proportion of fraction II was

~ 20I­0..a:o:fl 10et

Ly!:oozyme

oC - Chymotrypsinogt'n - A

Alcohol dt'hydrogl'l1ast'{d imt'r)Bovint' St'rum albumin

Alcohol dt'hydrogenast'( trimt'r)

Alcohol dt'hydrogt'nast' (PE'ntamer)

Alcohol dt'hydrogt'nase (tt'tramt'r)

10

9876-..:

5'0

><~ 4~W:;;c:

3

:3 :::lU!:l0~ 2

20

30

109

10 8;:;;7

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~ 4-':::l

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INDIAN J. TEXT. RES., VOL. 9, JUNE 1984

o Ovalbumin

\ Alcohol dehydrogenaseo'0 Pt'psin

\'" - C"'mo',,,.;oogM\o Trypsin

\0 Myoglobin\ Haemoglobin00 Lysozyme

,\Rlbonuclease'f Chymotrypsin0, Cytochromt'-C

o Chymotrypsin.\ I I

0·1 0'3 0·5 0·7 0,9 1-1

MOBILITY

placed on a carbon-coated copper grid, stained with

aqueous phosphotungstic acid (Ii,;) at pH 6, andexamined in a Philips EM 300 electron microscope at100 kV.

0·2 0'4 0'6 0,8 1'0 1·2MOBILITY

Fig. I-Plots of molecular weights of monomeric and crosslinkedoligomeric standard proteins against their electrophoretic mobilities

on 5% polyacrylamide gel

Fig. 2-Plots of molecular weights of standard proteins against theirelectrophoretic mobilities on 10% polyacrylamide gel

66

AHMADI: ISOLATION & CHARACTERIZATION OF TWO MAIN SUB-COMPONENT PROTEINS OF WOOL

(0)

(B)6

components A and B indicates that they are releasedduring the reduction of the wool. To be sure thatcomponents A and B are genuine structural units of theprotein, and that the proteins of lower molecularweights in the primary filtrate (Fig. 3) are not, sampleswere treated in a variety of ways designed to testwhether the extent of aggregation could be altered. Nochange in the pattern on the gel was observed by thefollowing tests: Omission of the reducing agent fromsolution C; a lO-fold increase in the concentration ofthe reducing agent; boiling the solution containingprotein and sodium dodecyl sulphate for 5 min;incubation of the mixture at 50°C for 6 hr; use ofsolutions free from dissolved oxygen; mixing thesupernatant immediately with reducing solution A (20ml) or solution C (20 ml).

The proportion of soluble protein extracted fromwool (which consists mainly of components A and B)was determined gravimetrically after it had beenprecipitated at pH 4.6. The average of fivedeterminations showed 32± 3% protein in theplasmolyzed protein and 33 ±4% protein in thefiltrate. Hence, components A and B together mustconstitute at least 60% of wool.

After the samples of components A and B had beenisolated by preparative electrophoresis, equalquantities of each were submitted to analyticalelectrophoresis. The pherograms showed that the areasunder the peaks were not the same, which means thatthe affinities of these substances for the dye mustdiffer.

The fact that these two components have differentaffinities for Coomassie brilliant blue enabled the ratioin which they aggregate to be determined; theyaggregate in the ratio 1:1 (in preparation).

The amino acid compositions of purifiedcomponents A and B and of the mixture of proteins inthe supernatant are given in Table 2. The helix content,calculated from the data of Davies 14, is 40% for mixedproteins, 52% for component A and 55% forcomponent B. Earlier values, based on themeasurement of optical rotatory dispersion, show thatthe helix content of the low-sulphur protein fraction(SCMK -A) is 50%15 and that of component 8 (whichcorresponds to component B) is 63%. Taking intoconsideration the approximations in the calculatedhelix contents, the concordance of the two sets ofresults suggests that components A and B are in factthe same as the SCMK-A components 7 and 8.

Components A and B have molecular weights verysimilar to those of the two main components ofSCMK-A. So, it is likely that these pairs ofcomponents are in fact the same. Aggregation ofcomponents A and B could not be reversed bytreatments that break disulphide bonds and secondary

•

Molecular weightsof components

1-100002-125003-150004-250005-300006-440007-580008-105000

(Al7

8

·0-'-'­~MObility

Fig. 5-Pherograms of the two cuts corresponding to the maxima inFig. 4 on 10% polyacrylamide gel [(a) fraction I; (b) fraction II]

h"OriginMobility

Fig. 6-Pherogram of soluble protein released during the reductionof wool

negligible. It seems that fraction II undergoes slowaggregation to form fraction I.

The molecular weights of the two main componentsA and B of the soluble protein are 58,000 and 44,000daltons respectively (coefficient of variation ± 4%means of 10 determinations).

The pherogram of the filtrate obtained on thetreatment of wool with solution A shows a series ofbands (Fig. 6), with molecular weights ranging from10,000 to 105,000 daltons. The presence of

67

INDIAN J. TEXT. RES., VOL. 9, JUNE 1984

y

2

1 Molecular weightsof componpnts

1-135002- 155003- 27000

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b.~UeX ~MObility

Fig. 8-Pherogram of the most concentrated cut from the column ofSepharose 68 on 15% polyacrylamide gel

0_10 20 30 40 5060 7075

TUBE NUMBER

Fig. 7-Elution pattern of limited tryptic digestion of protein in thesupernatant on Sepharose 68

~Z 20o~Cl.

gj 10<fl(II<[

daltons and a minor component of molecular weight27,000 daltons (Fig. ·8). These molecular weights arevery similar to those of fragments obtained by thelimited digestion of wool with trypsin 8 • So, it is likeiythat these are t>roduced by' the breakdown of.components A and B. '

E 40c~It>

~30<[

68

Fig. 9-Electron micrograph of protein supernatant (x 40,000 and five times enlarged) [Arrows indicate the twisted ropes]

associative links, indicating that aggregates are heldtogether by covalent links.

Electrophoretic analysis of the main fraction fromthe column of Sepharose 6B (Fig. 7) shows two maincompqnents of molecular weights 15,500 and 13,500

Table I-Amino Acid Compositions (Residues per 1000) of Com­ponents A and 8 and of the Mixture of Proteins in Supernatant

(Values are means of three determinations)

Amino acid Component· Protein

A 8

Nanine 77 72 64Arginine 78 80 83Aspartic acid 106 90 84Cysteinet 39 31 71Glutamic acid 193 162 145

Glycine 56 114 76Histidine 9 10 9Isoleucine 34 37 30Leucine 125 105 95

Lysine 32 41 33M;ethionine 4 3 4Phenylalanine 21 26 31Proline 30 33 50Serine 68 72 80Threonine 49 49 57

Tyrosine 21 12 32Valine 55 63 56

• Moleculfir weights of components A and 8 are 44,000 and 58,000daltons respectively.tComprising cysteine, half cysteine and cysteic acid.

AHMADI: ISOLATION & CHARACTERIZATION OF TWO MAIN SUB-COMPONENT PROTEINS OF WOOL

It has been reported eaJlier8 that a component ofmolecular weight 42,000 daltons, obtained by theoxidation of Merokeratin, could not be broken downagain by reduction. The tendency for irreversibleaggregation noted in the present work suggests that thecomponent of molecular weight 42,000 is formed byaggregation and not by the formation of disulphidecrosslinks. Therefore, it is necessary to take care inidentifying the cause of aggregation.

Electron microscopy of the plasmolyzed proteinreveals that it consists of a pair of loosely twistedfilaments, each of which is about 4 nm thick (Fig. 9);the length of the filament is indeterminate.

Acknowledgement

The author is grateful to Dr P.T. Speakman and DrE.V. Truter for valuable discussion.

References1 Crewther W G, in Proceedings, 5th Int Wool Text Res Con[,

Aachen, Vol I (Technische Hochschule, Aachen) 1975, 1.2 Crewther W G, Fraser R D B, Lennox F G & Lindley H, Adv

Protein Chem, 20 (1965) 191.3 Thompson E 0 P & O'Donnell I J, Aust J bioi Sci, 17(1964) 277.4 Jeffery P D, J Text Inst, 63 (1972) 9l.5 Thompson E 0 P & O'Donnell 11, Aust J bioiSci, 18(1965) 1207.6 Dowling L M & Crewther W G, Prep Biochem, 4 (1974) 203.7 Harrap B S & Gillespie J M, Aust J bioi Sci, 16 (1963) 542.8 Ahmadi B & Speakman P T, FEBS Leu, 94 (1978) 365.9 Weber K & Osborn M, in Theproteins, Vol 1, 3rd edn, edited by

H Neurathand R L Hill (Academic Press, New York). 1975,179.

10 Doty P & Edsall J T, Adv Protein Chem, 6 (1951) 37.11 Ahmadi B, Analyt Biochem, 97 (1979) 229.12 Darnall D W & Klotz 1M, Archs Biochem Biophys, 166 (1975)

65l.13 Davies G E & Stark G R, Proc natn Acad Sci USA, 66(1970) 651.14 Davies D R, J molec Bioi, 9 (1964) 605.15 Harrap B S, Biopolymers, 8 (1969) 187.16 O'Donnell I J, Aust J bioi Sci, 22 (1969) 471.

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