Enzymatic reduction of disulfide bonds in fibrin-o gen by the thioredoxin system I. Identification of reduced bonds and studies on reoxidation process

THROMBOSIS RESEARCH Printed in the United

Vol.

States

ENZYMATIC REDUCI'ION OF DISULFIDE BONDS IN FIBRIN-Om

4, PP* 55-75, 1974 Pergamon Press, Inc.

BY THE THIOREDOXINSYSTEM

I. IDENTIFICATIONOF REDUCED BONDS AND STUDIES ON REOXIDATIONPROCESS

Birger BlombPck, MargaretaBlomback,1Wolfgang Finkbeiner,Arne Holmgren, Barbara Kowalska-Loth and Gunilla Olovson ChemistryDepartment,Karolinska Institutet,Stockholm,Sweden and The New York Blood Center, New York, N.Y.

(Received

6.11.1973.

Accepted

by Editor H.A. Scheraga).

ABSTRACT

Disulfides in human fibrinogenor fibrin were reduced by the NADPH-dependent reduction system consistingof thioredoxinand thioredoxinreductase from Escherlchia coli. In this system a fast and selective reduction of only 5 out of a total of 28 dlsulfides in fibrinogenoccurred. The position of the susceptiblebrid s were located in reduced fibrinogenafter carboxymethylation with % -1odoaceticacid. Three of these bridges (Aa 28-Aa 28, y8-y8 and yg-y9) were symmetricaland located in the NH2-terminalportion of fibrinogen.One additionaldlsulfide was present in an internal part of the Aa-chain. The usefulness of the thioredoxinsystem as a "chemicalprobe" for surface oriented disulfides was demonstratedsince the bonds reduced were all In hydrophilic regions of fibrinogen.On reoxidatlonof reduced fibrinogenor fibrin highly cross-linkedpolymers were formed by disulfide exchange. Since human platelet extracts were found to possess a thioredoxin-like activity which in the presence of calf liver thioredoxinreductase could reduce insulin or fibrinogen,such a reduction system may play a role in physiologicalcross-linkingof fibrin.

INTRO~CTION Fibrin formationIs initiatedby limited proteolysisof fibrinogenby thrombln (cf.1).The binding sites involved in the initial polymerizationprocess have until recently been largely unknown. Recent studies with insolubilized fibrin monomer and a NH2-terminalfragment (N-DSK) of fibrin suggest that plasmlc fragmentD and N-DSK contain complementarystructures involved in align' Present address: InstytntBadafiJadrowych,Osrodek Zerafi,Ulica Dorodna 16, Warszawa 9, Poland. 55

56

FIBRINOGEN

REDUCTION-OXIDATION

Vol.4,No.l

ment of activated fibrinogen (2, 3). The initially formed polymer is subsequently cross-linkedby introductionof r-glutamyl - <-aminolysinebonds between chains of fibrin (cf.4). It has recently been suggestedthat disulfide exchange may play a role in polymerizationof fibrinogen (5). A disulfide exchange process could possibly be initiatedby a reductase enzyme. One such system which has been described is the thioredoxinsystem which was originallydiscovered in Escherlchiacalf (6, 7) and has subsequentlybeen identifiedin yeast (8, 9) and calf liver (10). The thioredoxlnsystem of E. coli consists of the small hydrogen transportprotein, thloredoxln (ll), and the flavoprotein,thioredoxinreductase.This system is capable of affecting pyridine nucleotide dependent reductionof exposed disulfidesby a combinationof reactionsI and II as shown below: thloredoxin reductase 2) Thioredoxin-S2 + NADPH + H+I *thioredoxin-(SH)2 + NADP' I. II. Thioredoxin-(SH)2+ X-S2 ,->

Thloredoxin-S2+ X-(SH)2, where X repre-

sents a disulfide containingcompound. The present report is the first in a series of studies on the influence of thioredoxinreductaseand thioredoxinon fibrinogenand fibrin. MATEFUArS

ANDMETHOIB

Enzymes and various reagents. Glutathionereductaseand lipolc acid dehydrogenase were obtained from Boehringer,Mannhelm, Germany.Nicotinamideadeninedinucleotldephosphate (NADPH)and 5-5'-dithlobls-(2-nltrobenzoic acid)(m) 35 3 were products of Signa, St. Louis, MO.. H-iodoacetlcacid and thiocyanatewere purchased from RadiochemicalCentre, Amersham, England. Cyanogen bromide was from Schuchardt,Minchen, Germany.Sephadex and Sepharose were from Pharmacia,Uppsala, Sweden. Acquacide I was from Calbiochem,Los Angeles, Ca.. All chemicalsused were of analytical grade. Apparatus.Beckman liquid scintillationcounter,Beckman 200, Palo Alto Ca.; Thin-layer scanner,Berthold, Wildbad, Germany; Fraction collector,UltroFlac 7COC, LXB-ProdukterAB, Stockholm,Sweden; Ultraviolet analyzer and recorder, ISCO UA-2, Lincoln, Neb.; Thin-layer electrophoresisapparatus,Desaga, Heidelberg, Germany; Polyacrylamidegel electrophoretlcapparatus,Shandon Scientific co., London, England; Ultraviolet spectrophotweters, Hitachi 101, Tokyo, Japan and.for kinetic enzymatic analysis Zeiss FM QII, Oberkochen,Germany;Amino acid analyzer,Technicon autoanalyzerTSM-1, Chauncey,New York, N.Y.; Centrl2

Footnote:Thloredoxin-S2and thioredoxln-(SH)2representthe oxidized and reduced form of thioredoxin,respectively.

Vol.4,No.l

FIBRINOGEN

REDUCTION-OXIDATION

57

fugs, MSE Super Minor Centrifuge,London, England. Fibrinogen (Fractionl-2) was prepared from human plasma as previously described (12) and had a coagulabilityof 94 - 97 $. Fibrin,

non cross-linked,was obtained from fibrinogen (5 mg/ml) in 0.15 M tris-

HCl buffer, pH 7.2, by treatment with thrombln (1 NIH unit/ml) for 2 hours at 20-22'~. In some experimentsthe fibrinogenwas clotted in the presence of EDTA (0.003 M). After extensivewashing the clots were dissolved in 0.15 M tris-HCl buffer, pH 7.2, containing4 M urea to a concentrationof 1 k (w/v) and stored until further use at -7O'C. Before use the fibrin solution was diluted with an equal volume of 0.15 M tris-HCl buffer, pH 7.2. No y-y dlmers or poly a structures were detected in reduced and alkylated fibrin by polyacrylamidegel electrophoresis in presence of sodium dodecyl sulfate. NH2-terminalamino acid analysis of the flbrlns showed that tyroslne and glyclne were the only amino acids and their molar ratio was 1:2, indicatingremoval of fibrinopeptidesA and B. Platelets.Blood was collected in ACD-solution (8.5 ml blood + 1.5 ml ACD-solution). The plateletswere harvested from 90 ml of platelet-richplasma by centrifugstlonat 2.200 g for 20 min at +4'. The pellet was resuspendedand washed three times In 0.13 M NaCl containing0.02 M trisodlum citrate.After washing the plateletswere suspended in 2 ml of the same solution as to give a platelet 6 count of around 125 x 10 per rmn3.On addition of ADP, the platelets showed the typical aggregationbehavlour.The plateletswere disintegratedin 2 ml of 0.10 M tris-HCl-0.002M EDNA buffer, pH 8.0, by either freezing - thawing three times or in a Potter-Elvehjemtube using 3C strokes with the teflon pestle. The homogenates were centrifugedat 2.200 g for 20 minutes and the supernatantswere withdrawn and tested for thioredoxinand thioredoxinreductase activity as described below. Rabbit anti HI2-DSK was prepared by foot-pad injection of the antigen as previously described (13). Thioredoxin from Escherichia coli B was prepared by method 2 as described previously (14). The preparationwas homogeneous.Thioredoxln reductase from E. _B3)

was a preparationobtained as previouslydescribed (15). Thloredoxin

reductase from Calf liver was a preparationwhich had been purified 500-fold by chromatographyon DEAE-cellulose,TEAE-celluloseand Sephadex C-200 (16). this Preparationwas essentiallyfree Of glutathionereductaseand lipoic acid dehydrogenase activities. 3

Footnote:This preparationwas a kind gift of Dr. Lars Thelander,Dept. of Biochemistry,Karolinska lnstltutet,Stockholm,Sweden.

58

FIBRINOGEN

REDUCTION-OXIDATION

Vol.4,No.l

Free sulfhydryl groups in fibrinogenor fibrin were determinedby the use of 55'-dlthiobis-(2-nltrobenzoic acid) (DTNB) with the exception that all solutions in the assay contained2 M urea (17). The number of dlsulfides reduced in fibrinogenor fibrin was determinedby following the disappearanceof NADPH by reading the change in absorbanceat 340 nm with time. As seen from reactions I and II (page 2), one mole of NADPH corresponds to the reductionof 1 mole of disulfide.From the total change In absorbance at 340 nm the number of disulfides reduced was calculatedusing the molar extinctioncoefficientfor NADPH of 6.2 x 103. Each of two cuvettes contained in a final volume of 0.500 ml of 0.15 M tris-HCl-0.001M EKYPAbuffer, pH 7.2 -4 the following:NADPH at a final concentrationof 2.1 x 10 M and 0.5-5 mg of fibrinogenor fibrin. To one cuvette was added thioredoxinto a final concentration of 1.0 x lo+ M and this cuvettewas used as blank in the spectrophotometer. -8 After zero time reading of the absorbanceat 340 rmu1.5 pg (4 x 10 M) of thloredoxin reductase from E. coli was added to both cuvettes and the enzymatic reaction was followedat 340 nm. Alkylation of free thiol groups after reduction in the thloredoxinsystem was carried out In an atmosphere of N2 for 30 minutes at room-temperaturewith 'Hiodoaceticacid containingcarrier (20 mCl/mmole) in a lo-fold molar excess over free SH-groups.The pH was adjusted to 8.6 - 9. The reaction mixture was about 20 ml. After alkylationan equal volume (20 ml) of glacial acetic acid was added and excess reducing agents and iodoaceticacid were removed by gel filtrationon Sephadex G-50 (3 cm' x 72 cm) equilibratedwith 50 $ acetic acid. The protein peak was freeze-dried.Other alkylationsmentioned in text were according to previouslypublished procedures (19). Thioredoxinactivity in platelet extracts and column fractionswas assayed by coupling the reduction of thioredoxinto the reduction of disulfides in lnsulin(or fibrinogen)(11). The incubationmixture had a volume of 0.14 ml. It contained 0.25 mg of bovine Insulin, 0.6 pole of MTA, 10 poles of N-2-hydroxyethyl plperazine-N'-2-ethane-sulfonlc acid buffer, pH 7.6,

0.11 pmole of NADPH

plus an allquot of platelet extract. The reaction was started by addition of 10 pg of thloredoxlnreductase from calf liver and the mixture was incubatedat 37' for 15 minutes. The reactionwas interruptedby addition of 0.50 ml of 6M guanidine hydrochloride-0.05 M tris-HCl buffer, pH 8.0;contalnlng 0.20 mg of DTMB and the sufhydryl group content was determinedby reading the absorbanceat 412 nm. An Incubationmixture without thloredoxinreductasewas used as blank. Fibrinogendeterminationwas performedby measuting ultravioletabsorbtionat " - 16.53 (19). 280 nm in alkaline urea, El_ Imrmnoelectrophoresls was according to a previouslydescribed technique (20).

Vol.4,No.l

FIBRINOGEN

REDUCTION-OXIDATION

59

NJ2-tennina analysis was performed using the phenylisothlocyanatetechnique of Edman as previouslydescribed (19, 21, 22). Amino acid analysis. Polyacrylamidegel electrophoresis(PAA) without and with sodium dodecyl sulphate (SDS), and Peptide mapping were according to techniques described in detail in previous publications(19, 22, 23, 24, 25).

RESULTS Enzymatic reduction of dlsulfide bonds in fibrinogenand fibrin. The NADPH dependent reduction of fibrinogenand fibrin in the presence of E. co11 thloredoxinreductaseand thioredoxinIs shown in Fig. 1. Under the conditionsof these experimentsa fast reduction of about 5-6 disulfide bridges of a total of 28-29 (26)

in fibrinogenoccurs within 20 minutes. Within experi-

mental error the same results were obtained both with the NADPH oxidationmethod and by direct determinationof free SH-groups.There was only a slight dlfference in the number of bonds reduced when the reduction of fibrinogenwas perfor-

FIG. 1 NADPH oxidation by fibrinogenin the presence of the thloredoxinsystem. Fibrinogen (1 mg/ml) in 0.15 M tris-HCl - 0.15 M NaCl buffer, pH 7.2, e---_--e; fibrinogen (1 mg/ml) in 0.15 M tris-HCl - 0.15 M NaCl - 2M urea, pH 7.2,x-x; fibrlnoepen cleaved with CNBr (1 m&l) in 0.15 M trls-HCl - 0.15 M NaCl, pH 7.2, -. The inCUbatiOn mixture contained 2 x 10-4 M NADPH. To the fibrinogenor fibrin cuvette was added thioredoxln-S2to a final concentrationof 1.0 x 10-5 M. The reaction was s arted by addition of thioredoxin reductaseto a final concentrationof 4 x 10'Q M.

60

FIBRINOGEN

REDUCTION-OXIDATION

Vo1.4,No.l

formed in the presence of 2 M urea. This concentrationof urea is known not to interferewith the action of the enzyme (18). In fibrin dissolved in 2 M urea about the same number of disulfide bridges as in fibrinogenare rapidly reduced by thioredoxin-(SH)2. In one experimentcyanogen bromide treated fibrinogenwas used as substrate for the enzyme system (Fig. 1). In this case between 76 and gC $ or between 22 and 25 of the totally available disulfide bonds were reduced.This shows that steric factors in the structureof fibrinogento a large extent determines the specificityof the enzyme system. Similar experimentswith fibrinogenin the systems glutathione-reductase-glutathioneand lipoic acid dehydrogenase- lipoic acid resulted in only a slow and small degree of reduction,under conditionswhich give extensive reduction of glutathioneandlipoic acid, respectively.In view of these results all further experimentswere performedwith the thioredoxinsystem. Identificationof reduced bonds. Fibrinogenand fibrin was reduced with the thioredoxinsystem for 20 minutes at room-temperature(cf. Fig. 1). Reduction of fibrinogenwas performed both with and without addition of urea (2M). Fibrin was always reduced in 2 M urea in order to keep it in solution. In order to obtain Informationon the na-

3 E = 0.4

-4

El Ly

0.3

-3

2 E

: s?

0.2

-2

g U

0.1

-1

0

v)

m

a

TUBE

NUMBER FIG.

(4.5 ml tractions)

2

Separation on Sephadex G-100 of CNBr-cleavageproducts of partially reduced fibrinogen.For details of reduction compare text and Fig. 1. For alkylation lOmoles of lodoaceticacid per mole of SH-groups were used. (comparereference 19). Cleavage with CNBr was as described previously (compare reference19). The column (5 cm2 x 92.5 cm) was equilibratedand developedwith 10 $ acetic acid. Flow rate: 22 ml/h. The sample (110.3 mg) was applied in 5.5 ml of 10 $ acetic acid. The fractionswere pooled as indicatedand lyophillzed.Yield of material In fractions:1, 24.0 mg; 2, 13.1 mg; 3, 16.7 mg. Adsorbance at 280 run -; radioactivity-------*

Vol.4,No.l

FIBRINOGEN

61

REDUCTION-OXIDATION

ture of the bonds reduced, the SH-groups,appearing after reduction,were alkylated with 'H-labellediodoaceticacid. The alkylated protein was subjectedto cleSvgl_

with cyanogen bromide in formic acid (19). The cleavageproducts were

separated on.SephadexG-100 (Fig. 2). The radioactivityoccurred in three

diS-

tinct.peaks.'Thesewere pooled and lyophili~ed. Peak I had approximatelythe same location (Kav = 0.15) on the chrcmatogram as the NR2-terminal"disulfideknot" (N-DSK),which contains the NH2terminal fragmentsof the Aa,

E@ and y-chains of fibrinogen (5, 19, 22). The

material In this peak was therefore mixed with purified N-DSK (22) and the mlxture was completelyreduced with dithiothreitoland afterwardsalkylated (19) with non-radioactive("cold") iodoacetlcacid. The fragmentsin the mixture were then separated on Sephadex G-100 (Fig. 3).

The radioactivityprofile of the

chromatogramas comparedwith that of the W-absorbance profile clearly indicated that disulfide bonds involvingthe Aa and y-chain of N-DSK had been reduced. These profiles for fibrinogenand fibrin were essentiallythe same. It should, however, be noted that the radioactivitypeak of the A&chain from fibrin (not shown In the figure) is somewhat shifted towards higher elution volumes which

is to be expected, since this chain fragmentdoes not contain fibrinopeptideA. The ratio of the radioactivityof Aa

or a-chain to y-chain In both fibrinogen

and fibrin was approximately1:2. This shows that, on the average, for every

g 0.08d g c 0.08f g 0.04-

TUBE

NUMBER

(2.0ml fractions)

FIG. 3 Separation on Sephadex G-100 of carboxymethylatedN-DBK. Tc 7.9 mg of N-DSK wa6 added 2.3 mg of Peak I material (cf. Fig. 2). The mixture was reduced, all@ated as described in reference19. The column (1.8 cm2 x 92 cm) was equilibrated and developedwith 10 $ acetic acid containing5 $ ethanol. Flaw rate: 7 ml/h. The sample (10.3 mg) was applied in 0.5 ml. The fracticnswere pooled a8 indicated and lyophilized.Adsorbance at 280 nm i radioactivity--------.

62

FIBRINOGEN

Aa 280Aa 28 bond reduced

REDUCTION-OXIDATION

both the ~898

and the yg-yg

Vol.4,No.

must also

1

have been clea-

ved. The smaller mdioactive peak eluted close to the void volume has about .. the same, Kav as the disulfide containing peptlde Hi2-IBK (see below page 10) and therefore

most likely

represents

The bonds being

reduced

in the peaks

(cf. Fig.

freeze-dried

and then subjected

nal peptlde and r-chains, ly published

spot

bonds in the y-chain, in the y-chain

For this

the Aa and y-chain digestion,

development

followed

radioactive

tryptic

chain

bond In the AU-chain respectively

was also

(cf.

confirmed

Fig.

were pooled,

by two dimenslomaps of the Aa

agreement fm@nents.

peptldes

material

maps they were stai-

The peptlde

22, 25) of these

in Peak I.

purpose

fm@nents

of the peptide

radioactivity.

peptlde

were those

wi,th previousIn both

fibri-

involving

the

and the No. 8 and 9 symmetri9).

The identity

by amino acid

analysis

of the tryptlc of the eluted

(25). Peak 2 (cf.

half

disulfide

identified.

to tryptlc for

of the latter

4a and 4b, are in complete

maps (19,

the only

No. 28 symmetrical

peptide

After

and scanned

peptlde

were next

containing

shown in Figs.

nogen and fibrin

cal

3)

mapping (25).

ned with ninhydrin

contaminants

Fig.

of the radioactivity

2) from both of Peak I.

fibrinogen

The radioactive

and fibrin, material

contained

roughly

of Peak 2 has

FIG. 4 Tryptlc peptide maps of chains of N-DSK. Tryptic digestion and thin-layer electrophoresis chromatography was carried out as described in reference 25. Of the The plates were scanned for radioactivity and digest 0.4 - 0.5 mg was applied. stained with ninhydrin. Yellow spots are marked ‘y”. Dotted circles Indicate weaker spots. Hatched circles Indicate radioactive spots. The vertical repeated scans over the plates are inserted. CPS = counts per second.

FIBRINOGEN

Vol.4,No.l

REDUCTION-OXIDATION

63

about the same effluent volume as Hol-DSK which is one of the major disulfide knots of fibrinogen(13,27)(cfFig.9). Peak 2 material (1.9 mg) was, therefore, added to purified Hol-DSK (7.2 mg) and the mixture reduced and alkylated with '(cold'iodoacetlcacid as describedabove (see legend Fig. 3). Gel filtration on Sephadex C-100 (1.8 cm2 x 92 cm) revealedthat the radioactivematerial had a lower K,,-value than the largest chain of Hol-DSK. It must thus be concluded that the thioredoxin-(SH)2susceptiblebond in Peak 2 is not part of Hoi-DSK. In order to further characterizethis peptlde, 4.6 mg of Peak 2 was subjectedto counter-currentdistribution(22). One radioactivepeak with low K-value (about 0.1) and one with hi& K

( about 6) was obtained.The latter

peak correspondsto Hol-DSK. It does not contain appreciableamounts of radloactivity and was therefore not further analyzed. The radioactivepeak was purified by gel filtrationon Sephadex G-100 (for details see legend to Fig. 3). The main radioactivepeak (85 k) had a K,, of 0.2. A smaller peak (15 %) appeared to consist of N-DSK and was not further studied. The main peak was essentiallyhomogeneous on polyacrylamidegel electrophoresis(Fig. 5a). The mobility was different from all hitherto known disulfide containingpeptides of CNBr-treatedfibrinogen (13, 27). Polyacrylamidegel electrophoresiswith SDS gave a molecularweight of 28,COC both before and after reductionwith mercap-

a

b FIG. 5

Polyacrylamldegel electropherogramand peptide map of HI2-DSK. a) Electrophoresis.The sample (50 )Ig)was applied and run In 5 $ acetic acid for 9C min. Gelmatrix:7.5 8. Staining: Coomasie blue. For further details see text and references23 and 24. b) Peptide map of tryptlc digest. Of the sample 0.4 mg was applied. For further details see legend to Fig, 4.

64

FIBRINOGEN

REDUCTION-OXIDATION

Vol.4,No.l

toethanol. Glutamic acid was found to be NH2-terminalresidue in the peptide. Paptide mapping of a tryptic digest of the peptide showed 12 ninhydrin positive spots (Fig. 5b). Sosnning of the plates for radioactivityrevealed that two .. spots containedapproximatelythe same amount of radioactivity.A third radioaotive spot containedonly smaller amounts of radioactivitywhich most likely representedcontaminantsof N-IXSKin the peptide. The results show that the thioredoxln-(SH)2susceptiblepeptlde In Peak 2 consists of a single polypeptide ohain with at least one lntrachaindisulflde bridge. Since this disulfide containingpeptlde has not been previouslyobserved in CNBr-treatedfibrinogen 4). we denote it as Hi2-IXSK Non-reducedHIP-DSK has In addition been isolated from CNBr,-treated flbrlnogen.After reductionand alkylationthis peptide has essentiallythe same electrophoreticmobility and on tryptic digestion produced the same peptide map as Hi2-DSK isolated from thioredoxin-(SH)2reduced fibrinogen.The molecular weltit (PAA-SDS)was also the same. The COW-terminal residue was homoserine whleh shows that HI2-DSK is not carboxyterminalin fibrinogen.Its amino acid compositionconfirmedthe presence of one disulfide bond and presented some interesting additional features inasmuch as it containeda high proportionof glyclne and proline and in this respect resembles collagen,resilin and elastln (28). It has recently been shown that an apparently identicalpeptlde (carboxymethylatedHi2-DSK) can be isolated from CNBr-treatedA-chain of human fibrinogen (a). This Indicatesthat HI2-DSK is located in the AC&chain. Further support for this localizationcomes fraa the results of invnunoelectrophoresis. Anti Hl2-DSK reacts with AC&chain but not with R@ and y-chain of fibrinogen.It should be mentioned that anti Hl2-DSK also reacts with whole fibrinogen. Since Hi2-ASK.is a monomeric structure the question arises whether one or two Hl2-DSK are reduced in the Mbrinogen dimer. The amount of radioactivity of Peak 2, which is predominantlyassociatedwith Hi2-DSK, support the conclusion that on the average for every reduced Aa 28-Aa 28 bond one disulfide in each of two Hi2-DSK Is also split (Pig. 9). Thls conclusionIs deduced from the fact that the relative radioactivityof Peak 2 appears to be at least twice as hi& as the radioactivityof the AC&chain in Peak I, which contributesto about 30 $ of the radioactivityof Peak I (see separationof chains shown in Fig. 3). 4

Footnote: Prevl~uslydescribeddisulflde containing peptldes are : N-DSK (Hll'DSK),l-DSK, Ho2-DSK and Ho3-DSK. Ho and Hi denote the hydrophobicand hydr0pMl10 nature, respectively,of the peptides during counter-currentdistrlbutlon. Previouslyused names for Hol-Ho)-DSKwere Hl-H3-DSK (13, 27).

FIBRINOGEN

Vol.4,No.l

REDUCTION-OXIDATION

65

The Kav-value of Peak 3 (cf. Fig. 2), which contributesto about 10 $ of the total radioactivityin the chromatogramindicatedthat thioredoxin-(SH)2 susceptiblebonds might also be located in other disulfide containingregions of fibrinogen.Polyacrylamidegel electrophoresisof Peak 3 material showed that the radioactivitywas mainly associatedwith three bands and to approxlmately the same extent in each. The mobilities of these bands do not exclude the possibilitythat other bonds involvingAa-chain of N-DSK, chains of Hol-DSK, Ho2- and Ho3-DSK have also been partially reduced. Propertiesof reduced fibrinogenand fibrin. In order to study some propertiesof fibrinogenreduced In the thioredoxln system it was separated from the reactionmixture by gel filtration.In a typical experiment25 mg of reduced fibrinogen (see legend Fig. 1 and Methods) in 5 ml of solution was applied on a Sephadex G-50 column (1.8 cm2 x 92 cm) equilibrated with 0.15 M tris-CHl buffer, pH 7.2,at a flow rate of 20 ml/h. In order to avoid unintentionalreoxldationduring the separationprocess all buffers were flushed with N2 and deareated several times and kept during the whole procedure under N2. The gelsuspensionwas also flushed with N2 and deareated before pouring into the column filled with N2. The column was equilibratedwith at least one bed volume of equilibrationbuffer before use. A sample (7-10 ml) of the protein peak appearing at the void volume was collectedin a centrifugetube under N2*

In order to avoid contaminationwith thioredoxin,only the first 2/3 of the

TABLE I Number of disulfide bridges per mole reduced fibrinogenand fibrin. Reduction time: 20 minutes. For further details see text and experimentalsection. Fibrinogen lADPH SH-groups/2, lssay before after column column

Fibrinogenin 2M urea NADPH SH-groups/2, assay before after column column

Fibrin in 2M urea NADPH SH-groups/2, assay before after column COlumI

5.0

5.5

4.8

6.5

7.5

6.8

5.5

5.5

4.0

7.0

6.5

5.5

5.8

6.3

5.5

5.5

5.5

4.5

5.5 6.0

6.0 6.0

5.0

4.5

6.5

5.5

5.5

7.0

5.8

5.0

6.0

4.8

4.0

5.0

4.5

5.5

6.5

5.5

5.2

5.9

4.9

lean: 5.9

6.0

5.1

5.6

6.7

5.9

66

FIBRINOGEN

REDUCTION-OXIDATION

Vol.4,No.l

peak was used. Preliminaryexperimentshas shown that thioredoxinstarted to elute at elutlon volumes correspondingto the descendingpart of the void volume peak. The protein concentrationin the pooled fractionwas between 1.5 and 2 mg per ml. Fibrinogenand fibrin in 2 M urea solution were also reduced and isolated as describedabove with the exceptionthat all buffers contained2 M urea. The number of disulfides reduced in fibrinogenand fibrin is shown in Table I. It Is evident that there is no significantdifference in number of reduced bonds in the two proteins. In the presence of 2 M urea essentiallythe same or possibly slightly higfiervalues were obtained.The number of SH-groups in the reaction mixture before gel-filtrationwas, as would be expected, somewhat higher than after removal of reagents. The reactivitywith thrombln (2.5 NIH units/ml) of reduced fibrinogen (0.17 $) in 0.15 M tris-HCl buffer, pH 7.2, was determined in one experiment. Before reductionthe fibrinogenat the same concentrationclottedwithin 19 and 24 seconds.After reductionno clot was obtained within ZO minutes. In order to determine the molecular size of reduced fibrinogenand fibrin they were filteredthroua Se&arose 4B columns in 4 M urea solution (Fig. 6). Both proteins had the same K,,-value as non-reduced fibrinogen.These experiments indicatedthat under these conditionsreduced fibrinogenand fibrin do

a

C

b

E gj

1.0

0.1

0.1

P z P 4:

0 20 30 40 5 TUBE NUMBER (l.Sml fractiis)

FIG. 6 Separation on Sepharose 4B of fibrinogen,reduced fibrinogenand fibrin.After reduction excess reagents and thioredoxlnwere removed by gel-filtrationon Sephadex G-50 (see text). The reduced sample was then applied to a Sepharose column (1.8 cm2 x 51 cm), equilibratedand developedwith 0.15 M tris-CHl buffer, pH 7.2, containing4 PIurea in an atmosphere of N2. a, fibrinogen (10 me/ ml), 1 ml applied; b, reduced fibrinogen (1.7 mg/ml), 0.5 ml applied; c, reduced fibrin in 2 M urea (0.7 m&ml>, 1 ml applied.

FIBRINOGEN

Vol.4,No.l

REDUCTION-OXIDATION

67

not dissociate. Polymer formation. Reduced fibrinogenor fibrin (the latter always in 2 M urea) are easily reofidisedon exposure to oxygen. Concomitantwith reoxidationformationof : intermediateand highly cross-linkedPolymers occurs. Reduced fibrinogenor fibrin was prepared as describedabove. The centrifuge tube containing7-10 ml of protein solution (1.5to 2 mg/ml) was provided with a magnetic stirrer and gently flushed with O2 during stirring for 20 hours. Samples were taken before and at different intervalsduring the reoxidation. Table II shows the change in concentrationof soluble fibrinogenand the disappearanceof free SH-groups during reoxidation.Free SH-groups disappear

TABIE II Free SH-groups and fibrinogenconcentrationduring reoxldationof reduced fibrinogen. For details see Text and Methods. All samples after reoxidationwere centrifugedto remove insolublematerial.The supernatantwas analysed for free SH-groups and protein concentration.

Fibrinogen, nmoles/ml Reduced fibrinogen after gel filtration.

SH-groups, nmoles/ml moles/mole

5.5

57.0

10.5

After

1 hour

5.1

10.0

2.0

reoxi-

2 hours

5.0

4.6

0.9

dation

3

-(I-

4.6

2.0

0.4

-1' -

0.3

0

0

for

20

rapidly. Fibrinogen is convertedto insolublepolymers at an appreciablyslower rate. However, small amounts of visible thread-likeaggregatesappear already after 5 minutes. After 20 hours of reoxidationless than 10 $ of the fibrinogen remains in solution. The polymerizedprotein (lo-20 mg) formed after 20 hours, was removed by centrifugationand washed for 60 minutes; once with 2 ml of 0.15 M trls-HCl buffer, pH 7.2, which did not remove appreciableamounts of protein, and twice with 2 ml of the same buffer containing6 M urea. The insolublepolymers constituted 70 - 80 $ of the fibrinogenoriginallypresent before oxidation.When fibrinogenor fibrin in 2 M urea solutionwere reduced and oxidlsed as descrlbed above the disappearanceof SH-groupswas slower and the yield of Insoluble

68

FIBRINOGEN REDUCTION-OXIDATION

Vo1.4,No.l

polymersafter 20 hours of reoxldation was also lower. The proteinleft In the supernatant after removalof the insolublepolymer&were concentrated (by dialysisagainstAcquacide)and subjectedto chromatography . on Sepharose4B (cf.Mg. 6). In additionto a peak with the same Kav as fibrinogen, a void volumepeak of varyingsize appeared.The lattermost likelyrepresentssolubleintermediate polymers.Therewere more intermediate polymerspresentwhen reductionand reoxldatlon had been performedin 2

M

urea

solution. Pronertiesof Insolublepolymers. The insolublepolymers(about 10 mg) were suspendedin 2 ml of 8 M urea (or 8M guanidlnium HCl) in 0.05 M trls buffer,pH 7.2, and stirredfor 24 hoursat room-temperature. Proteindetermination on the supernatants after centrlfugation showedthat about 20 $ had dissolved.The Insolublepelletwas next suspendedin 8 M guanidinium HCl in 0.05 M tris buffer,pH. 7.2, and treatedas describedabove.About 30 $ of the proteinwent into solutionin this solvent, The remainingpolymers(about5 mg) dissolveeasilyin 8 M guanldlniwn HCl on reductionwith dithlothreltol (3). Polyacrylamide gel electrophoresls of reduced and alkylatedpolymersfrom fibrinogenis shown In Fig. 7a and b. It is evident that the Aa, Bj3and y-chainsof fibrinogen are releasedon reductionand that no y-y dimersor Aa-chain polymers,typicalfor FactorXIII catalyzedcrosslinkingare present.This experimentgivessupportfor the conclusion that a dlsulfideexchangemechanismIs Involvedin polymerformation. FIG. 7 Polyacrylamide gel electrophoresis of reducedand alkylatedpolymersfrom fibrinogen.a) reducedand alkylatedsamples run in 5 $ aceticacid for 90 min. Gelmatrix:7.5 $. b) reducedand alblatedsamplesrun in 0.1 M phosphate buffer,pH 7.0, containing 0.1 $ SDS. Gelmatrix:7 $. Left: polymerformedon reoxidation of reducedfibrinogen. Right: fibrinogen. Staining:Coomasleblue. For furtherdetails see text and references23 and 24.

Thioredoxin activityof platelets. It appearsthat human plateletscontaina thloredoxin-like substance which in the preeence of calf liverthloredoxinreductaseand NADFH can reduce insulin(TableIII),Inorderto determinewhetherthe thioredoxln-like activity was released by thranbin an aliquot of a platelet preparationwas incubatedwith

Vol.4,No.l

FIBRINOGEN

REDUCTION-OXIDATION

69

TABLE III activity was determinedby Thloredoxinactivity of human platelets.'Phioredoxin the insulin-coupledassay. All tubes containedan incubationmixture with the additions specified below in a final volume of 0.14 ml. For experimentaldetails see Methods. Additions

mo!?$fzact

Platelet extract + thioredoxin reductase (calf liver) 10 ).'g

3.0

Platelet extract

0.0

Thioredoxln reductase (calf liver) 10 pg

0.0

Supernatantof platelets incubated with thrombin + thioredoxin reductase (calf liver) 10 rg

0.1

bovine thrombin (20 NIH units/ml for 5 hours). No release of thioredoxinactivity In the supernatantoccured.The results of these experimentsare shown in Table III. In an experimentunder the same conditions,except for replacement -5 of insulin with fibrinogen (concentration:2.4 x 10 M), the platelet extract was found to reduce 1.5 disulfidesper mole of fibrinogen.Further evidence for the presence of thioredoxin-likesubstancesin plateletswas obtained by chromatography of the crude platelet extract on a column of Sephadex G-50 (Fig. 8). The material having reducing activity in the presence of calf liver thioredoxin reductasewas eluted with a Kav-value of approximately0.5 which is the same value as determined for thioredoxin (m.w. 12,OCC). No evidence was obtained in

4

FIG. 8

A

1.2 -

-06

: :,

E c

:

4

I

6 6 10 12 EFFLUENT VOLUME

ts 5c 52 -O.L( rt Z$ 8g go -020: z-u

14 16 (ml 1

Chromatographyof crude platelet extract on a column of Sephadex G-50. The column (0.6 cm2 x 32 cm) was equilibrated with 0.10 M tris-HCl-0.002M EDPA, pH 8.0. The sample (1.0 ml) of crude platelet extract was applied and fractions of 0.50 ml were collectedat a flow late of 2 ml/h. The protein content of fractionswas determinedby reading the absorbanceat 280 nm lhioredoxinactivity xd was determined on 100 ~1 aliquots of the fractions with the insulin coupled assay. For further details see methods.

70

FIBRINOGEN

REDUCTION-OXIDATION

Vo1.4,No.l

this experiment for the occurrenceof the enzyme, thioredoxinreductase,in the platelet extracts.

DISCUSSION It has been shown previouslythat in the presence of glutathioneand glutathionereductaseabout 4, not identified,bonds were reduced in fibrinogen (31, 32). Our study confirms the susceptibilityof disulfide bonds in fibrinogen to reduced glutathioneand lipolc acid, but also shows that their action is slow as compared with thioredoxin-(SH)2.It is, however, interestingthat the total number of disulfide bonds reduced in fibrinogenis about the same in the glutathioneand thioredoxinsystems. Five bonds appears to be reduced at a fast rate In fibrinogenby thioredoxin-(SH)2(Fig. 9). They involve the three symmetricalbridges in N-DSK (ACY

28-ACe8,r8-y8 and y+rg) and two bonds in a not yet identifiedpart of

the A&chain. The deduction of number of reduced bonds was derived from the relative distributionof radioactivityIn the different chain fragmentsafter CNBr-treatment,and it agreed with the number determinedby both NADPH disappearance and appearance of thiol-groupsduring the reduction.Since all the symmetrical disulfides in N-DSK were found to be reduced, monomers of N-DSK must be present after reduction.However, the K,,-value of N-DSK after reduction (Peak 1 in Fig. 2) as comparedwith Hi2-DSK (m.w. 28,000),

which has approximately

the same molecularweight as monomeric N-DSK suggested that it may be present in dlmeric form. It is therefore possible that monomeric N-DSK may show an

BP if Ad

!__________I 1q /Li I

0

1 r---------

I

II!

BP

I

--------1

I 1

I

r

Ad

i Hi2-DSK i ! (1 S-S) : ;_________L___________________,_____---1------__--~---_----~ ; HIS-DSK

:

: (IS-S)

:16-7

Hoi-3 DSK S-51

:

;

N-‘DSK

(11 is-9

: Hoi-3 DSK

jl6-7

S-S)

FIG. 9 Schematic representationof the fibrinomenmolecule illustratingchain arrangesusceptible ments, and the location of "disulfideknots". l'hioredoxln-(SH)2 bonds are indicatedwith bars.

FIBRINOGEN

Vol.4,No.l

71

REDUCTION-OXIDATION

anamolous behaviour on gel filtration.Another possibilityis that reduced NDSK is kept in dimeric form by bonds not split under the acidic conditionsused in gel filtration.A third possibilityis that alkylationafter reductionwas not complete and that random reoxidationof symmetricalbonds resulted in production of a large proportionof dimers. However, it seems unlikely that this would invalidate conclusionsdrawn with regard to number of bonds reduced. Tn this connectionit is interestingto note that Sepharose filtration of reduced (not alkylated) fibrinogenand fibrin failed to show species of fibrinogen

of lower

molecular size than the dimeric form. Budzynski and Stahl af-

ter reduction of fibrinogenwith mercaptoethanol(P), did not either observe any change in effective volume on gel-filtration.Our findingswould indicate that there are either at least one more symmetricaldisulfide bond in fibrinogen in addition to those already described in N-DSK or that the monomers in reduced fibrinogenare held together by other bonds than S-S bridges. The Possibility that reoxidationhas occurred during gel filtrationon Sepharose 43 is less likely in view of our experiencewith similar gel filtrationson Sephadex G-50 (see Table I). The results of the present study show in particularthat reductionsystems may be useful as "chemical probes' for tertiary structural features in a protein. It seems likely that the susceptiblestructuresare surface oriented in proteins.Thioredoxin-(SH)2can reduce almost all disulfldes in low molecular weight proteins like insulin (6, 7) or in fragmentsof denatured fibrinogen such as those present in CR&-treated fibrinogen.The actual reductant,thioredoxin-(SH)2,renders a high degree of specificityto the system when protein substancesare being reduced.All the disulfides reduced at a fast rate by thioredoxin-(SH)2are In the hydrophilicregions (N-DSK and HI2-DSK) of the protein as judged by the behaviour of these compoundsin counter-currentdistribution systems (22, 27). All disulfidesbeing part of hydrophobicstructures in fibrinogen (Hol-Ho4-DSK)appear not to be reduced by the system, indicating that these structuresare burled in the protein. It is interestingthat antibodiesproduced against one of the hydrophobic knots (Hoi-DSK)do not react with intact fibrinogen,which enforces the notion that this structure,to a large extent, is hidden. Furthermore,our present results have shown that antibodiesto the hydrophilic"knot", Hi2-I>SK,reacts readily with fibrinogen.On the other hand, antibodies raised a@nst

N-DSK

(also a hydrophilic knot) which is susceptibleto reductionby thioredoxin-(SH)2 do not react to any appreciableextent with native fibrinogenindicatingthat the antigenic structures of N-DSK are hidden in fibrinogen.The fact that three

FIBRINOGEN

72

REDUCTION-OXIDATION

Vo1.4,No.l

out of 11 disulflde bonds in N-DSK react with thioredoxin-(SH)2suggests that these parts of N-DSK ars surface oriented in the fibrinogenmolecule. It may be that.the susceptiblebonds represent surface structures of N-DSK of low immunogenelty or that they are located in a cleft in the molecule not available to the a&body.

It should be mentioned that the bond reduced (No, 28) in the Aa-

chain fragment of N-DSK is in close proximityto the hydrophilic fibrinopeptide A, and antibodiesto this portion of N-DSK appear not either to react readily with fibrinogen (33). The results of the present investigationraises the importantquestion as to what extent disulfide exchange reactionsmay play a physiologicalrole in cross-linkingof fibrinogenor fibrin. Reoxidationof reduced fibrinogenis accompaniedby formationof a highly cross-linkedpolymer. Since the individual chains of fibrinogenare released on reductionand alkylatlon of the polymer, dlsulfide exchange must have been operative in this process. Reductase systems involvingthioredoxlnare found in many ormnisms includingmammals (10, 34). In this study we found a thioredoxin-likeactivity associatedwith human platelets. Experimentsattemptedat the isolation of the active substanceare now in progress.The results by gel filtrationon Sephadex G-50 seems to exclude glutathione as the active principle.We have not yet been able to demonstratethe presence of a thioredoxinreductase-likeenzyme in platelet extracts.However, it is known that thioredoxinreductase is present in mammalian liver (16) and it cannot be excluded that the enzyme is present in blood or in the blood vessel wall. Compartmentalizationof reducing agent (thloredoxin)and enzyme would, from the teleologicalpoint of view, be advantageousin a physiologicalcrosslinking reaction, since reductionand subsequent reoxidationwith polymerization would start subsequentto aggregationand desintergrationof plateletsat sites of lesions in the vessel wall. It is likely the thrombin catalized formation of fibrin at such sites and perhaps also Factor XIII mediated cross-linking preceed the reductase induced cross-linking. ACKNOWlEDGU%N!PS The authors express their gratitude for skilful technical assistance to Mrss. Elvy Andersson,Birgit Hessel, Helga Messel, Sonja Soderman,Ebba Sijrensenand Lisbeth Therklldsenand to Dr. Bohdan Kudryk for help in preparation of antibodiesand Immunologicalanalysis. This work was supportedby grants from The Swedish Medical Research Council (Nos. 13X-2475-07,19X-520-10and 13X-3529-03),Magnus Bergvalls Stlf'telse and The National Institutes of Health (No. 5 R01 HT.07379~08).

Vol.4,No.l

FIBRINOGEN

REDUCTION-OXIDATION

73

REFERENCES

1.

BLQMB;(iCK,B.Fibrinogento fibrin transformation.In: Blood Clotting E vmology W.H. Seegers (Ed.), New York and London, Academic Press, 1;:7, p.14;.

2.

B. Adsorption of plasmic fragKUDRYK, B., REUTERBY, J. and BI.OMBACK, ment D to thrombin modified fibrinogen- Sepharose. ThrombosisRem, 2, 297, 1973.

3.

KUDRYK, B., COLIEN, D. and BLOMBACK, B. Evidence for polymerization sites in fibrinogen. In preparation,1973.

4.

LORAND, L. The fibrin-stabilizingfactor system of blood plasma. Ann. New York Acad. Sci., 202, 6, 1972.

5.

BLOMBACK, B. and BLCMBACK, M. The molecular structure of fibrinogen. Ann. New York Acad. Sci., x)2, 77, 1972.

6.

IAURENT, T.C., MOORE, E.C. and REICHARD, P. Enzymatic Synthesis of DeoxyribonucleotidesIV. Isolationand characterizationof thioredoxin, the hydrogen donor from Escherichia coli B. J. Biol. Chem., m, 3436, 1964.

7.

MOORE, E.C., REICHARD, P. and THELANDER, L. Enzymatic Synthesis of Deoxyribonucleotides. V. Purificationand properties of thloredoxln reductase from Escherichia coli B. J. Blol. Chem., a, 3445, 1964.

8.

GONZALEZ PORQUE, P., BAIDESTEN,A. and REICHARD, P. Purificationof a thioredoxinsystem from yeast. J. Biol. Chem., a, 2363, 1970.

9.

GONZAIEZ PORQUE, P., BAIDESTEN,A. and REICHARD, P. The involvement of the thioredoxinsystem In the reduction of methionine sulfoxlde and sulfate. J. Biol. Chem., a, 2371, 1970.

10.

ENGSTR@l, N.-E., HOLMGREN,A., LARSSON, A. and S@ERH&T,, S. Isolation and characterizationof calf liver thioredoxin. J. Biol. Chem., in press, 1973.

11.

HOIMGREN, A. Thioredoxin.6. The amino acid sequence of the protein from Escherichia co11 B. Eur. J. Biochem., 6, 475, 1968.

12.

BLOMBACK, B. and BLOMBACK, M. Purificationof human and bovine fibrinogen. Arkiv Kemi, l0, 415, 1956.

13.

GhLUND, B., K(IWALSKA-LCTH,B.,GRm, N.J. and BLOMBACK, B. Plasmlc degradationproducts of human fibrinogenI. Isolation and characterization of fraepnents E and D and their relation to "disulfide knots". ThrombosisResearch, &.,371, 1972.

14.

HOIMGREN, A. andREICiiARD,P. Thloredoxln 2. Cleavage with cyanogen bromide. Eur. J. Biochem., 2, 187, 1967.

15.

THELANDER, L. Thloredoxin reductase.Characterizationof a hornogeneous preparationfrom Escherichia co11 B. J. Biol. Chem., 247, 852, 1967.

FIBRINOGEN

74.

REDUCTION-OXIDATION

Vol.4,No.l

16.

HOIMGHEN, A. Purificationand characterizationof thioredoxinreductase from calf liver. Mmnuscrlpt in preparation,1973.

17.

ELIMAN, G.L. Tissue sulfhydryl groups. Arch. Biochem. Biophys.,82,

70, 1959. 18.

HOLMGHEN, A. Reversible chemicalmodificationof the tryptophan residues of thioredoxinfrom Escherichia coli B. Eur. J. Blochem., 26,

528, 1972. 19.

BLOMBACK, B., HESSEL, B., IWANAGA, S., REUTEHBY, J. and BICMBACK, M. Primary structure of human fibrinogenand fibrin. I. Cleavage of fibrinogen with cyanogenbromide. Isolationand characterizationof NH2terminal fragmentsof the a( "A")chain. J. Biol. Chem., 247, 1496,

1972. 20.

KWALSKA-m, B., &HDDJND, B., E-G, N. and BLOMBACK, B. Plasmic degradationproducts of human fibrinogen.II. Chemical and immunological relation between fragmentE and N-DSK. ThrombosisResearch,1,

423, 1973. 21.

IWANAOA, S., WALL&I, P., GH&'DAHL,N.J., HENSCHEN,A. and BLCMBACK,B. On the primary structure of human fibrinogen.Isolationand characte-

rization of N-termlnal fra@nents from plasmic digests. Eur. J. Bioa., 8, 189, 1969. 22.

BLOMHACK, B., GF&DAHL, N.J., HESSEL, B., IWANAGA, S. and WALLJ?N,P. Primary structure of human fibrinogenand fibrin. II. Structural studies on NH2-terminalpart of y-chain. J. Biol. Chem., 248, 5806, 1973.

23.

BHUMMEL, M.C. and MONTGOMERY,R. Acrylamide gel electrophoresisof the S-sulfo derivativesof fibrinogen. Analyt. Biochem., ,Z& 28,

1970. 24.

McDGNAC& J., MESSEL, H., McDGNAQI, R.P. Jr., kXJHAN0,G. and BLGMBACK, B. Molecular weight analysis of fibrinogenand fibrin chains by an improved sodium dodecylsulfategel electrophoresismethod. Biochim. Biophys. Acta, x, 135, 1972.

25.

BIQMBACK, M., BLCMBiiCK, B., MAMMEN, E.F. and PRASAD, A.S. Fibrinogen Detroit - a molecular defect In the N-terminaldisulfide knot of human fibrinogen. Nature, 2l8, 19, 1968.

26.

HENSCHEN,A. Number and reactivityof disulfide bonds in fibrinogen and fibrin. Arkiv Keml, 22, 355, 1964.

27.

COLL,EN,D., GAHDIHND,B., GHGNDAHL,N.J., KUDRYK, B. and BLOMBACK,B. Partial characterizationof hydrophobicdisulfide knots in human fibrinogen. IVth Int. Congress Thromb. Haemostasls,June 19-22, 1973, Vienna. Abstract 336.

28.

HUDALL, K.M. Comparativebiology and biochemistryof collagen.In: Treatise on collagen.B.S. Gould (Ed.), London and New York, Academic Press 1968,p. 83.

Vol.4,No.l

FIBRINOGEN

REDUCTION-OXIDATION

75

Personal communication. 1973.

29.

MURANO, G.

30.

MURANO, G., WIMAN, B., BLCMBACK, M. and BLOMBACK, B. Preparationand isolation of the S-carbomethyl derivative chains of human fibrinogen. Febs Letters, 14, 37, 1971.

31.

DAVIDSON, B.E. and HIRD, F.J.R. The reactivityof the disulphide bonds of purified proteins in relationshipto primary structure. Biochem. J., l& 473, 1967.

32.

BUDZYNSKI,A.Z. and STAHL, M. Partial reductionof bovine fibrinogen by some sulphydrylcompounds. Biochim. Biophys. Acta, 175, 282, 1969.

33.

NOSSEL, H.L., YOUNGER, L.R., WILNER, G.D., PROCUPEZ,T., CANFIELD, R. E. and BUTLER, Jr., V.P. Radioimmunoassayof Human FibrinopeptideA. Proc. Nat. Acad. Sci., 68, 2350, 1971.

34.

HERRMANN, E.C. and MOORE, E.C.

Purificationof thioredoxin from rat novikoff ascites hepatoma. J. Biol. Chem., 248, 1219, 1973.