cytochromne c from two protozoa, euglena gracilis and

#wchem. J. (1915); 147, 291-302Printed in Great Britain

Purification, Properties and Amino Acid Sequence of AtypicalCytochromne c from Two Protozoa, Euglena gracilis and Crithidia oncopelti

By GRAHAM W. PETTLIGREW,* JOHN L. LEAVER, TERENCE E. MEYER*and ANDREW P. RYLE

Department ofBiochemistry and Department ofMolecular Biology,University ofEdinburgh, Edinburgh EH8 9AG, U.K.

(Received 27 September 1974)

A basic cytochrome was isolated from the phytomastigophorean protozoan Euglenagracilis and a similar protein from the zoomastigophorean protozoan Crithidiaoncopetti. In both cases chromatography on CM-cellulose in first the reduced and thenthe oxidized form proved to be an efficient means of purification. The two cytochromescan be classed in the cytochrome c family but they have certain atypical features. Thea peak of the absorption spectrum is shifted towards the red and is asymmetrical. Thepyridine ferrohaemochrome has an a-peak maximum, intermediate between that ofc-type cytochromes and proteins containing protohaem IX. The test for free vinyl groupswas positive. The amino acid sequences of the two cytochromes were determined.Attention is drawn in the text to those parts of the evidence that are less satisfactory.Both sequences are homologous with the family of cytochrome c, but are unusual inhaving only one cysteine residue so that the haem is attached through only one thioetherbond. Detailed evidence for the amino acid sequences of the two proteins has beendeposited as Supplementary Publication SUP 50042 (70 pages) at the British Library(Lending Division) (formerly the National Lending Library for Science and Technology),Boston Spa, Wetherby, Yorks. LS23 7BQ, U.K., from whom copies can be obtained onthe terms indicated in Biochem. J. (1975) 145, 5.

Cytochromes c from a wide range of eukaryoticsources have been purified. They form a close family.of proteins with very similar properties (Margoliash& Schejter, 1966). They contain covalently boundhaem, attached to the apoprotein through twothioether bonds involving two cysteine. residues(Theorell, 1938). They are readily extracted from cellsand are interchangeable in, their ability to restorerespiration in mammalian mitochondria depletedof their native cytochrome c (Byers et al., 1971).Cytochromes from a few Protozoan sources have

been purified, Hill-et al. (1971) and Kusel etal. (1969)have investigated cytochrome c-555 from Crithidiafasciculata. Euglena cytochrome c-558 was purifiedby Perini et al. (1964) and Meyer & Cusanovich(1972). It was found that both proteins formedpyridine ferrohaemochromes with atypical spectra,the a-peak maxima being at 553 nm. This is in

* Present address: Department of Chemistry, Univer-sity of California, San Diego, La Jolla, Calif. 92037,U.S.A.

Vol 147

contrast with the 550nm peak of the pyridineferrohaemochrome of all other c-type cytochromesand the 556-558nm peak of all those proteinscontaining protohaem IX as prosthetic group.

ExperimentalMaterials

Organisms. Crithidia oncopelti and the Guttman'cured' strain of Crithidia oncopelti were obtainedfrom Dr. B. A. Newton, Molteno Institute ofParasitology, University of Cambridge, Cambridge,U.K., and Euglena gracilis, strain z, was obtainedfrom Dr. N. Jardine, Department of Biochemistry,University of Edinburgh, Edinburgh, U.K.

Chromatographic materials and enzymes. Thesewere obtained from the sources listed in Ambler &Wynn (1973). Silicone fluid MS550 was obtained fromBDH Chemicals Ltd., Poole, Dorset, U.K.

Chemicals. e-N-Monomethyl-lysine was obtainedfrom a hydrolysate of Salmonella flagellin which wasagift from Dr. R. P. Ambler, University ofEdinburgh

9i

0. W. PBTfIGREW, J. L. LIAVIER, T. E. MIYIYk AND A. P. RYL1

(Ambler & Rees, 1959). e-N-Dimethyl-lysine was agift from Dr. G. Leaf, Department of Biochemistry,University of Glasgow, Glasgow, U.K. a-N-Acetyl-lysine and cytochrome c (horse heart, grade JII) wereobtained fiom Sigma (London) Chemical Co.,London S.W.6, U.K. Evans bacteriological peptonewas obtained from BDH Chemicals Ltd.

Methods

Growth of organisms. Euglena gracilis was grownas described by Pettigrew (1974). Crithidia oncopeltiwas grown axenically in 3% bacteriological peptone(Evans), 0.5% glucose, 0.5% NaCI (all w/v) with aninitial pH of 7.4. Stock solutions (lOml) were main-tained in 25ml conical flasks at 25°C and subculturedevery 3-5 days. For bulk growth the organisms weretransferred to lOOml volumes of medium, incubatedat 25°C for 3 days, and then inoculated into 1.5 litrevolumes of medium in 2 litre conical flasks. Theflasks were shaken on an orbital shaker at approx.25°C for 3-5 days and then harvested in a Alfa-Lavalcentrifuge (LAB 102 B-25). The packed cells werestored frozen at -20°C. The yield was approx. 60gof packed wet cells/10 litres of medium.

Purification of cytochromes. Essentially the samemethod was used to prepare Euglena cytochromec-558 and Crithidia cytochrome c-557. Euglena has atough pellicle and it was necessary to use a Manton-Gaulin pressure cell to achieve good cell breakage,which was estimated microscopically (Pettigrew,1974).

Crithidia cells (200g) were frozen and thawed twiceand suspended in 4vol. of ice-cold 0.1 M-NaH2PO4-Na2HPO4, pH7.0. Extraction was allowed toproceed for I h at 0-4°C. Centrifugation at 25000gfor 30min yielded a cloudy pink supernatant. Thecell debris was extracted again with 4vol. of 0.1 M-NaH2PO4-Na2HPO4, pH7.0, and after centrifuga-tion the combined supernatants were acidified topH 5.5 by the addition of 2M-acetic acid with stirring.The heavy white precipitate was separated bycentrifugation and the supernatant was subjectedto gel filtration at 0-40C on a column (9cm x 40cm)of Sephadex G-25 (medium grade) equilibrated in0.01 M-NaH2PO4-Na2HPO4, pH 7.0. The purificationprocedure for Euglena cytochrome c-558 up to thegel-filtration step is very similar and is described byPettigrew (1974). After the gel-filtration step theEuglena extract was passed through a column ofDEAE-cellulose in order to adsorb the cytchrome f.Thereafter the purification procedures for Euglenacytochrome c-558 and Crithidia cytochrome c-557were identical. The procedure was based on thedifferent chromatographic behaviour of the reducedand oxidized proteins, which allowed a 'diagonal'approach to be applied to their purification (Hartley,

1970). The chromatographic properties on CM-cellulose of reduced and oxidized cytochrome chave been described by Dixon &Thompson (1968).The cytochrome, in solution in 0.01 M-NaH2PO4-

Na2HPO4, pH7.0, was adsorbed on to a column(2cmxl0cm) of CM-cellulose (Whatman CM-23)which had been equilibrated in the same buffer, andthe cytochrome was eluted in a single step with thephosphate buffer containing 0.5M-NaCl. After gelfiltration on Sephadex G-25 equilibrated in 0.01 M-NaH2PO4-Na2HPO4, pH 7.0, to remove the salt, thecytochrome was fully reduced by the addition of atenfold molar excess of neutralized ascorbic acid andthen adsorbed on to a column (1.0cm x 10cm) ofCM-cellulose (CM-52) equilibrated with 0.01 M-NaH2PO4-Na2HPO4, pH 7.0. Elution was carried outwith 0.04M-NaH2PO4-Na2HPO4, pH7.0. The pinkfractions were diluted with 1 vol. of water, the cyto-chrome was oxidized with the minimum amount offerricyanide and then re-adsorbed on CM-cellulose:under the same conditions as for the chromatographyin the reduced form. Chromatography was again'carried out with 0.04M-NaH2PO4-Na2HPO4, pH 7.0.The eluate was diluted with I vol. of water and the

cytochrome was concentrated by adsorption on asmall column (0.5cm x 3 cm) ofCM-cellulose (CM-52)and a single-step elution with 0.01 M-NaH2PO4-Na2HPO4, pH7.0, containing 0.5M-NaCl. Thecytochrome was usually stored at 0-40C in thissolution or alternatively precipitated in 90%-satd.(NH4)2SO4 and stored as a slurry. The yields aregiven in the Results section.

Electrophoresis in polyacrylamide gels. Polyacryl-amide-gel columns (0.5cm x 7cm) were prepared bythe methods of Davis (1964). The conditions usedwere 10% (w/v) acrylamide, 0.2% (w/v) NN-methyl-enebisacrylamide in 0.05M-acetic acid-sodium ace-tate, pH 5.0. Electrophoresis was carried out at5mA/gel with loadings of protein of 3 nmol or0.1 nmol. Staining was by 0.2% (w/v) Amido Blackin methanol-water-acetic acid (5:5:1, by vol.) andthe same solvent was used to destain the gel columns.Approximate estimation of molecular weight. A

formula weight can be derived from the proposedamino acid sequence (Pettigrew, 1972) and aminimummolecular weight can be obtained from the aminoacid composition of the proteins based on iron con-tent. Gel filtration in a column (50cmx1.5cm) ofSephadex G-75 (superfine grade) equilibrated withO.1M-Tris-HCI, pH7.5, containing 0.5M-NaCl wasused to estimate molecular size (Andrews, 1964).A standard curve was obtained by plotting elutionvolumes of known proteins relative to that ofe-Dnp-lysine against log (mol.wt.).

Electrophoresis in 10% (w/v) polyacrylamide gelscontaining sodium dodecyl sulphate was carried outas described by Dunker & Rueckert (1969). Cyto-chrome c (horse heart) and myoglobin were run as

1975

292

EUGLENA AND CRITHIDIA CYTOCHROME c

molecular-weight markers in co-electrophoresis withthe unknown.

Iron determination. The method ofCameron (1965)was used.

Spectrophotometry. Spectra were recorded on aUnicam SP. 1800 spectrophotometer or manuallyon a Unicam SP. 500 spectrophotometer.

Pyridine ferrohaemochromes were prepared bythe method of Falk (1964). The final concentrationof pyridine was 2.1 M and ofNaOH was 0.17M.

Test for free vinyl groups. When vinyl groups onhaem molecules are saturated with hydrazine hydratea shift in the pyridine ferrohaemochrome spectrumcan be observed (Fischer & Gibian, 1941). Thespectrum of the protein or haemin in 0.75ml ofpyridine and 3 ml of hydrazine hydrate was recorded.The solution was heated at 100°C for 15min and thespectrum was recorded again.Removal ofhaem from cytochrome. (1) Haem was

removed from protein by the method of Sletten et al.(1968). The protein (approx. 1 mol) was dissolvedin 0.01 M-HCI and finely ground HgCI2 was added toa concentration of 12mg/ml. The solution wasincubated overnight in the dark at approx. 18°C.

After this incubation the solution was sometimesturbid but clarified after addition of an equal volumeof 1.8% (v/v) HCI in acetone. Diethyl ether was usedto extract the haem (three times with equal volumesof ether) and during this process the apoproteinprecipitated. The ether solution of haem (whichprobably contains most of the acetone and HgCl2)was washed several times with water and concen-trated under a N2 stream. Any experiments to beperformed on the haem were usually planned for thesame day, as the haemin was unstable and agedpreparations gave different chromatographicpatterns.

(2) The method described by Ambler & Wynn(1973) was used to remove the haem from cytochromec-557 and c-558 in preparation for amino acidsequence studies. Protein concentration was 0.5-1.0,umol/ml. HgCl2 was added as a fine powder togive a concentration of 10mg/ml. Protein, freeze-dried from 5% (v/v) formic acid, was normally usedas starting material but considerable difficulty wassometimes experienced in removing the haem fromcytochrome c-558. More consistent results wereobtained if the protein was precipitated from solutionat pH 7.0 by the addition of ethanol to give an 80%(v/v) solution (Margoliash et al., 1962). The precipi-tate was washed with ethanol, dried and dissolved inthe urea solution for removal of the haem group.

Chromatography of the isolated haemin. Thechromatography system used was that described byChu & Chu (1955). Whatman no. 1 filter paper wasdipped in light petroleum (b.p. 40-60°C) containingsilicone fluid MS550 (10%, v/v) and allowed to dryhorizontally. Ascending chromatography on this

Vol. 147

support was carried out in water-propan-1-ol-pyridine (55:1:4, by vol.). The chromatograms werestained for haem with the o-tolidine spray (Flatmark,1964). Haematohaemin was prepared by HgCI2cleavage ofcytochrome c(horse heart), andprotohaemIX was obtained by precipitation of haemoglobinwith acetone containing 2% (v/v) HCI and extractionof the haem into ether.Amino acid analysis. The method of Spackman

(1963) was used, with a Locarte amino acid analyser.Samples for analysis (approx. 50nmol of protein or10nmol of peptide) were prepared and hydrolysedby the methods of Ambler & Brown (1967).

Trimethyl-lysine was prepared from a-N-acetyl-lysine by the method ofDe Lange et al. (1969). It washomogeneous as judged by amino acid analysis andpaper electrophoresis at pH 6.5. On the Locarte20cm single-column programme, trimethyl-lysinewas not usually visible owing to the ammonia peak.On the 50cm column operated at 63°C, equilibratedand run in 1.0M-sodium citrate, pH 6.65, all methyl-ated lysines were completely separated.Tryptophan was measured by the spectrophoto-

metric method of Goodwin & Morton (1946) bytaking readings at 294.4 and 280nm. Because ofinterference from the haem group, solutions ofapoprotein were used, but it must be borne in mindthat the protein had then been exposed to acidicconditions which might be expected to result inpartial destruction of the tryptophan. A portion ofthe protein solution, used for the spectrophotometricexamination, was taken for amino acid analysis andthe number of molecules of tyrosine and tryptophanper molecule could be calculated by using the formu-lae of Goodwin & Morton (1946). Tryptophan wasnot usually quantified in peptides. A qualitativeindication of tryptophan was obtained by theEhrlich reaction on paper (Smith, 1953) and chymo-tryptic peptides containing tryptophan were oftendigested with carboxypeptidase A, and the productswere analysed by paper electrophoresis at pH2.0.

Cysteine was measured as cysteic acid afteroxidation of apoprotein or peptide with performicacid (Hirs, 1967) or was reduced and carboxymethyl-ated by the method of Crestfield et al. (1963). Themercaptoethanol and iodoacetic acid were presentin 100-fold molar excess over protein (30umol to0.3,umol). The carboxymethylated protein wasseparated from excess of reagents by gel filtration ona column (2cmx25cm) of Sephadex G-25 (finegrade), equilibrated and run in 0.2M-acetic acid in thedark.

Cysteic acid and carboxymethylcysteine weredetermined by amino acid analysis.

N-Terminal and C-terminal regions of proteins.Proteins were labelled with dansyl chloride essentiallyas described by Gray (1 972a). Dried protein (20nmol)was dissolved in 0.5ml of 0.5M-NaHCO3 containing

293

G. W. PETTIGREW, J. L. LEAVER, T. E. MEYER AND A. P. RYLE

8M-urea, and 0.5ml of dansyl chloride in acetone(20mg/ml) was added. After incubation at 37°C for2hthe protein was precipitated by the addition ofacetone. After centrifugation the precipitate waswashed with acetone and then hydrolysed in 6M-HClat 105°C overnight. The dried hydrolysate wasexamined by electrophoresis at pH4.36 (Gray, 1972a;Ambler & Brown, 1967).The method of Porter (1957) was used for di-

nitrophenylation, and dinitrophenyl-amino acidswere separated by chromatography on polyamidelayers (Wang & Wang, 1968). One cycle of theEdman degradation (Gray, 1972b) was performedon cytochrome c-557.

Digestion of proteins or peptides with carboxy-peptidase A was carried out as described by Ambler(1972).

Methods of sequence determination. Establishedmethods were used for the digestion of proteins andthe separation of peptides (Ambler, 1963; Ambler &Wynn, 1973). The amino acid sequences of purepeptides were investigated by using the Edmandegradation (Gray, 1972a). The dansyl method (Gray,1972b) was used for identification of N-terminalresidues. Dansyl-amino acids were separated onpolyamide layers by the system of Ramshaw et al.(1970).

Acetyl groups were identified by hydrazinolysis ofa peptide and treatment of the acetylhydrazine withdansyl chloride (Schmer & Kreil, 1969). Acetyl-dansyl-hydrazine was identified by chromatographyon polyamide layers (Ramshaw et al., 1970).

2.0

E .0-

0

2.0 r

E I.0

0

10 20 30Fraction no.

7 (b)

I0 20 30Fraction no.

Fig. 1. Chromatography of (a) ferrocytochrome c-557 and(b)ferricytochrome c-557 on CM-cellulose

The conditions were as described in the text with a column(1Ocm x 1cm) of Whatman CM-52. Fractions of volume1 ml were collected. In (a) fractions 5-15 were pooled foroxidation and rechromatography. o, E280; *, E412-

ResultsPurification ofcytochromes

Profiles of the chromatographic separations of thereduced and oxidized species of cytochrome c-557are shown in Figs. l(a) and l(b). A similar patternwas obtained for cytochrome c-558 under the sameconditions. The approximate yields of pure cyto-chrome c-557 and cytochrome c-558 per lOOg wetweight ofcells were 2,umol and 0.2,umol respectively.Both cytochromes gave single bands after electro-

phoresis in polyacrylamide gels. Two gels were runfor each cytochrome, one containing 3 nmol ofprotein and the other 0.1 nmol of protein. The latterquantity- was easily visible after staining.

Preparations of both proteins showed less than 4%binding ofCO, although this percentage was increasedslightly if the protein had been previously freeze-driedfor storage.

Margoliash & Schejter (1966) have emphasizedthat multiple chromatographic forms of cytochromec can occur in preparations of the protein. Tests ofpurity must therefore not only distinguish non-cytochrome components but also detect non-nativecytochrome fractions.

Crithidia cell extract which had been kept at pH4.5overnight contained two forms of cytochrome c-557.One of these forms, cytochrome c-557(A), did notadsorb on to CM-cellulose in 0.02M-NaH2PO4-Na2HPO4, pH7.0, whereas the second, cytochromec-557 (B), behaved identically with the single speciesisolated after extraction at pH 7.0. The spectralparameters of forms A and B were very similar andneither combined with CO.

Glutamic acid or glutamine was identified as theN-terminal amino acid of cytochrome c-557 (A) andthe amino acid composition was consistent with theloss of the four N-terminal amino acids (G. W.Pettigrew, unpublished work).Because of the difference in amino acid composi-

tions of form (A) and form (B), and because form (A)was recovered in decreasing yield as the exposureto acid pH was shortened, we consider that form (A)is an artifact of the preparation (henceforth referredto as modified cytochrome c-557) and that form (B) isnative protein.

1975

294


Molecular weight

Assuming that the proteins contain only haem andamino acids, minimum molecular weights can becalculated from the amino acid composition relatedto the iron content, and formula weights can becalculated from the proposed sequences.Cytochrome c-557 and c-558 have formula weights

12510 and 11856 respectively and because of thegood correspondence of amino acid compositionsbased on iron content and on sequence (Table 4),the minimum molecular weights approximate tothese values.An estimate of molecular size was made for each

protein by using the gel-filtration method. Cyto-chrome c-558 had an apparent molecular weight of13 000, but cytochrome c-557 was considerably moreexcluded from the gel matrix, and a value of 16000

was obtained. This latter result is similar to thatobtained by Hill et al. (1971) for cytochrome c-555from Crithidiafasciculata.

Because of this disagreement with the formulaweight in the case of cytochrome c-557, electro-phoresis in sodium dodecyl sulphate-polyacrylamidegels was used to obtain a second estimate of themolecular weight. In this system, cytochrome c-557migrated with a slightly greater mobility (1.04) thancytochrome c (1.0). Myoglobin had a mobility of0.87. Dunker & Rueckert (1969) found that in thissystem cytochrome c (horse heart) had an apparentmolecular weight of 13 600compared with the formulaweight of 11700. If this value of 13 600 is used forcytochrome c (horse heart), then the apparent mole-cular weight of cytochrome c-557 in the sodiumdodecyl sulphate-polyacrylamide system approxi-mates to its formula weight.

419nm.0

(a).9

.8 _ Reduced spectrum

412 nm

.7 I

56 Oxidized spectrum

.5 /

.3 ~1

.2 'V / 2 557 nm

.1 ,524nm

350 400 450 500 550 600Wavelength (nm)

E o.5s

iced spectrum

lized spectrum

558 nm

526 nm

400 450 500Wavelength (nm)

550

Fig. 2. Visible spectra ofreduced and oxidized cytochrome c-557 (a) and cytochrome c-558 (b)

Concentrations of cytochrome used were (a) 7pM and (b) 6juM. Reduction was with sodium dithionite, oxidation withpotassium ferricyanide.Vol. 147

I.

0.

0.

0.

0.

E o.

0.

0.

0.

0.

600

295


Table 1. Spectralparameters ofcytochrome c-557, c-558 and c

Extinction coefficients are based on iron content of the solutions.

Cytochrome c-557 Cytochrome c-558 Cytochrome c (horse)

Maxima Extinction Maxima Extinction Maxima Extinction(nm) (mM) (nm) (mM) (nm) (mM)

Reduced 557 24.7 Reduced 558 26.3 Reduced 550 29524 15.0 526 15.5 521 15.5419 140 422 158 415 135

Oxidized 532 12.5 Oxidized 530 10.5 Oxidized 528 11.2412 100 412 101 408 109275 24.1 280 30.0 280 23.2

Ratio Ratio RatioEsorct (red.)/a 5.65 Esoret (red.)Ia 6.0 Esoret (red.)/° 4.65odE275 1.02 o/E280 0.88 a/E2so 1.25

Table 2. a-Peak maxima ofpyridine ferrohaemochromes ofcytochromes and haemins

The solutions contained pyridine (2.1 M) and NaOH (0.17M).oc-Peak maximum of pyridineferrohaemochrome (nm) SmM Reference

Cytochrome c (horse heart) 550 29.1 Morton (1958)Proteins containing protohaem IX 557-558 34.4 Morton (1958)Cytochrome c-557 553 29.95 This workCytochrome c-558 553 This workHaemin from cytochrome c 547-548 This workHaemin from cytochrome c-557 552 This work4(2)-Hydroxyethyl-2(4)-vinyldeuterohaem IX 552 Porra & Jones (1963)Haematohaemin 549 Porra & Jones (1963)

Spectralparameters ofthe cytochromesThe visible spectra of cytochromes c-557 and c-558

are shown in Figs. 2(a) and 2(b). Maxima andextinction coefficients are listed in Table 1 and arebased on iron content of the solution used formeasurement.The spectrum of the a peak of cytochrome c-557

does not change through the purification. Cellshomogenized three times in 0.25% (v/v) HCI inacetone and resuspended in 0.01 M-NaH2PO4-Na2HPO4, pH7.0, showed the same a-peak absorp-tion maximum and shoulder as pure protein.The characteristics of the oa peak of the pyridine

ferrohaemochromes of cytochrome c-557 and cyto-chrome c-558 are compared in Table 2 with those ofcytochrome c (horse heart) and cytochromes b.The position of the a-peak absorption maximum

of the pyridine ferrohaemochrome before and afterheating with hydrazine hydrate and pyridine areshown in Table 3 for cytochrome c (horse heart),haemoglobin, cytochrome c-557 and cytochromec-558.

Table 3. ac-Peak maxima ofpyridineferrohaemochromes inhydrazine hydrate

The solutions contained 0.75ml of pyridine and 3ml ofhydrazine hydrate. Heating was at 100°C for 15min.

a peak of pyridineferrohaemochrome inhydrazine hydrate (nm)

Before Afterheating heating

Cytochrome c 551 551Haemoglobin 557 548-549Cytochrome c-557 554 551Cytochrome c-558 553 550Haemin from cytochrome c 548 548Protohaem IX 557 548-549Haemin from cytochrome c-557 553 549

Properties of the haemin isolated from cytochromec-557The absorption peaks of the haemin in ether were

at 640nm, 535nm and a double Soret peak with

1975

296


maxima at 404 and 370nm. The correspondingspectrum of haemin isolated from cytochrome c(horse heart) had absorption maxima at 637, 532,400 and 370nm. The pyridine ferrohaemochrome ofthe haemin from cytochrome c-557 had an a-peakabsorption maximum at 551-552nm, compared withthat of cytochrome c (horse heart) at 547-548nm(Table 2). The corresponding parameters for4(2)-hydroxyethyl-2(4)-vinyl deuterohaem IX andhaematohaemin are 552 and 549nm respectively(Porra & Jones, 1963).

Typical RF values obtained for chromatographyof the isolated haemins were 0.91 for haematohaemin(horse heart cytochrome c), 0.84 for the haemin fromcytochrome c-557 and 0.60 for protohaem IX. Inseveral repeated experiments these values were notreproducible, but the pattern of separation remainedthe same. In aged preparations of the haemins fromcytochrome c-557 and cytochrome c, a slower-streak-ing material was observed with approx. RF 0.3.The presence of free vinyl groups on the isolated

haemin was tested by using hydrazine hydrate. Theresults are shown in Table 3.

Amino acid compositionThe amino acid compositions of cytochrome c-557

and c-558 are shown in Table 4, where they are

compared with the residues identified in the proposedsequences. The composition of Euglena cytochromec-558 is very similar to that given by Meyer &Cusanovich (1972).

Cysteic acid was determined in separate hydroly-sates of both cytochromes after removal of the haemgroup and oxidation with performic acid. Forcytochrome c-557, 1.07 and 1.08mol of cysteic acid/7mol of leucine and for cytochrome c-558, 0.92molof cysteic acid/5mol of leucine were recovered. Aftercarboxymethylation of apocytochrome c-557,0.85mol of carboxymethylcysteine/7mol of leucinewas found.Tryptophan and tyrosine were estimated by the

method of Goodwin & Morton (1946). The resultswere 3.19mol of tyrosine and 0.97mol of tryptophanper mol of cytochrome c-557 and 5.2mol of tyrosineand 2.07mol of tryptophan per mol of cytochromec-558. After removal ofthehaem group ofcytochromec (horse heart) under the same conditions a ratio oftyrosine to tryptophan of 3.81 was obtained. Thereare four tyrosine and one tryptophan residues in thehorse heart sequence.Cytochrome c-557 contained 1.72mol of trimethyl-

lysine/lOmol of lysine and cytochrome c-558 con-tained 1.04mol of trimethyl-lysine/12mol of lysine.Small amounts of monomethyl- and dimethyl-lysine

Table 4. Amino acid compositions ofCrithidia cytochrome c-557 and Euglena cytochrome c-558Hydrolyses for 20 and 90h were performed on portions ofa solution ofeach protein. The iron content ofeach solution wasdetermined. The analysis results are derived from average values ofthe 20 and 90h hydrolysis, except for those ofthreonineand serine, which were extrapolated to zero time of hydrolysis, and those of isoleucine and valine, for which the 90h valueswere taken. The results in the analysis (1) columns were calculated relative to the iron content of the parent solutions. Theresults in the analysis (2) columns were calculated relative to the sums of the amounts of leucine, phenylalanine, histidineand arginine divided by 18 for cytochrome c-557 and by 14 for cytochrome c-558. Values in the sequencecolumns are residuetotals from the proposed sequences. Analyses of cysteine and tryptophan were performed separately and the methods aredescribed in the text.

Cytochrome c-557

Analysis (2)10.24.14.08.99.3

14.012.86.91.94.06.92.85.12.0

10.11.74.0

1.11.0

Sequence10449914137247352102411

Cytochrome c-558

Analysis (1) Analysis (2) Sequence9.5 9.2 95.0 4.9 57.1 6.9 79.3 9.0 94.4 4.3 49.9 9.7 10

12.2 11.8 125.2 5.0 52.0 1.9 24.1 4.0 45.1 5.0 55.0 4.8 53.1 3.0 32.0 1.9 2

12.3 12.0 121.0 1.0 14.2 4.1 4

0.9 12.1 2

Analysis (1)9.84.03.98.79.1

13.712.46.71.83.96.72.75.01.99.81.73.8

AspThrSerGluProGlyAlaValMetIleLeuTyrPheHisLysMe3LysArgCysTrp

Vol. 147

297


were detected in the hydrolysate ofcytochrome c-557and these results are discussed below.

N-Terminal groups ofcytochrome c-557 and c-558

Dansyl-glutamate was identified after dansylationof the modified form of cytochrome c-557 discussedabove. The only a-dansyl-amino acid spot identifiedin the hydrolysate of native cytochrome c-557treated with dansyl chloride was a faint spot ofdansyl-arginine. No a-dinitrophenyl-amino acid wasidentified in the hydrolysate of dinitrophenylatednative cytochrome c-557. There was, however, adecrease of approx. 1 mol of arginine/mol in thehydrolysate of the dinitrophenylated protein. Asingle Edman cycle was carried out on apoprotein andamino acid analysis showed a loss of 0.3 mol ofarginine/mol.The amino acid sequence evidence discussed below

suggests that the N-terminus ofthe protein is blocked.It is possible that these positive results for argininemay be due to 'raggedness' at the N-terminus. If,during isolations of the protein limited proteolysishad occurred at the amino side of arginine (-6) aswell as the observed cleavage at the carboxyl sidethat gives rise to 'modified' form of cytochromec-557, then the resultant fraction of modified proteinmay not have been separated from intact proteinduring purification and some N-terminal argininewould be detected. This cannot, however, account forthe loss of one arginine residue per molecule in thehydrolysate of the dinitrophenylated protein.No a-dansyl-amino acid was identified in the

hydrolysate of cytochrome c-558 treated with dansylchloride.

C-Terminal regions of cytochrome c-557 and c-558

The results of digestion of the two proteins withcarboxypeptidase A are given in Table 5.

Determination of the amino acid sequences of cyto-chrome c-557 andc-558 anddeposition ofsupplementarydata

The peptides from tryptic and chymotrypticdigestion of cytochrome c-557 and tryptic, chymo-tryptic and peptic digestion ofcytochrome c-558 havealready been discussed (Pettigrew, 1972, 1973; Linet al., 1973). This evidence has now been extended toinclude thermolytic digestion of the two proteins.The detailed evidence has been deposited in theBritish Library (Lending Division) (formerly theNational Lending Library for Science and Techno-logy) Boston Spa, Yorks. LS23 7BQ, U.K., forstorage on microfiche as Supplementary PublicationSUP 50042.

Table 5. Carboxypeptidase A digestion ofcytochrome c-557(a) and c-558 (b)

Apocytochrome c-557 (approx. lOnmol) and a portion ofa solution of apocytochrome c-558, the protein contentof which was accurately known from amino acid analysis,were digested with lO,g of carboxypeptidase A for 60minas described by Ambler (1972). The solutions wereacidified after incubation by addition of a few drops of1 M-acetic acid and dried in vacuo. The residues were takenup in sodium citrate buffer, pH2.0, and applied to theanalyser column. For cytochrome c-557 digestion, theresidues per molecule were calculated relative to lysinereleased, equal to 1. For the cytochrome c-558 digestionthe residues per molecule are based on the total amount ofprotein digested (12nmol).

(a)AspSer+ Asn+ GlnGluAlaIleLeuTyrLys

Interpretation:

Amino acidsreleased(nmol)

1.53.12.03.91.9

10.41.86.5

0.3

Residues/mol0.230.480.310.600.301.60.281.0

0.5 1.0

Arg(Ala-Asp-Leu-Ile-Ala-Tyr-Leu-Glu-)Asn(Leu-Lys)91

AspThrAlaMetIleLeuTyrLys

(b)9.47.05.25.5

10.05.85.3

11.2

100

0.80.590.440.470.850.490.450.95

Interpretation:0.43 0.52 0.8

Asp(lle-Ile-Ala-Tyr-Met-Lys)(Thr-Leu-Lys)Asp

100

The evidence comprises: (1) a description anddefinition of the conventions and abbreviations usedin recording the information; (2) electrophoreticmaps of the digestion mixtures; (3) tables summariz-ing the amino acid compositions of peptides andcertain parameters of purification, i.e. the electro-phoretic mobility at pH6.5, the relative elutionvolume on gel filtration through Sephadex G-25and the final yield; (4) the descriptions of individual

1975

298


Eq4 U) of1

:: Ez X

E-;.X1 M 4 ,

H > , Z E4 2 O

I' X]>4 >4 >H

~~H H H

40 P4 Pq 0

> E-4 E- n a4 t

z Sz 1 :<XMXn~~~~~~~~~~~~~0XEO

0 > cC

E En E4 1:3: :] X

F: < <

L) c) u

cx ()I 01

0 0t 04 H) H1 H

U) 01

X~~~~~~~~~~~~~~~~~~- E-fifi E-

| X >1>4

H~~ ~

O z X

HX Z Z

aV@ > ~~~~~~~~~~~~~~~E-E-4H4 E

< 3: 3 3x ~~~~>:> E-4

H H

<

Z Z

,,~~~~~(9

L1)01OD n z z

0) 01 c¢4¢LO LZn

u <

a) 0Ed 4~1 Z4 0 0

H 414 E) .I11 H9

0 0 La

> >1 0

Hl 1u

Vol. 147

peptides for each enzymic digestion including dansyl-.Edman evidence, secondary digestion of peptides andassignment of amide groups.The sequences of the two proteins are shown in

Fig. 3 where they are aligned with the sequence ofhorse cytochrome c.

In a few regions, the evidence for the amino acidsequence is poor or incomplete. These points areemphasized below.

Evidencefor the sequence of cytochrome c-557

Studies on the N-terminus of this cytochrome aredescribed above. They indicate that at least a fractionof the purified preparation contained N-terminalarginine. This result is difficult to explain in view ofthe peptide evidence discussed below and also becausethe four arginine residues expected from the analysisof whole protein are already positioned in thesequence.No terminal amino group could be detected

in the tryptic peptide [Pro(_9)-Arg(6)j or thechymotryptic peptide [Pro(_9)-Phe(10o] by the nin-hydrin, isatin, dansyl or dansyl-Edman methods.The electrophoretic mobility of this tryptic pep-tide at pH6.5 was 0.9 (relative to lysine =A 1). Thisindicates that the peptide has a net positive chargeof two rather than one (Offord, 1966), a surprisingresult in view of the apparently blocked a aminogroup.

Peptide [Pro(_9)-Arg(6j)] was digested withthermolysin and one ninhydrin-positive peptide wasrecovered with composition (Ala-Arg). No otherstaining material was detected with the chlorinationmethod (Rydon & Smith, 1952).A peptide containingproline and trimethyl-lysine and with an electro-phoretic mobility atpH 6.5 of0.64was recovered fromthe paper by cutting the whole sheet into strips. Afterhydrazinolysis of this material (Akabori et al., 1952)and treatment with dansyl chloride, dansyl-trimethyl-lysine was identified.

Carboxypeptidase B released arginine from peptide[Pro(_9)-Arg(-6)].A ninhydrin-positive peptide with composition

(Pro-Lys) was isolated in low yield (4%) from thetryptic digestion ofcytochrome c-557. No N-terminalamino acid was identified with dansyl chloride bute-Dns-lysine was recorded. The electrophoreticmobility at pH 6.5 is not very much lower than thatofa dipeptide (0.65 compared with 0.75 for Ser-Lys).

This result is consistent with the pattern of partialmethylation found in the amino acid analysis oftryptic peptide [Pro(_9)-Arg(6)] and chymotrypticpeptide [Pro(_9)-Phe(1O)]. The analysis of the latterpeptide indicated that residue (-8) is approx. 7%monomethyl-lysine, 14% dimethyl-lysine, 63% tri-methyl-lysine and 16%.lysine. Therefore the tryptic

299


peptide (Pro-Lys) may derive from that fraction ofthe protein which was not methylated at lysine (--8).Thus the evidence for the sequence of the

N-terminal region is unsatisfactory and the natureof the blocking group is unknown.One other result with cytochrome c-557 should be

emphasized and that is the recovery of a dipeptide(Ala-Leu) in 2% yield from the thermolysin digestionofthe protein. This peptide could not be positioned inthe proposed sequence and it was thought that it mayderive from transpeptidation of amino acids. Nosuch dipeptide was found, however, in an incubationmixture of alanine (2,umol), leucine (2,umol) andthermolysin (1 mg).The evidence for the purity of the protein prepara-

tion indicates that levels of contamination with asingle protein of greater than about 3% are unlikely.Assuming that the dipeptide (Ala-Leu) was recoveredin approx. 20% yield after purification a recovery of2% would represent a 10% molar level of (Ala-Leu)in the digestion mixture. The possibility that theproposed sequence is incorrect in this respect isconsidered unlikely as the amino acid compositionof the protein indicates 7 residues of leucine permolecule and 7 leucine residues have been locatedon the basis of good evidence. A dimorphism ateither an'alanine or a leucine in the sequence cannotbe ruled out but no evidence from amino acid analysesor dansyl-Edman results could be found to supportthis hypothesis.

Evidencejfor the sequence ofcytochrome c-558

Some difficulty was encountered in the dansyl-Edman degradation of the tryptic peptide [Gly(23)-Arg(38)] of cytochrome c-558 (Pettigrew, 1973). Inthree experiments, only one degradation gave cleanresults up to tryptophan-33. In the other two experi-ments, results were obtained which indicated thatacidolysis of the peptide chain at residue 28 hadoccurred. Three separate secondary digestions ofpeptide T5 were performed and there was no evidenceto suggest that there is a mistake in the sequence.However, only one thermolysin peptide from this

region was recovered, namely [Val(24)-Ser(31)]. in thisrespect both the thermolysin and the pepsin digestionsofcytochrome c-558 were unsatisfactory. The proteinused in these digestions still contained haem althoughit had been subjected to cleavage with HgCI2.Both digestion mixtures contained brown material ofhigh molecular weight which was not fractionated andwhich interfered with the electrophoresis of peptides.However, thermolysin peptides were obtained inreasonable yield from the region containing cysteine-17, indicating that the treatment with HgCI2 did infact cleave the thioether bond.Thus it is possible that the observed loss of a

large section of the molecule may be due to the

attachment of the haem group to a secondary siteon the peptide chain.

Acetyl-dansyl-hydrazine was identified after treat-ment of tryptic peptide [Gly(1)-Arg(5)] with hydra-zine. In a separate experiment, this peptide wasincubated with 6M-HCI at 100°C for 30min and themixture was fractionated by electrophoresis atpH6.5 and 2.1. The results were consistent withthe sequence (acetyl-Gly-Asp-Ala-Glu-Arg), but nopeptide containing the glycine residue was identifiednor was acetyl-glycine.

Discussion

The method of chromatography on an ion-exchange column in the reduced and then the oxidizedform yielded protein of a high degree of purity. Whencytochrome c (horse heart) with a E55o/E280 ratio of0.94 was subjected to the same procedure a productwith E550/E28o ratio of 1.23 was obtained by usingchromatography in 0.07M-NaH2PO4-Na2HPO4,pH7.0. This is within the range 1.2-1.25 quoted byMargoliash & Walasek (1967) for pure protein. Themost stringent test of purity was considered to be thecorrespondence between the amino acid compositionsof the two cytochromes derived from acid hydrolysisof the whole proteins and those derived from theamino acid sequences. The values in Table 4 can bemanipulated by using individual amino acids ratherthan iron as a reference and a closer approximationto integral values can be obtained (analysis 2).The elucidation of a unique sequence with no unfittedpeptides is not, in this case, considered an indicationof purity, since contaminants of up to 10% wouldprobably not be detected as peptides with themethodology used. CO binding by the pure prepara-tions was low, indicating the presence of only a smallfraction of deamidated or polymeric forms, and thegel electrophoresis supports this conclusion. Forsequence work this is a relatively unimportantconsideration (unless the percentage of deamidationis very high) but for other purposes, e.g. crystallizationand enzymic studies, the presence of such modifiedforms can greatly affect results.

Featiures ofthe spectra ofcytochromes c-557 and c-558

The peaks ofthe visible spectra ofboth cytochromesc-557 and c-558 are shifted to the red in comparisonwith horse heart cytochrome c. Such shifts are notunknown in c-type cytochromes (Kamen & Horio,1970), but all mitochondrial cytochromes c studieduntil now possess a symmetrical a peak at 550nm. Theasymmetry of the a peaks of the two protozoancytochromes is of interest, as cytochrome c-555 fromCrithidia fasciculata possesses a symmetrical a peak(Hill et al., 1971; Kusel et al., 1969). Cytochromec-555 is presumably a close relative of cytochrome

1975

300

EUGLENA AND C:RI 'HIDIA CYTOCHROME e

c-557, and comparative studies may reveal a basis forthis spectral dissimilarity. On the other hand, thesimilarities in the properties of the cytochromes fromthe two species of Crithidla rules out the possibilitythat cytochrome c-557 is derived from the bacterialendosymbiote of Crithidia oncopelti (Chang &Trager, 1974).Some preliminary experiments were performed on

the Guttman strain of Crithidia oncopelti, 'cured' ofits endosymbiote, but the cytochrome isolated fromthese organisms had an a peak at 555nm and anamino acid composition resembling that of cyto-chrome c-555 from Crithidiafasciculata. This supportssuspicions that the 'cured' strain of Crithidia oncopeltiis, in fact, Crithidiafasciculata (Newton, 1968).The low purity indices [ratios ofE ( peak reduced) to

E280] of cytochromes c-557 and c-558 are due in bothcases to a decreased a-peak absorption and also,with cytochrome c-558, to a greatly increased absorp-tion at 280nm. This is consistent with the presence ofa second tryptophan residue in cytochrome c-558.The pyridine haemochrome spectrum of a haemo-

protein is diagnostic of the type of haem it contains(Enzyme Nomenclature Recommendations, 1965).The absorption maximum of the pyridine ferro-haemochrome a peak of both cytochromes c-557 andc-558 lies between that of all c-type cytochromes andall proteins containing protohaem IX. This ledMeyer & Cusanovich (1972) to postulate that thehaem of cytochrome c-558 was not haem c but4(2)-hydroxyethyl-2(4)-vinyldeuterohaem and that itwas bound to the protein through only one thioetherbond.The shifts in the pyridine ferrohaemochrome

spectra of both the cytochrome c-557 and c-558proteins and their derived haemins on treatment withhydrazine hydrate are consistent with the presenceof single vinyl functions on the haem groups. Thehaem cannot be removed from either protein bytreatment with acetone containing 2% (v/v) HCI andthe successful cleavage with heavy-metal salt indicatesthe presence of a thioether bond.The prosthetic group in both cytochromes c-557

and c-558 is therefore probably monosubstitutedprotohaem IX with one unsaturated vinyl group.However, rigorous proof of the haem structure wouldrequire chemical characterization of the isolatedhaemin. The evolutionary conservatism ofcoenzymesand the likelihood of a common origin for cyto-chromes c-557, c-558 and the mitochondrial cyto-chromes c from plants, animals and fungi argue infavour of the simple difference outlined above.

Structure ofthe haem-binding site oJ cytochromes c-557and c-558

Both cytochrome c-557 and c-558 lack the cysteineresidue at position 14 which forms a thioether link

Vol. 147

with the haem group in cytochromes c. This resultis compatible with the body of evidence presentedabove for the presence of an unsaturated vinyl groupon the haem. The alanine at residue 14 implies atleast two base changes in the protozoan cytochromesc since their divergence from the other eukaryoticcytochrome c genes.The possibility that alanine-14 is an artifact should

be considered. Cysteine can give rise to dehydro-alanine under certain conditions (Patchomik &Sokolovsky, 1964). However, when cytochrome c-557was treated with sodium sulphite, which convertsdehydroalanine into cysteic acid (Wiener et al.,1966) no cysteic acid was found after acid hydrolysisin vacuo. Also, the spectrum of Crithidia cells afterextraction with acid acetone shows an asymmetricala peak at 557nm, so that the spectrum is not modifiedduring purification.

Lastly, enzymic studies indicate that cytochromec-558 is very similar to horse cytochrome c in itsreaction with mammalian cytochrome c reductaseand cytochrome oxidase preparations (Davis et al.,1972). Thus, if the atypical haem-binding is anartifact, it is an artifact that has no effect on thebiological activity of the molecule.

Amino acid sequences ofcytochrome c-557 and c-558

The aligned sequences are shown in Fig. 3. Thenovel changes that occur in these two proteins havebeen discussed (Pettigrew, 1972, 1973). The totals ofresidues located in both sequences show goodcorrespondence with the amino acid composition(Table 4).Approx. 100mg of cytochrome c-557 and 75mg of

cytochrome c-558 were used to determine the aminoacid sequences. These are relatively small quantitiescompared with many of the sequence investigationsof cytochromes c, but are much larger than theamounts used in the characterization of some plantcytochrome c sequences (Thompson et al., 1971).

Because of the small quantities obtained, some ofthe analytical systems used, in particular peptidepurification on paper, were employed near the limitsof their sensitivity. Amino acid-analysis valuestended to be less satisfactory than those of studiesusing the same techniques but on larger quantities ofprotein (e.g. Ambler & Brown, 1967).

Because of the small amounts of pure peptideobtained, thorough characterization was often notpossible, and thus corroborative evidence fromseveral sources was necessary. Dansyl-Edmanevidence was relied on a great deal and thus a rela-tively small percentage of the peptide bonds wereshown to be suspectible to enzymic hydrolysis (53 outof 110 in the case of cytochrome c-557 and 59 out of101 in the case of cytochrome c-558).

301

302 G. W. PETTIGREW, J. L. LEAVER, T. E. MEYER AND A. P. RYLE

The evidence of the anmino acid sequences supportsthe conclusion that both cytochrome c-557 and c-558are members of the cytochrome c family althoughthese are exceptions to the rule that the haem-bindingsite of c-type cytochromes follows the patternCys-X-Y-Cys-His.

In the case of cytochrome c-558 the novel changesin the sequence have not impaired reactivity withmammalian reductase and oxidase preparations.In the case of cytochrome c-557, Keller et aL. (1973),using n.m.r. spectroscopy, have shown that the struc-ture and properties of the haem crevice are verysimilar to that of horse cytochrome c. The n.m.r.spectra contain features consistent with the absenceof a thioether bond and the presence of a freevinyl group.

G. P. is indebted to Dr. R. P. Ambler for encouragementand expert guidance. We thank Mr. J. McGowan forassistance with amino acid analysis and Mr. T. Bruce forgrowing Euglena.

References

Akabori, S., Ohno, K. & Narita, K. (1952) Bull. Chem.Soc. Jap. 25, 214

Ambler, R. P. (1963) Biochem. J. 89, 349-378Ambler, R. P. (1972) Methods Enzymol. 25, 262-272Ambler, R. P. & Brown, L. H. (1967) Biochem. J. 104,

784-825Ambler, R. P. & Rees, M. W. (1959) Nature (London)

184, 56-57Ambler, R. P. & Wynn, M. (1973) Biochem. J. 131,

485-498Andrews, P. (1964) Biochem. J. 91, 222-233Byers, V., Lambeth, D., Lardy, H. A. & Margoliash, E.

(1971) Fed. Proc. Fed. Amer. Soc. Exp. Biol. 30, 1286Cameron, B. F. (1965) Anal. Biochem. 11, 164-169Chang, K. P. & Trager, W. (1974) Science 183, 531-532Chu, T. C. & Chu, E. J. (1955) J. Biol. Chem. 212, 1-7Crestfield, A. M., Moore, S. & Stein, W. H. (1963)J. Biol.

Chem. 238,622-627Davis, B. J. (1964) Ann. N. Y. Acad. Sci. 121, 404-427Davis, K. A., Hatefi, Y., Salemme, F. R. & Kamen, M. D.

(1972) Biochem. Biophys. Res. Commun. 49, 1329-1335De Lange, R. J., Glazer, A. N. & Smith, E. L. (1969)

J. Biol. Chem. 244, 1385-1388Dixon, H. B. F. & Thompson, C. M. (1968) Biochem. J.

107, 427-431Dunker, A. K. & Rueckert, R. R. (1969) J. Biol. Chem.

244, 5074-5080Enzyme Nomenclature Recommendations (1965) pp.

18-24, Elsevier, AmsterdamFalk, J. E. (1964) Porphyrins and Metalloporphyrins,

Elsevier Press, Amsterdam

Fischer, H. & Gibian, H. (1941) JustusLiebigs Ann.- Chem.548, 183-194

Flatmark, T. (1964) Acta Chem. Scand. 18, 1517-1527Goodwin, T. W. & Morton, R. A. (1946) Biochem. J. 40,

628-632Gray, W. R. (1972a) Methods Enzymol. 25, 121-138Gray, W. R. (1972b) Methods Enzymol. 25, 333-344Hartley, B. S. (1970) Biochem. J. 119, 805-&22Hill, G. C., Chan, S. K. & Smith, L. (1971) Bioehim.

Biophys. Acta 253, 78-87Hirs, C. H. W. (1967) Methods Enzymol. 11, 197-199Kamen, M. D. & Horio, T. (1970) Annw. Rev. Biochem. 39,

673-700Keller, R. M., Pettigrew G. W. & Wuithrich, K. (1973)FEBS Lett. 36, 151-156

Kusel, J. P., Suriano, J. R. & Weber, M. M. (1969)-Arch.Biochem. Biophys. 133, 293-304

Lin, D. K., Niece, R. L. & Fitch, W. M. (1973) Nature(London) 241, 533-535

Margoliash, E. (1962) J. Biol. Chem. 237, 2161-21-74Margoliash,- E. & Schejter, A. (1966) Advan. Protein Chem.r

21, 113-286Margoliash, E. & Walasek, 0. F. (1967) Methods Enzymol.

10, 339-348Margoliash, E., Kimmel, J. R., Hill, R. L. & Schmidt,W. L. (1962) J. Biol. Chem. 237, 2148-2150

Meyer, T. E. & Cusanovich, M. A. (1972) Biochim.Biophys. Acta 267, 383-387

Morton, R. K. (1958) Rev. Pure Appl. Chem. 8,161-220Newton, B. A. (1968) Annu. Rev. Microbiol. 22, 109-130Offord, R. E. (1966) Nature (London) 211, 591-593Patchornik, A. & Sokolovsky, M. (1964) J. Amer. Chem.

Soc. 86, 1206-1212Perini, F., Kamen, M. D. & Schiff, J. A. (1964) Biochim.

Biophys. Acta 8, 74-90Pettigrew, G. W. (1972) FEBSLett. 22, 64-66Pettigrew, G. W. (1973) Nature (London) 241, 531-533Pettigrew, G. W. (1974) Biochem. J. 139, 449-459Porra, R. J. & Jones, 0. T. G. (1,963) Biochem. J. 87,

186-192Porter, R. R. (1957) Methods Enzymol. 4, 221-237Ramshaw, J. A. M.,Thompson, E. W. &Boulter, D. (1970)

Biochem. J. 119, 535-539Rydon, H. N. & Smith, P. W. G. (1952) Nature (London)

169, 922-923Schmer, G. & Kreil, G. (1969) Anal. Biochem. 29, 186-192Sletten, K., Dus, K., de Klerk, H. & Kaamen, M. D. (1968)

J. Biol. Chem. 243, 5492-5506Smith, I. (1953) Nature (London) 171,431-4Spackman, D. H. (1963) Fed. Proc. Fed. Amer. Soc. Exp.

Biol. 22, 244Theorell, H. (1938) Biochem. Z. 298,242-267Thompson, E. W., Notton, B. A., Richardson, M. &

Boulter, D. (1971) Biochem. J. 124, 787-791Wang, K.-T. & Wang, I. S. Y. (1968) J. Chromatogr. 27,

318-320Wiener, H., White, W. N., Hoare, D. G. & Koshland,D. E. (1966) J. Amer. Chem. Soc. 88, 3851-3859

1975

cytochromne c from two protozoa, euglena gracilis and

Documents