THE JOVENAL OF BIOI,O~ICAL C~WISTBY Vol. 2’34, No. 12, Deoember 1969

Print.&. in U.S.A.

Cytochromes c, and c, of Azotobacter uineZandii=

Chromatographic Purification, Crystallization,

and a Study of Their Physical Properties*

NORBERT P. NEutirrNt AND R. H. BURRIS

From the Department of Biochemistry, College of Agriculture, University of Wisconsin, Madison 6, Wisconsin

(Received for publication, May 11, 1959)

In 1956, Tissi&res and Burris (1) and Tissi&es (2) described the purification and certain properties of cytochromes c; and cz isolated from the nitrogen-fixing bacterium, Awtobacter wine- ZundG. Because of their spectral similarities to cytochromes c and cl of mammalian tissues, it was suggested that the bacterial pigments may perform similar functions. This paper reports modifications of the purification procedure and some of the physi- cal properties of the purified pigments. In addition, crystalline preparations have been obtained.

METHODS

Organism and Culture Method-Azotiacter vinelandii, Wis- consin strain 0, was grown with vigorous aeration for 16 to 24 hours at 30” in Burk’s nitrogen-free medium (3).

Extract&m and Purafiation of Cytochrmes cd and ccThe pro- cedure used for extraction of the cells was essentially that de- scribed by Tiiieres (2) but included some modifications. After clari6cation of the butanol-water extract with basic lead acetate, the excess lead was precipitated together with the cytochromes by ammonium sulfate added to approximately 70% saturation. Extraction of the precipitate with phosphate buffer dissolved the cytochromes without dissolving the lead sulfate; these modifica- tions increased the recovery of cytochromes. The partially puri- fied mixture of cytochromes was subjected to chromatography on calcium phosphate gel and then on carboxymethyl cellulose. A total yield of 15 to 25 mg of cytochrome c6 and 30 to 50 mg of cytochrome c4 was obtained from 200 g of bacteria (dry weight basis).

Calcium Phosphate Gel-This was prepared as described by Tiselius et al. (4). The cytochrome solution was dialyzed against 0.001 M phosphate buffer of pH 6.8 and passed through a short, wide column of the gel (example described in Fig. 1). The ma- terial which passed through in about 3 column volumes was con- centrated by adding ammonium sulfate to 70% saturation and dialyzing the precipitate against 0.001 M sodium acetate-acetic acid of pH 5.0.

Carboxymthyl CeUulo8e-Preparations of this exchanger, con-

* Published with the approval of the Director of the Wisconsin Agricultural Experiment Station. This investigation was sup- sorted in Dart bv the Research Committee of the Graduate School with fund; supplied by the Wisconsin Alumni Research Founda- tion and by grants from the Rockefeller Foundation and the Na- tional Science Foundation.

t Present address, The Rockefeller Institute, New York 21, New York.

taiuing 0.5 to 0.7 milliequivalent of tit&able carboxyl groups pe g, were obtained with the procedure of Peterson and Sober (5) with Whatman standard grade cellulose powder. The column was equilibrated with 0.001 Ed sodium acetate-acetic acid of pH 5.0, and after addition of the sample in a small volume, develop- ment was performed by gradient elution with a solution whose concentration approached a final value of 0.05 M in sodium ion; alternatively, 0.02 M sodium acetate-acetic acid buffer was used as the developing solvent. Pooled fractions were concentrated by adding ammonium sulfate to 70 $$ saturation. All procedures were performed at O-5’, except for the crystallization which was done at room temperature.

Analytical Procedures--Nitrogen was determined by the method of Johnson (6) to establish the protein content of purified cyto- chromes and to provide a basis for calculating their percentage iron; protein was assumed (2) to equal total nitrogen X 6.25. To determine the approximate concentration of protein in frac- tious eluted from columns, aliquots were analyzed with the Lowry modification of the Folin phenol reagent (7) or by measurement of absorption at 270 to 280 rnb in a Beckman DU spectrophotom- eter. The anthrone reagent of Morris (8) served for the determi- nation of carbohydrate with glucose as a standard. Analysis for total iron was performed on cytochrome samples dialyzed for 48 hours against glass distilled water, with a method essentially as described by Dr. Helmut Beinert. This involved a digestion of the cytochrome with sulfuric acid and hydrogen peroxide, ad- justment of the pH with acetate buffer previously freed from iron with “bathophenanthroline,” addition of 4,7-diphenyl-l , lo- phenanthroline (“bathophenanthroline”), and extraction of the colored complex with isoamyl alcohol for calorimetric analysis. Cytochrome concentrations were determined by measuring ab- sorption of the reduced form at 550 to 555 rnp, with a millimolar extinction coefficient of 23.3 (2). Spectra were measured with a Gary model 11 recording spectrophotometer. A Hartridge re- version spectroscope served for the estimation of approximate locations of absorption bands.

Ekctrophoresis and Se&nent&on-The Spinco model H elec- trophoresis apparatus -with schlieren optics was used for moving boundary experiments. Solutions were dialyzed for 24 hours before analysis. The schlieren patterns were photographed on Kodak Royal Pan Film with a Corning 2418 6lter in front of the light source. Mobil&s were calculated from descending bound- aries as described by Alberty (9). Sedimentation studies were performed with a Spinco model E ultracentrifuge with schlieren optics. Exposures of 60 seconds on Kodak spectroscopic plates

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December 1959 N. P. Neumann and R. H. Burris

with a Corning 2418 filter were found suitable. Lyophilized samples were dissolved in the buffer, and samples in solution were dialyzed against the buffer for 24 hours before analysis.

RESULTS

Purifccation-Calcium phosphate gel has a low affinity for cyto- chromes c4 and cg, and this property was used to advantage for the removal of protein impurities as shown in Fig. 1. Columns adsorbed variable amounts of cytochromes, but the bulk of the pigments passed through in three column volumes. The small amount of adsorbed cytochromes (these can be cluted at higher ionic strengths) probably consisted of complexes with other pro- teins, since these fractions contained higher amounts of total protein relative to the quantity of cytochrome present. An ex- amination of the material from the main peak showed that the ratio of absorption at 270 rnp to absorption at 550 rnp was higher for the fractions from the trailing edge than for the .fractions from the leading edge. This suggests that protein-protein dii- placement occurred; such displacement would give a somewhat better purification than could be expected from a batchwise ad- sorption and elution.

Passage through a column of carboxymethyl cellulose separated the crude cytochrome solution obtained from the calcium phos- phate gel column into a number of red bands. Over 90% of the total pigment was recovered in two major fractions. Fig. 2 rep- resents a typical column. The sample was added to the column in acetate buffer of pH 5.0 (0.001 M in sodium ion) and formed a narrow dark red band at the top. The column was developed with a gradient of sodium ion; to a constant volume mixing cham- ber containing 100 ml of 0.001 M buffer was added 0.01 M buffer from a reservoir. The solution in t,he reservoir was changed to 0.05 M at Fraction 18 and 0.25 M at Fraction 73. Material from the fastest moving band (Peak I) was opalescent and yellow, and it gave a strongly positive test for carbohydrate with anthrone. It had a surprisingly high iron content, although spectral analy- sis indicated no cytochrome bands. Data for the various frac- tions are given in Table I. The two major fractions (Peaks II and V) had the spectral properties of cytochromes c5 and ~4, re- spectively, and the physical studies reported in this paper were done with these samples. Rechromatography of these samples on separate columns yielded a single peak in each case, demon- strating that the multiple bands separated from the crude extract by columns of carboxymethyl cellulose could not be attributed to compounds arising from the main constituents by alteration on the column.

Cytoehrome c6 purified in the above fashion crystallized readily from solution, when solid ammonium sulfate was added slowly until a slight turbidity was reached. The pH was maintained close to 7 by the continual addition of concentrated ammonium hydroxide. Crystals, in the form of thin pink needles, were ob- tained from solutions of cytochrome c4 only once. Figs 3 and 4 show the rosette crystals of the oxidized form of cytochrome c5 and the plate-like crystals of the reduced form, respectively.

Properties of Isolabd Pigments-Fig. 5 gives a spectrum of cyto- chrome c6. The spectrum of c4 is not shown, as it is similar to that given in Tissieres’ paper (2). The spectral properties of the two pigments are summarized in Table II.

When the purified samples were subjected to moving boundary electrophoresis, the patterns shown in Fig. 6 were obtained. The cytochrome c4 contained about 7 ‘% of a rapidly moving colorless impurity. The colored boundary split into two peaks for each

x 0.8 .% ii 0.6 8

0 270 mp m 551 m/r

O.&M - 0.5M ]

Fraction number FIG. 1. Chromatography of crude cytochromes on a 1.6 cm di-

ameter 3 cm high column of calcium phosphate gel at 25”. Flow rate was 8 ml per hour and the fraction size was 4 ml. Load on column, 9 mg of prot.ein. Elution was performed with pH 6.8 phosphate buffer increasing stepwise in concentration from 0.001 Y to 0.05 Y.

20 40 60 so Fraction number

FIG. 2. Chromatography of cytochromes (purified with calcium phosphate gel) on a 44 X 1.9 cm column of carboxymethyl cellulose at 5”. Flow rate was 20 ml per hour. Fraction size was 5 ml, except Fractions 12 to 16 which contained a total of 750 ml. Elu- tion was performed with a buffer gradient as described in the text. The optical density recorded at 556 mp should not be interpreted in absolute terms, for samples were measured in 18 mm diameter test tubes in a calorimeter, and the compounds were not reduced with Na&04 before measurement. Protein was measured by the Lowry method (7) with cytochrome c (Sigma Chemical Company) as a standard.

pigment. The measured mobiities are given in Table III. The splitting of the boundary does not indicate that each preparation is a mixture of cytochromes cd and ~5, for samples removed from both the ascending and descending limbs of the electrophoresis cell had absorption peaks which were in identical positions in the visible region after reduction with Na$Sz04.

Studies of sedimentation velocity in 0.1 ionic strength buffer of pH 5.0 revealed a single sedimenting boundary for each pig- ment. Sedimentation coefficients (corrected to water and 20”) of 2.4 S and 1.34 S were obtained for cytochromes c4 and cg, re- spectively. The value of 2.4 S was independent of the concen- tration of cytochrome CP between the limits tested (0.26 to 1.1%). Cytochrome cg was tested at a single concentration of 0.51%.

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3288 Cytochromes c4 and 12, of Axotobacter vinelandii Vol. 234, Xo. 12

TABLE I

Properties of fractions obtained by chromatoyraphy of extracts

from Azotobacter vinelandii on carboxymethyl cellulose

Peak IrOn a-Band

I II (cd

III IV v (cd

VI

%

1.55 0.48

0.33 0.11 0.50 0.24

none 554-555 mp 554-555 rnp

550-551 mp 0.79 553-554 mp 1.82

Optical density (270 mp) Optical density @-band)

0.88

FIG. 3. (top) Crystalline oxidized cytochrome cg FIG. 4. (bottom) Crystalline reduced cytochrome cg

Several attempts were made to determine the molecular weight of crystalline cytochrome c5 by sedimentation equilibrium measure- ments in 0.1 ionic strength acetate buffer of pH 5.0. Studies made with protein concentrations of 0.26 to 0.75yo and centri- fuge speeds of 12,590 and 20,410 r.p.m. yielded schlieren patterns which indicated the presence of aggregated material. The re- fractive index gradient was very steep at the bottom of the cen- trifuge cell and very shallow in the middle and near the air- liquid interface at the top. The very broad boundary observed in the sedimentation velocity run indicates the presence of sev- eral molecular species. Representative patterns are shown in Fig. 7.

9 h 1.6 - -Reduced .S 1 UJ /I 5 1.2 - a Oxidized-/ 1

300 350 400 450 500 550

Wave length - m,u

FIG. 5. Spectrum of crystalline cytochrome cg

TABLE II

Spectral properties of cytochromes cq and cj isolated by chromatography on carboxymethyl cellulose

I ‘4 I’ cs

a-Band (reduced). p-Band (reduced). -r-Band (oxidized). T-Band (reduced). &Band (reduced). . Ratio (-r/a) (reduced) Ratio (01/p) (reduced). Ratio (a/270 m).

550-551 mp

522 rnr 409 mp 414 mj.4

314 mp 6.5

1.3 1.27

-I

-

554-555 rnp

524 rnb 414 mp

418 mp 318 mp 6.0

1.4 1.19

TABLE III

Electrophoretic mobilities of puri$ed cytochromes cd and cg

Mobility

Fast component Slow component -

cnz2 Yell-’ sec.7

Cytochrome c$.. -2.35 X 1O-6 -1.89 x 10-s Cytochrome cg.. -2.36 X 1O-6 -1.87 X 10-e

C4 75Ji; 75 min. _I.

230 min. 2z - 220 min. . C 5 220min.

--AA L 643 min. 643 min

0 b Ascending 0

FIG. 6. Electrophoresis of cytochromes cd and cg in tris(hy- droxymethyl)aminomethane acetate buffer, pH 8.6, 0.1 ionic strength.

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December 1959 N. P. Neumann and R. H. Burris

DISCUSSION

The use of chromatography on columns of carboxymethyl cellu- lose in place of paper elcctrophoresis for the separation and purify- cation of cytochromes cd and cb has yielded a preparation of c4 which is purer both on the basis of spectral properties (lower ratio of optical density at 270 mp to the optical density at 550 to 551 mp) and iron content than that described by Tissieres (2). A major advantage of this modification has been the relatively large amount of material which can be fractionated at one time.

The large number of cytochrome components obtained from the column is a reflection of the high resolving power of carboxy- methyl cellulose. Since the extensive adoption of chromato- graphic methods for protein fractionation, it has become evident that many enzyme preparations can be separated into a number of enzymatically indistinguishable components whose only ob- vious difference lies in their chromatographic behavior. Nozaki et al. (10) have demonstrated that during isolation of yeast cyto- chrome c, several chromatographic species appear during extrac- tion with a variety of mild reagents. The small amounts of the minor components obtained with the azotobacter cytochromes have hindered any extensive studies of their properties.

The presence of variable amounts of carbohydrate gum in a number of batches of harvested cells presented a difficulty in this work. The gum seriously interfered with some of the fractiona- tion procedures and prevented adsorption of the cytochromes to carboxymethyl cellulose. Preliminary results indicate that most of this gum can be removed from cytochrome solutions by adsorb- ing it on diethylaminoethyl cellulose.

The two major components which have been isolated cor- respond closely in spectroscopic properties to the cytochromes c4 and c5 described by TissiZres (2). The slight differences in the positions of the absorption maxima of our preparations and those of Tissieres are not fully understood, but they may reflect dif- ferences in calibration or experimental technique of measurement. The properties of cytochrome cd are altered in some fashion dur- ing chromatographic purification, or an impurity accompanies the fraction, for if cytochrome c4 from a column of carboxymethyl cellulose is precipitated with ammonium sulfate, a part of it be- comes insoluble in distilled water or phosphate buffer. The ma- terial from the column is enzymatically active (the cytochrome c reductase system in the electron-transport particle preparation of Bruemmer et al. (11) was used with succinate as substrate to measure enzymatic activity) before ammonium sulfate precipita- tion, and it does not react with carbon monoxide as determined from spectrophotometric analysis; hence, the pigment is not de- natured extensively by the chromatographic procedure. How- ever, the insoluble fraction carries cytochrome c4, because its sharp a-band is visible in a suspension of the precipitate observed with a microspectroscope. It may be that the pigment becomes associated with an insoluble impurity.

Cytochrome c5, on the other hand, remains completely and readily soluble after repeated crystallizations from ammonium sulfate. If the crystals of the reduced form stand at room tem- perature over a period of weeks, oxidation gradually occurs, and the pigment becomes modified as evidenced by an increase in absorption in the ultraviolet region relative to that in the region of 550 rnp. According to Hagihara et al. (12), mammalian cyto- chrome c is more stable in the reduced form, and probably the same is true for the bacterial pigment.

FIG. 7. Ultracentrifuge pattern for cytochromes c4 and cg. A and ATE, cytochrome cq centrifuged for 9 and 73 minutes. Bp and BTS, oytochrome cg centrifuged for 9 and 73 minutes. Samples were dialyzed for 24 hours against a solution containing 0.1 M

KCI, 0.01 M potassium acetate, and acetic acid to bring the pH to 5.0. Protein concentration, 0.53% in A and 0.51% in B. Cen- trifuge speed, 59,780 r.p.m.

The anomalous electrophoretic behavior of the purified pig- ments may indicate the presence of so called “modified” forms similar to those which have been obtained chromatographically on Amberlite IRC-50 from mammalian cytochrome c prepara- tions (13). On the other hand, it may simply reflect a difference in mobilities of the oxidized and reduced forms.

On the basis of iron contents (Table I), the minimum molecular weights of cytochromes c4 and c5 are 11,200 and 11,600, respec- tively. Apparently, cytochrome c5 undergoes molecular associa- tion at pH 5, whereas c4 does not. These differences indicate the need for obtaining accurate molecular weights based upon physi- cal data.

It has been tempting to emphasize the spectroscopic similari- ties between these bacterial pigments and the two mammalian cytochromes, c and cl, for there may be a parallel in the functions which they perform. The molecular constitutions of the bac- terial cytochromes and cytochrome c and cl may, however, be quite different.

SUMMARY

1. A modified procedure, employing chromatography on car- boxymethyl cellulose, has been described for the isolation of cyto-

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3290 Cytochrmes c4 and cs of Azotobacter v&elan&i Vol. 234, Ko. 12

chromes cq and c5 from the nitrogen-fixing microorganism, ,4zo- cultures of Azotobacter uinclandii, and to Dr. 1~. Jlagihara for

tobacter vinelandii. helpful discussions regnrding crystallization techniques.

2, Cytochromes c4 and c6 have been obtained in crystalline form.

3. The purest preparations of cytochromes cd and c5 contained 0.50 and 0.48 % iron, respectively. Calculated molecular weights on the basis of I atom of iron per molecule are 11,200 and 11,600, respectively.

4. Each cytochrome gives a single moving boundary during sediment&ion. The broad boundary for cs indicated the prcs- cnce of aggregates.

5. Electrophorctic experiments demonstrated the presence of about 7% impurity in cytochrome c1 prepared by the method described. The main colored boundary for each pigment splits into two peaks during electrophoresis; the significance of this is discussed.

AcknowZedgment-The authors are indebted to Dr. R. Xl. Bock and Dr. R. L. Baldwin for their valuable advice and assistance in performing the sedimentation and electrophoretic studies, to Dr. 81. J. Johnson and his associates for aid in growing large scale

REFERENCES

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2. TISSI&RES, A., Biochem. J., 64, 682 (1956). 3. NEWTON, J. W., WILSON, P. W., AND BURRIS, R. H., J. Biol.

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Norbert P. Neumann and R. H. BurrisPurification, Crystallization, and a Study of Their Physical Properties

ChromatographicAzotobacter vinelandii: of 5c and 4cCytochromes

1959, 234:3286-3290.J. Biol. Chem. 

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