Proc. NatL Acad. Sci. USA Vol. 79, pp. 2447-2450, April 1982 Biochemistry

Affinity chromatography-purification of c binding (Saccharomyces cerevisiae/oxidase/reductase/) ANGELO AZZI, KURT BILL, AND CLEMENS BROGER Medizinisch-chemisches Institut, Universitat Bern, 3000 Bern 9, Switzerland Communicated by E. Margoliash, December 18, 1981 ABSTRACT An efficient affinity chromatography procedure with the resin, making the binding site inaccessible. for the isolation of mitochondrial and re- The technically simplest solution was found by using Sac- ductase is described. Saccharomyces cereviiae cytochrome c was charomyces cerevisiae cytochrome c. It contains, at position used as a , bound to a thiol-Sepharose 4B gel through cys- 107, a residue (16) not essential for enzymatic activity. teine-107. In this way, the site of interaction of cytochrome c with This group of S. cerevisiae cytochrome c was covalently bound cytochrome oxidase and reductase remained unmodified and to an activated thiol-Sepharose (Fig. 1). Because cysteine-107 available for binding to a number of partner enzymes. The pro- most probably is located at the rear part ofthe , the front cedure is adequate for the purification ofall those having and top areas remain free for binding with high affinity and in common the property of binding with high affinity to cyto- specificity to partner enzymes. chrome c-e.g., cytochrome c oxidase, reductase, and peroxidase, , and reaction centers of photosynthetic bacteria. MATERIALS AND METHODS Several enzymes have the property of interacting with cyto- Materials. Triton X-100 was obtained from Fluka, cyto- chrome c. They are cytochrome c oxidase, cytochrome cl, cy- chrome c type VIII (S. cerevisiae) and type VI (horse heart) were tochrome b5, sulfite oxidase, and cytochrome c peroxidase. The from Sigma, activated thiol-Sepharose 4B was from Pharmacia reaction centers ofphotosynthetic bacteria interact probably in (it consists ofa Sepharose 4B covalently linked to a glutathione a similar way with cytochrome c. Moreover, these enzymes bind spacer with its cysteine residue activated by a 2-thiopyridyl to the same or a similar surface region of cyt6chrome c (1). group), and Affi-gel 10 was from Bio-Rad. Coenzyme Q1 was The region of cytochrome c at which the binding occurs in- a gift from Hoffman-LaRoche (Basel, Switzerland). All other cludes mainly lysine residues 8, 13, 27, 72, and 87 and it is de- reagents used were of the highest purity commercially available. fined as the front (exposed edge) and top left part of the Preparation of Mitochondria and Mitoplasts. All manipu- molecule (2). This conclusion has been reached from a study of lations were performed at 0-4°C. Mitochondria were prepared the enzymatic reactivity ofcytochrome c molecules chemically from bovine heart according to Smith (18) and stored at -80°C. modified at single but different lysines (3). With cytochrome They were depleted of cytochrome c by the method of Jacobs c oxidase, the enzymatic activities ofthe single-lysine modified and Sanadi (19). Cytochrome c-depleted mitoplasts were stored c (4) follow the order 13 < 72 < 87 < 8 < 27; with at -80°C ifnot used immediately; after storage, before use they the reductase (5) the order is 86 = 87 < 13 < 72 < 27 (the were homogenized in a glass/Teflon homogenizer. The mito- numbers represent the residue that has been chemically mod- plasts were suspended in 50 mM Tris HCl (pH 7.2) containing ified). In essential agreement with the above conclusion are the 1% Triton X-100. The concentrations ofcytochromes aa3, b, and data of Smith et al and Ahmed et al. (6, 7) who used trifluo- cl were calculated by using the extinction coefficients (reduced roacetyl- and trifluoromethylphenylcarbamoyl derivatives of minus oxidized) of 12 mM-1 cm-f1 (605-630 nm), 25.6 mM- cytochrome c. Rieder and Bosshard (8, 9) used another ap- cm1 (562-577nm), and20.1 mM-l cm- (553-540O, respectively. proach-namely, differential chemical labeling-and con- Analyses. The protein concentration was estimated by using cluded that the same lysine residues are involved similarly in the method of Wang and Smith (20) or of Spector (21). Poly- the interaction of cytochrome c with all cytochrome c binding acrylamide gel electrophoresis was carried out for cytochrome enzymes (1). c oxidase and for cytochrome c reductase according to Laemmli Interestingly, the lysine residues involved in binding are the (22) on 14% gels. Slab gels were scanned in a special gel scanner most chemically reactive (10, 11). The high affinity of the in- attached to the Aminco DW-2a spectrophotometer (23) at 400 teraction between cytochrome c and its partner enzymes (12, minus 430 nm to localize absorption (cytochrome cl) and 13) and the definition of the area involved in the binding have were photographed after staining with Coomassie blue. Bovine been necessary prerequisites for devising a chromatographic serum albumin, carbonic anhydrase, , and cyto- procedure that uses cytochrome c as an affinity ligand. This chrome c were used as molecular weight standards (67,000, permitted the purification of a number of enzymes specifically 29,000, 17,000 and 12,000, respectively). interacting with cytochrome c. In such a procedure, the area Phosphorus was determined according to Chen et al. (24). involved in the binding of cytochrome c to cytochrome c oxi- The enzymatic activity ofcytochrome c reductase was measured dase, reductase, peroxidase, etc. has to be left available (14). by using the method of Wan and Folkers (25). The activity of Thus, the most obvious technique of linking cytochrome c to the oxidase was assayed polarographically according to Rosevear a CNBr-activated Sepharose gel had to be discarded (15) be- et al. (26) with lauryl maltoside as detergent. cause the functionally important lysine residues would react RESULTS The publication costs ofthis article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertise- Preparation of the Affinity Gel. S. cerevisiae cytochrome c ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact. (75 mg) was dissolved in 25 ml of50 mM Tris HCl (pH 7.2) and 2447 Downloaded by guest on September 23, 2021 2448 Biochemistry: Azzi et al. Proc. Natl. Acad. Sci. USA. 79 (1982)

IS-SN~~~' &O+ HSt>* S St

GLUTATHIONE CYTOCHROME GLUTATHIONE CYTOCHROME c SPACER CYOHO ESPACER CTCRM SEPHAROSE SEPHAROSE

FIG. 1. Scheme of the reaction of S. cerevisiae cytochrome c with activated thiol-Sepharose 4B, shown as a side view (17) of horse heart cyto- chrome c with a cysteine substituted in position 107 to simulate the situation with S. cerevisiae cytochrome c. Square, heme; solid circle, ; open circles, functionally important lysine residues. At position 13 in yeast cytochrome c, an arginine replaces a lysine of horse heart cytochrome c. The region delimited by the arc is that involved in the binding of cytochrome c to other proteins.

added to a suspension of25 ml ofswollen and washed activated, bound to the swollen gel was 0.125 Amol/ml. The stability of thiol-Sepharose 4B suspended in the same buffer. After the the gel has been found to depend on the loading material which, suspension was stirred overnight, most ofthe cytochrome c was in some cases (presence of reducing substances?), produced a bound to the gel, as judged from the practically complete ab- small release ofcytochrome c. Proteolytic degradation may also sence of color in the supernatant after sedimentation of the occur in the absence ofinhibitors ofbacterial growth, although Sepharose. Then, 50 ml of 1.5 mM 2-mercaptoethanoV50 mM in this condition we were able to use several columns for periods Na acetate, pH 4.5, was added to block nonreacted thiol groups. of at least a month. Regeneration of the gel has been possible The suspension was poured into a column (2 x 20 cm) and (27). washed with 1 liter of 50 mM Tris HCl, pH 7.2/1% Triton X- Two control gels were prepared. One used thiol-activated 100/1 M NaCl/1 mM K ferricyanide. The washing had the ef- Sepharose 4B, treated as above, except that cytochrome c was fects of removing noncovalently bound cytochrome c and oxi- omitted. The second, with a N-hydroxysuccinimide active ester dizing the bound fully. derivative of crosslinked agarose beads (Affi-Gel 10), reacted Finally, the gel was equilibrated with 50 mM Tris HCl, pH with horse heart cytochrome c via its amino groups in 0.1 M 7.2/1% Triton X-100. The amount of cytochrome c remaining bicarbonate buffer at pH 8.0.

1.5 1.5

1.0 .- 0 E 0)

0.5

0.0 10 20 30 40. 50 60 70 80 90 100 Fraction

FIG. 2. Elution pattern of cytochrome c oxidase and reductase. Elution was obtained with 50 mM Tris'HCl, pH 7.2/0.1% Triton X-100 and a linear 0-75 mM NaCl gradient (broken line). The concentration of NaCl can be calculated by multiplying they axis by 100. *, Protein; 0, ; i, cytochrome aa3. Downloaded by guest on September 23, 2021 Biochemistry: Azzi et al. Proc. Natl. Acad. Sci. USA 79 (1982) 2449

Binding ofCytochrome c Oxidase and Reductase. Mitoplasts Origin (2 mg/ml in 50 mM Tris HCl at pH 7.2) prepared from beef heart mitochondria (28) and depleted ofcytochrome c (19) were solubilized by addition of1% Triton X-100. After centrifugation, 400 ml of the solution containing 1.1 mg of protein per ml was applied to the affinity gel. During loading, absorbances of cy- tochromes aa3, b, and cl did not appear in the eluate until sat- -mmono __. I "Mma uration of the column was reached. * Alternatively, the gel could be added to the Triton X-100 I - _ II extract and subsequently poured into a column. The control gels did not bind appreciable amounts of cytochromes. - III Elution of Cytochrome c Oxidase and Reductase. As a con- " IV sequence ofwashing with 50 mM Tris HCl, pH 7.2/0. 1% Triton II-- Nou X-100, 70% ofthe applied protein, 60% ofthe , and 50% -V ofthe heme b + cl absorbance could be detected in the eluate. III -- vow Subsequently, a linear NaCl gradient (0-175 mM) was applied to the column containing the gel. At a flow rate of 35 ml/hr, 5-ml fractions were collected and analyzed for their protein and IV-e-l cytochrome contents. Elution of cytochrome c oxidase was - VI around 30 mM NaCl; that of the b/c1 complex was around 90 mM. The oxidase appeared in the first 150 ml of eluate followed VI-- _ - VII by 75 ml of eluate in which both cytochrome c oxidase and re- VI- ductase were present and by 200 ml containing only cytochrome - VIII c reductase (Fig. 2). The recovery of the heme a after elution VII- was about 60% of the amount retained by the gel, that ofheme b + cl was 75%, and the rest was found in mixed fractions. Analysis of the Eluted Cytochromes. The fractions around the center ofthe elution peaks were pooled (15-25 and 60-80), Ox. Std. Red. FIG. 4. Polypeptide patterns of cytochrome c oxidase and of the b- cl complex on 14% polyacrylamide gels (22). Ox., cytochrome c oxidase; Std., molecular weight standards; Red., cytochrome c reductase.

concentrated on an Amicon filter (PM-30), and analyzed. The spectra obtained (reduced minus oxidized) have the character- a) istics of pure cytochrome c oxidase and b-c1 complex (Fig. 3). Q Absolute spectra ofcytochrome c oxidase were also measured. -o 0 They fulfilled the purity criteria of Lemberg (29). ~0 The polyacrylamide gel electrophoretic patterns are shown in Fig. 4. Seven and eight major polypeptides for cytochrome c oxidase and reductase can be observed, respectively. The heme content ofthe oxidase (=10 nmoVmg ofprotein), its activity (>100 electrons/sec per molecule of oxidase), and its lipid content (35 mol/mol ofenzyme) define this preparation as one of high purity (30). Similarly, with the reductase 6.5 nmol of heme b and 3.3 nmol ofheme cl were present per mg ofprotein, characteristics of a good preparation (31). The enzymatic activity (S-mol of cy- tochrome c reduced per mol of reductase per sec) w~ com- pletely antimycin sensitive. The relative amount of subunit V (Fig. 4) with respect to the other subunits was variable and par- a) alleled the activity of the complex. More subunit V was ob-

0 served when solubilization and elution were done in the pres- ence of 1 mM MgCl2; there was less in the presence of 1 mM 0 EDTA. Subunit V is the polypeptide corresponding to the iron- protein of the b-c1 complex (32). DISCUSSION The rationale for coupling cytochrome c to a gel, which can be used for purification ofa number ofcytochrome c binding pro- 500 550 teins by affinity chromatography, proved to be correct. From Wavelength, nm the same considerations it is now clear why those procedures FIG. 3. Difference spectra (dithionite reduced minus oxidized) of in which cytochrome c is linked to the gel via its essential E- cytochrome c oxidase and reductase obtained by affinity chromatog- amino groups did not lead (5, 33) to a useful affinity chroma- raphy. (A) Cytochrome c oxidase (fractions 15-25 of Fig. 2). (B) Cy- tography method. It appears from Fig. 1 that the region of cy- tochrome b-cl complex (fractions 60-80 of Fig. 2). tochrome c that binds to its partner enzymes is opposite to the Downloaded by guest on September 23, 2021 2450 Biochemistry: Azzi et al. Proc. Natl. Acad. Sci. USA 79 (1982)

Table 1. Proteins for which preliminary purification attempts by 1. Rieder, R. & Bosshard, H. R. (1980) J. BioL Chem. 255, the yeast cytochrome c Sepharose 4B affinity chromatography 4732-4739. procedure have been successful 2. Dickerson, R. E. & Timkovich, R. (1975) in The Enzymes, ed. Boyer, P. D. (Academic,,New York), Vol. 11k, pp. 397-547. Source 3. Brautigan, D..L., Ferguson-MNiller, S. & Margoliash, E. (1978)J. Bovine heart mitochondria, rat liver Biol. Chem. 253, 130-139. Cytochrome c oxidase 4. Ferguson-Miller, S., Brautigan, D. L. & Margoliash, E. (1978)J. mitochondria, maize mitochondria, Biol Chem. 253, 149-159. Rhodopseudomonas spheroides, 5. Speck, S. H., Ferguson-Miller, S., Osheroff, N. & Margoliash, Paracoccus denitrificans, Bacillus E. (1979) Proc. NatI Acad. Sci. USA 76, 155-159. subtilis 6. Smith, H. T., Staudenmayer, N. & Millett, F. (1977) Biochem- b-cl complex Bovine heart mitochondria, rat liver istry 16, 4971-4974. mitochondria 7. Ahmed, A. J., Smith, T. H., Smith, M. B. & Millett, F. (1978) cl Bovine heart mitochondria Biochemistry 17, 2479-2483. Cytochrome c peroxidase S. cerevisiae 8. Rieder, R. & Bosshard, H. R. (1978) J. Biol. Chem. 253, 6045-6053. 9. Rieder, R. & Bosshard, H. R. (1978) FEBS Lett. 92, 223-226. region that links the protein to the column. Although the 10. Wada, K. & Okunuki, K. (1969)J. Biochem. (Tokyo) 66, 249-272. scheme of Fig. 1 was obtained by using the protein backbone 11. Bisson, R., Azzi, A., Gutweniger, H., Colonna, R., Montecucco, ofhorse heart cytochrome c, the highly conservative C. & Zanotti, A. (1978)J. Biol Chem. .253, 1874-1880. 12. Brautigan, D. L., Ferguson-Miller, S. & Margoliash, E. (1978) sequence and the resulting similar three-dimensional structure Methods EnzymoL 53, 128-164. of cytochrome c molecules from different sources permit such 13. Weiss, H., Juchs, B. & Ziganka, B. (1978) Methods EnzymoL 53, an extrapolation. Yeast cytochrome c has a high affinity for 98-112. mammalian cytochrome c binding enzymes, thus satisfying an 14. Bill, K., Casey, R. P., Broger, C. & Azzi, A. (1980) FEBS Lett. important requirement in affinity chromatography. 120, 248-250. The data indicate also that the complex and the cyto- 15. Ozawa, T., Okumura, M. & Yagi, K. (1975) Biochem. Biophys. b-cf Res. Commun. 65, 1102-1107. chrome c oxidase from beef heart mitochondria have different 16. Dickerson, R. E., Takano, T., Eisenberg, D., Kallai, 0. B., Sam- affinities for S. cerevisiae cytochrome c, thus permitting elution son, L., Cooper, A. & Margoliash, E. (1971) J. BioL Chem. 246, of them separately. The conclusion that this technique repre- 1511-1555. sents a real affinity purification procedure for cytochrome c 17. Dickerson, R. E. (1972) Sci. Am. 226, 58-72. oxidase and reductase was reached by using control columns. 18. Smith, A. L. (1967) Methods EnzymoL 10, 81-86. Thus, purification ofcytochrome c oxidase and reductase is not 19. Jacobs, E. E. & Sanadi, D. R. (1960)J. BioL Chem. 235, 531-534. 20. Wang, C. S. & Smith, R. L. (1975) AnaL Biochem. 63, 414-417. the consequence ofunspecific hydrophobic or polar interactions 21. Spector, T. (1978) Anal. Biochem. 86, 142-146. of the detergent-protein complexes with the gel (34, 35). 22. Laemmli, U. K. (1970) Nature (London) 227, 680-685. The simple technique described above, which can also be 23. Broger, C., Allemann, P. & Azzi, A. (1978)J. AppI Biochem. 1, used on a micro scale, lends itself to a number of applications. 455-459. Proteins interacting specifically with eytochrome c have been 24. Chen, P. S., Toribara, T. & Warner, H. (1956) Anal. Biochem. isolated from crude extracts in this laboratory. Some examples 28, 1756-1758. 25. Wan, Y. P. & Folkers, K. (1978) Methods Enzymol. 53, 591-599. are listed in Table 1. The existence of a simple, rapid purifi- 26. Rosevear, P., VanAken, T., Baxter, J. & Ferguson-Miller, S. cation procedure for cytochrome c oxidase should facilitate iso- (1980) Biochemistry 19, 4108-4115. lating preparations from different organisms which may help in 27. Brocklehurst, K., Carlsson, J., Kierstan, P. Y. & Crook, E. M. understanding the structural and functional role ofthe complex (1974) Methods Enzymol. 34, 531-544. polypeptide composition in eukaryote cytochrome c oxidases. 28. Lenaz, G. & MacLennan, D. H. (1967) Methods Enzymol 10, The column also offers a possibility of extracting subunits of 499-504. 29. Lemberg, M. R. (1969) Physioi Rev. 49, 48-121. multipolypeptide complexes such as cytochrome c oxidase and 30. Hartzell, C. R., Beinert, H., van Gelder, B. F. & King, T. E. reductase, by modifying the elution buffer in terms of pH, de- (1978) Methods Enzymol. 53, 54-66. tergent type, and concentration and presence- of chaotropic 31. Nelson, B. D. & Gellerfors, P. (1978) Methods Enzymol. 53, agents. Finally, such a technique may be valuable in obtaining 80-91. extremely pure samples of enzymes usable for crystallization. 32. Trumpower, B. L. & Katki, A. G. (1979) in Membrane Proteins in Energy Transduction, ed. Capaldi, R. A. (Dekker, New York), The collaboration of Drs. R. Gennis, P. Heinrich, C. Leaver, and B. pp. 89-200. Ludwig in preliminary purification experiments using different starting 33. Weiss, H. & Kolb, J. (1979) Eur. J. Biochem. 99, 139-149. materials is much appreciated. This study was supported by Schweize- 34. Nagasawa, T., Nagasawa-Fujimori, H. & Heinrich, P. C. (1979) rischen Nationalfonds Grant 3.228.077. C.B. is the recipient of a fel- Eur. J. Biochem. 94, 31-39. lowship from Schweizerischen Gesellschaft fur chemische Industrie. 35. Rosen, S. (1978) Biochim. Biophys. Acta 523, 314-320. Downloaded by guest on September 23, 2021