- Email: [email protected]
[6] Affinity chromatography purification of cytochrome-c oxidase from bovine heart mitochondria and other sources
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[6]
(Kay = 0.55; Fig. 4C) also contain dimers. From the estimated sedimentation coefficient of 9.6 S for the monomer-detergent complex (species 3), a net molecular weight for the protein is calculated as 170,000 (Table III). This value differs from the theoretically expected one (200,000). However, it is neither possible to estimate directly the Stokes radius of the monomeric complex by light-scattering measurements nor to evaluate the diffusion coefficient by sedimentation analysis. Therefore the calculated molecular weight, which is based on an assumed Stokes radius of 61 ~,17 is less accurate and an error of 20% has to be admitted in this case.
[6] A f f i n i t y C h r o m a t o g r a p h y P u r i f i c a t i o n o f C y t o c h r o m e - c Oxidase from Bovine Heart Mitochondria and Other Sources B y CLEMENS BROGER, K U R T BILL, a n d ANGELO A Z Z l
In the mitochondrial respiratory chain cytochrome-c oxidase catalyzes the electron transfer from ferrocytochrome c to molecular oxygen. The molecular details of the interaction of the enzyme with its electron donor, cytochrome c, have been studied by using various labeling techniques. It has been established that the "front side" of cytochrome c, which contains the exposed heme edge, is in contact with the oxidase,l,2 and that subunit II of the enzyme complex is interacting with cytochrome c 3 through a domain on its surface which has also been mapped. 4 It has been recognized for a long time that the specific interaction between cytochrome-c oxidase and its electron donor could be useful in developing an affinity chromatography purification technique for cytochrome-c oxidase based on cytochrome c immobilized on a gel matrix. Essentially two types of affinity gels have been used which differ in the way cytochrome c is linked to the gel: The first was developed before details about the interaction of the oxidase with its electron donor were known. In this method cytochrome c is attached to the Sepharose through lysine residues which are mainly located on the front side of the protein and which are now known to be important for the interaction of this protein with the oxidase. In the development of the second method the i S. Ferguson-Miller, D. L. Brautigan, and E. Margoliash, J. Biol. Chem. 253, 149 (1978). 2 R. Rieder and H. R. Bosshard, J. Biol. Chem. 253, 6045 (1978). 3 R. Bisson, A. Azzi, H. Gutweniger, R. Colonna, C. Montecucco, and A. Zanotti, J. Biol. Chem. 253, 1874 (1978). 4 R. Bisson, G. C. M. Steffens, R. A. Capaldi, and G. Buse, FEBS Lett. 144, 359 (1982).
METHODS IN ENZYMOLOGY, VOL. 126
Copyright© 1986by AcademicPress, Inc. All rightsof reproductionin any formreserved.
[6]
AFFINITY CHROMATOGRAPHY PURIFICATION OF CYTOCHROME OXIDASE
65
new information about the interaction between the two proteins has been taken into consideration by attaching cytochrome c through its rear side. The application of the two gels for the purification of enzymes which interact specifically with cytochrome e is straightforward. In general, the solubilized proteins are loaded at low salt concentration to a column containing the gel and, after washing the column, they are eluted by increasing the ionic strength of the buffer. Various detergents, mainly of the nonionic type, have been used in the solubilization step and during the chromatography procedure. If the solubilization step is optimized and the protein of interest can be released from the membrane in a rather selective way, it is possible in many cases to purify it to homogeneity in one affinity chromatography step. The cytochrome c gels described below can be used for several purifications. They are washed after every use with 1 M NaC1 and detergent and subsequently stored at 4°.
Horse Heart Cytochrome c Linked to CNBr-Activated Sepharose 4B Materials
For the preparation of 4 ml of gel: Freeze-dried CNBr-activated Sepharose 4B (1 g) (Pharmacia Fine Chemicals AB, Uppsala, Sweden) 200 ml of 1 m_M HCI 10 ml of 0.1 M NaHCO3, pH 8.3 20 ml of 0.1 M NaHCO3, pH 8.0, containing 0.5 M NaC1 Horse heart cytochrome c (4 mg) (Sigma Chemical Company, St. Louis, MO 63178; type VI) 50 ml of 1 M ethanolamine/HC1, pH 8 400 ml of 1 M NaC1 200 ml of 0.1 M sodium acetate, pH 4.0 200 ml of 0.1 M sodium borate, pH 8.0 Preparation 5
CNBr-activated Sepharose 4B (1 g) is swollen and washed for 15 min on a sintered glass filter with about 200 ml of 1 mM HC1, yielding about 4 ml of swollen gel. Alternatively, Sepharose 4B can be activated with cyanogen bromide, as published in this series: Part of the active groups are hydrolyzed by shaking the gel at room temperature for 4 hr in about 10 ml of 0.1 M NaHCO3, pH 8.3. The gel is then incubated at room tempera5 H. Weiss, B. Juchs, and B. Ziganke, this series, Vol. 53, p. 98. 6 I. Parikh, S. March, and P. Cuatrecasas, this series, Vol. 34, p. 77.
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TABLE I PURIFICATION OF CYTOCHROME-C OXIDASE FROM VARIOUS SOURCES BY AFFINITY CHROMATOGRAPHY ON HORSE HEART CYTOCHROME c-CNBr-AcTIVATED SEPHAROSE 4B
Source Bovine heart
Neurospora crassa Rat liver
Comment Further purification of isolated enzyme One-step purification Main purification step Main purification step Final purification step
Detergent
References
Deoxycholate
a
Laurylmaltoside Triton X-100 Tween 80, Triton X-100 Laurylmaltoside
b c d b
a T. Ozawa, M. Okumura, and K. Yagi, Biochem. Biophys. Res. Commun. 65, 1102 (1975). b D. A. Thompson and S. Ferguson-Miller, Biochemistry 22, 3178 (1983). c H. Weiss and H. J. Kolb, Eur. J. Biochem. 99, 139 (1979). d R. J. Rascati and P. Parsons, J. Biol. Chem. 254, 1586 (1979).
ture for 2 hr in 5 ml of 0.1 M NaHCO3, pH 8, containing 0.5 M NaCI and 4 mg of horse heart cytochrome c (1 mg per milliliter of swollen gel). The nonbound cytochrome c is removed by washing the gel with 10 ml of coupling buffer, and the remaining active groups are reacted at 4° for 1 hr with 50 ml of 1 M ethanolamine-HCl, pH 8. The gel is then washed at 4 ° in turn with 1 M NaC1, 0.1 M sodium acetate, pH 4, 1 M NaCI, and 0.1 M sodium borate, pH 8, and subsequently with the buffer which will be used for the affinity chromatography procedure, e.g., 50 mM Tris-HCl, pH 7.4. Applications These are summarized in Table I. A detailed procedure for the purification of cytochrome-c oxidase from Neurospora crassa has been published in an earlier volume in this series: and the procedures for the purification of the enzyme from other sources can be found in the original literature as indicated in Table I. Comments The above-described procedure is not applicable to all starting materials and with all detergents. For example, it was reported that the bovine heart and yeast enzymes cannot be purified in Triton X-100 by using this method. 7 It wa~ assumed that Triton X-100 might not be able to disperse 7 H. Weiss and H. J. Kolb, Eur. J. Biochem. 99, 139 (1979).
[6]
AFFINITY CHROMATOGRAPHY PURIFICATION OF CYTOCHROME OXIDASE
67
the enzyme completely. On the other hand, it was shown that bovine heart cytochrome-c oxidase dispersed in Triton X-100 can be purified by using the affinity gel described below. It has to be stressed that cytochrome c may be attached to the CNBr-activated gel through some of its lysine residues which are known to be crucial for the interaction with the oxidase. This drawback can apparently be overcome, at least partially, by inactivating some of the CNBr-activated groups of the gel, as mentioned above, in order to avoid multipoint attachment of cytochrome c. Indeed the gel is useful in some applications.
Yeast Cytochrome c Linked to Activated Thiol-Sepharose 4B Materials
For preparation of 30 ml of gel: Freeze-dried activated thiol-Sepharose 4B (8 g) (Pharmacia Fine Chemicals AB, Uppsala, Sweden) 75 ml of 50 mM Tris-HC1, pH 7.2 Cytochrome c (90 mg) from Saccharomyces cerevisiae (Sigma Chemical Company, St. Louis, MO 63178; type VIII) 50 ml of 50 mM sodium acetate, pH 4.5, containing 1.5 mM 2-mercaptoethanol 1 liter of 50 mM Tris-HCl, pH 7.2, containing 1 M NaCI, 1% Triton X-100, and 1 mM potassium hexacyanoferrate(III) Preparation s
Activated thiol-Sepharose 4B (8 g) is swollen and washed at room temperature for 15 min with 50 mM Tris-HCl, pH 7.2, yielding about 30 ml of swollen gel. Alternatively, the gel can be prepared from Sepharose 4B according to a method published in a previous volume in this series. 9 The gel is suspended in 50 mM Tris-HCl, pH 7.2, and 3 mg of S. cereoisiae cytochrome c per milliliter of swollen gel are added in the same buffer. The final volume should be about 50 ml. The suspension is shaken gently overnight at 4°, after which time practically all the cytochrome c is bound to the gel. The almost colorless buffer is removed and the gel is incubated for 30 min in 50 ml of 50 mM sodium acetate, pH 4.5, containing 1.5 mM 2-mercaptoethanol, in order to inactivate residual active groups. Under these conditions no covalently bound cytochrome c is removed 8 A. Azzi, K. Bill, and C. Broger, Proc. Natl. Acad. Sci. U.S.A. 79, 2447 (1982). 9 K. Brocklehurst, J. Carlsson, M. P. J. Kierstan, and E. M. Crook, this series, Vol. 34, p. 531.
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[6]
HOOO-CI-I
9 )rz
F16. 1. (A) Schematic drawing of yeast cytochrome c linked to activated thiol-Sepharose 4B. The hatched square in cytochrome c represents the heme plane and the numbers indicate the lysine residues, which are important in the interaction of this protein with cytochrome-c oxidase. Notice that cytochrome c is attached through a cysteine which is located on the opposite side of the molecule. (B) Schematic drawing of yeast cytochrome c linked to Affi-Gel 102 through the heterobifunctional reagent SMPB. The spacer between the gel and cytochrome c is longer as compared to the gel above and not sensitive to reducing agents.
from the gel. The gel is then washed with 1 liter of 50 mM Tris-HCl, pH 7.2, containing 1 M NaCI, 1% Triton X-100 (the detergent to be used for the purification procedure described below), and 1 mM potassium hexacyanoferrate(III) in order to remove not covalently bound cytochrome c and to reoxidize the gel fully. The amount of cytochrome c remaining bound to the gel is about 125 nmol/ml. A schematic drawing of the gel is shown in Fig. 1A.
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AFFINITY CHROMATOGRAPHY PURIFICATION OF CYTOCHROME OXIDASE
69
As an example for the use of the affinity chromatography technique, the purification of cytochrome-c oxidase from bovine heart mitochondria is presented in detail. Materials
The following are used for preparation of 30 ml of affinity gel: Bovine heart mitochondria isolated according to a standard method, such as described in Ref. 10, about 1 g of protein I00 ml of I0 mM Tris-HCl, pH 7.2, containing 150 mM KCI 1 liter of 10 mM Tris-HCl, pH 7.2 500 ml of 50 mM Tris-HC1, pH 7.2 Triton X-100 stock solution, 25% in water 200 ml of 50 mM Tris-HCl, pH 7.2, containing 1% Triton X-100 200 ml of 50 mM Tris-HCl, pH 7.2, containing 0.1% Triton X-100 100 ml of 50 mM Tris-HCl, pH 7.2, containing 0.1% Triton and 50 mM NaC1 The entire procedure is carried out at 4°. The mitochondria are suspended in 50 ml of 150 mM KCI buffered with 10 mM Tris-HCl, pH 7.2, and depleted of their outer membrane and cytochrome c by dilution into 10 volumes of 10 mM Tris-HCl, pH 7.2, and subsequent centrifugation at 27,000 g for 20 min. The pellet of mitoplasts is suspended, diluted, and centrifuged again, and then taken up in 50 mM Tris-HC1, pH 7.2, at a protein concentration of 2 mg/ml. Triton X-100 is added to a final concentration of 1%. After incubation for about 30 min the suspension is centrifuged at 27,000 g for 1 hr. The supernatant has a protein concentration of about 1.1 mg/ml. About 400 ml of this solution are applied at a flow rate of 35 ml/hr to the affinity column (2 × 10 cm), equilibrated in 50 mM TrisHC1, pH 7.2, containing 1% Triton X-100. Only cytochrome-c oxidase and reductase bind to the gel; the other proteins are eluted without retardation. After loading, the column is washed with 50 mM Tris-HC1, pH 7.2, containing 0.1% Triton X-100 until the eluate is free of protein and hemes. Elution of the cytochrome-c oxidase is carried out by adding 50 mM NaC1 to the washing buffer or by applying a gradient of NaCI from 0 to 150 mM in the same buffer. The enzyme is eluted at a salt concentration of 50 mM or less, whereas cytochrome-c reductase remains bound to the column and may be eluted at higher salt concentration. The eluted enzyme can be concentrated by using an Amicon filter PM30. Ratios of 10 nmol heme a per milligram protein are reached with this preparation. The enzyme contains about 30-40 mol of phospholipid per mol of heme aa3. It to A. L. Smith, this series, Vol. 10, p. 81.
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TABLE II PURIFICATION OF CYTOCHROME-C OXIDASE FROM VARIOUS SOURCES BY AFFINITY CHROMATOGRAPHY ON YEAST CYTOCHROME c-AcTIVATED THIOL-SEPHAROSE 4B Source Bovine heart Bacillus subtilis Rhodopseudomonas sphaeroides Paracoccus denitrificans
Comment
Detergent
References
One-step purification One-step purification Main purification step
Triton X-100 Laurylmaltoside Triton X-100
a b c
Final purification step
Triton X-100
d
a A. Azzi, K. Bill, and C. Broger, Proc. Natl. Acad. Sci. U.S.A. 79, 2447 (1982). b W. de Vrij, A. Azzi, and W. N. Konings, Ear. J. Biochem. 131, 97 (1983). c R. B. Gennis, R. P. Casey, A. Azzi, and B. Ludwig, Ear. J. Biochem. 125, 189 (1982). M. Solioz, E. Carafoli, and B. Ludwig, J. Biol. Chem. 257, 1579 (1982).
consists of 12-13 polypeptides, as in the traditional preparations, as analyzed by polyacrylamide gel electrophoresis according to Kadenbach et al. 11 The spectral characteristics of the enzyme are identical to the best conventional preparations. Further Applications
Table II summarizes further applications of the method for the purification of cytochrome-c oxidases from various sources. The general procedures are similar to the one described in detail above. Some technical details can be found in the original literature as indicated in Table II. Comments
It is important to block activated thiol groups after coupling cytochrome c to the gel in order to prevent cysteine-containing proteins to bind covalently to the resin. It was observed that under some circumstances a gel with lower substitution of cytochrome c has a relatively higher capacity for binding of cytochrome-c oxidase (Bernd Ludwig, personal communication). This might be due to less steric hindrance. The method is very useful also for microscale purifications and can be used for the preparation of other proteins which interact specifically with cytochrome c, as cytochrome-c reductase 8 or photosynthetic reaction ~i B. Kadenbach, J. Jarausch, R. Hartmann, and P. Merle, Anal. Biochem. 129, 517 (1983).
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AFFINITY CHROMATOGRAPHY PURIFICATION OF CYTOCHROME OXIDASE
71
centers, which in fact have been copurified with cytochrome-c oxidase from Rhodopseudomonas sphaeroides.12 The preparation of the affinity gel just described is extremely simple and its applicability is wide; it cannot be used, however, under reducing conditions because the S-S bridge between cytochrome c and the column would be broken. Recently, a new type of gel has been proposed 13which not only is stable under reducing conditions, but also has a higher capacity for cytochrome-c oxidase, probably as a consequence of less steric hindrance due to a longer spacer. The preparation of this gel is also simple and uses commercially available chemicals. It is described below.
Yeast Cytochrome c Linked to Affi-Gel 102-SMPB
Materials For preparation of 15 ml of gel: 15 ml of Affi-Gel 102 (Bio-Rad, Richmond, CA 94804) 500 ml of 25 mM sodium phosphate, pH 7.4 75 ml of anhydrous dioxane 20 mg of succinimidyl-4-(p-maleimidophenyl)butyrate (SMPB) (Pierce Chemical Company, Rockford, IL 61105) 12 mg of cytochrome c from S. cerevisiae (Sigma Chemical Company, St. Louis, MO 63178; type VIII) 20 ml of swollen Sephadex G-25 (Pharmacia Fine Chemicals AB, Uppsala, Sweden) 200 ml of 25 mM sodium phosphate, pH 7.4, containing 1 M NaC1
Preparation 13 Swollen Affi-Gel 102 (15 ml) is washed on a sintered glass funnel with 25 mM sodium phosphate, pH 7.4. After being drained from buffer, the gel is washed three times with about 10 ml of anhydrous dioxane and finally suspended in 5 ml of dioxane. SMPB (20 rag) is dissolved in 2 ml of dioxane and added to the gel. The suspension is stirred under nitrogen for 3 hr in the dark. The gel is washed with dioxane to remove unbound SMPB and subsequently with several small portions of 25 mM sodium phosphate, pH 7.4. Finally, it is suspended in l0 ml of the buffer. Yeast cytochrome c (12 mg) is dissolved in the same buffer, reduced by the addition of sodium dithionite, and passed through a Sephadex G-25 column (2 × 20 cm) equilibrated in the same buffer. About 900 nmol of the 12 R. B. Gennis, R. P. Casey, A. Azzi, and B. Ludwig, Eur. J. Biochem. 125, 189 (1982). 13 K. Bill and A. Azzi, Biochem. Biophys. Res. Commun. 120, 124 (1984).
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reduced cytochrome c are added to the gel, and the suspension is stirred for 12 hr at 4 ° in the dark. The gel is washed free of nonbound cytochrome c with 25 mM sodium phosphate, pH 7.4, containing 1 M sodium chloride. More than 80% of the added cytochrome c remains bound to the resin. A schematic drawing of the gel is shown in Fig. lB.
Comments This gel has originally been used for measuring the binding affinities of cytochrome-c oxidase and reductase for oxidized and reduced cytochrome c. It will result in the useful purification of cytochrome-c oxidases from various sources not only because of its high capacity, but also because it can be used under reducing conditions where the binding affinity for cytochrome-c oxidase is higher.
[7] R e s o l u t i o n o f C y t o c h r o m e - c O x i d a s e By
ANGELO
AzzI,
CLEMENS BROGER, K U R T B I L L ,
and
MICHAEL J. CORBLEY
Cytochrome-c oxidase from bovine heart mitochondria is a multisubunit complex consisting of 13 polypeptides ~ and four prosthetic groups, two hemes and two coppers. 2 There have been mainly two approaches to study the role of the individual subunits in the function of the enzyme and the location of the prosthetic groups. Bacterial cytochrome-c oxidases 3 have been found to consist of a smaller number of subunits than mammalian enzymes, and in some cases it has been shown that they possess immuno-cross-reactivity with the largest subunits of the mitochondrial enzymes. In a second approach, bovine heart cytochrome-c oxidase has been split into subcomplexes and their polypeptide content and prosthetic groups have been analyzed. Alternatively, cytochrome-c oxidase has been depleted specifically of certain subunits and the function of the depleted complex has been studied. The purification of bacterial cytochrome-c oxidases by using affinity chromatography is described in ani B. Kadenbach, J. Jarausch, R. Hartmann, and P. Merle, Anal. Biochem. 129, 517 (1983). M. Wikstr0m, K. Krab, and M. Saraste, in "Cytochrome Oxidase, A Synthesis," p. 55. Academic Press, 1981. 3 B. Ludwig, Biochim. Biophys. Acta 594, 177 (1980).
METHODS IN ENZYMOLOGY, VOL. 126
Copyright© 1986by AcademicPress, Inc. All rightsof reproductionin any formreserved.
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(Kay = 0.55; Fig. 4C) also contain dimers. From the estimated sedimentation coefficient of 9.6 S for the monomer-detergent complex (species 3), a net molecular weight for the protein is calculated as 170,000 (Table III). This value differs from the theoretically expected one (200,000). However, it is neither possible to estimate directly the Stokes radius of the monomeric complex by light-scattering measurements nor to evaluate the diffusion coefficient by sedimentation analysis. Therefore the calculated molecular weight, which is based on an assumed Stokes radius of 61 ~,17 is less accurate and an error of 20% has to be admitted in this case.
[6] A f f i n i t y C h r o m a t o g r a p h y P u r i f i c a t i o n o f C y t o c h r o m e - c Oxidase from Bovine Heart Mitochondria and Other Sources B y CLEMENS BROGER, K U R T BILL, a n d ANGELO A Z Z l
In the mitochondrial respiratory chain cytochrome-c oxidase catalyzes the electron transfer from ferrocytochrome c to molecular oxygen. The molecular details of the interaction of the enzyme with its electron donor, cytochrome c, have been studied by using various labeling techniques. It has been established that the "front side" of cytochrome c, which contains the exposed heme edge, is in contact with the oxidase,l,2 and that subunit II of the enzyme complex is interacting with cytochrome c 3 through a domain on its surface which has also been mapped. 4 It has been recognized for a long time that the specific interaction between cytochrome-c oxidase and its electron donor could be useful in developing an affinity chromatography purification technique for cytochrome-c oxidase based on cytochrome c immobilized on a gel matrix. Essentially two types of affinity gels have been used which differ in the way cytochrome c is linked to the gel: The first was developed before details about the interaction of the oxidase with its electron donor were known. In this method cytochrome c is attached to the Sepharose through lysine residues which are mainly located on the front side of the protein and which are now known to be important for the interaction of this protein with the oxidase. In the development of the second method the i S. Ferguson-Miller, D. L. Brautigan, and E. Margoliash, J. Biol. Chem. 253, 149 (1978). 2 R. Rieder and H. R. Bosshard, J. Biol. Chem. 253, 6045 (1978). 3 R. Bisson, A. Azzi, H. Gutweniger, R. Colonna, C. Montecucco, and A. Zanotti, J. Biol. Chem. 253, 1874 (1978). 4 R. Bisson, G. C. M. Steffens, R. A. Capaldi, and G. Buse, FEBS Lett. 144, 359 (1982).
METHODS IN ENZYMOLOGY, VOL. 126
Copyright© 1986by AcademicPress, Inc. All rightsof reproductionin any formreserved.
[6]
AFFINITY CHROMATOGRAPHY PURIFICATION OF CYTOCHROME OXIDASE
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new information about the interaction between the two proteins has been taken into consideration by attaching cytochrome c through its rear side. The application of the two gels for the purification of enzymes which interact specifically with cytochrome e is straightforward. In general, the solubilized proteins are loaded at low salt concentration to a column containing the gel and, after washing the column, they are eluted by increasing the ionic strength of the buffer. Various detergents, mainly of the nonionic type, have been used in the solubilization step and during the chromatography procedure. If the solubilization step is optimized and the protein of interest can be released from the membrane in a rather selective way, it is possible in many cases to purify it to homogeneity in one affinity chromatography step. The cytochrome c gels described below can be used for several purifications. They are washed after every use with 1 M NaC1 and detergent and subsequently stored at 4°.
Horse Heart Cytochrome c Linked to CNBr-Activated Sepharose 4B Materials
For the preparation of 4 ml of gel: Freeze-dried CNBr-activated Sepharose 4B (1 g) (Pharmacia Fine Chemicals AB, Uppsala, Sweden) 200 ml of 1 m_M HCI 10 ml of 0.1 M NaHCO3, pH 8.3 20 ml of 0.1 M NaHCO3, pH 8.0, containing 0.5 M NaC1 Horse heart cytochrome c (4 mg) (Sigma Chemical Company, St. Louis, MO 63178; type VI) 50 ml of 1 M ethanolamine/HC1, pH 8 400 ml of 1 M NaC1 200 ml of 0.1 M sodium acetate, pH 4.0 200 ml of 0.1 M sodium borate, pH 8.0 Preparation 5
CNBr-activated Sepharose 4B (1 g) is swollen and washed for 15 min on a sintered glass filter with about 200 ml of 1 mM HC1, yielding about 4 ml of swollen gel. Alternatively, Sepharose 4B can be activated with cyanogen bromide, as published in this series: Part of the active groups are hydrolyzed by shaking the gel at room temperature for 4 hr in about 10 ml of 0.1 M NaHCO3, pH 8.3. The gel is then incubated at room tempera5 H. Weiss, B. Juchs, and B. Ziganke, this series, Vol. 53, p. 98. 6 I. Parikh, S. March, and P. Cuatrecasas, this series, Vol. 34, p. 77.
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TABLE I PURIFICATION OF CYTOCHROME-C OXIDASE FROM VARIOUS SOURCES BY AFFINITY CHROMATOGRAPHY ON HORSE HEART CYTOCHROME c-CNBr-AcTIVATED SEPHAROSE 4B
Source Bovine heart
Neurospora crassa Rat liver
Comment Further purification of isolated enzyme One-step purification Main purification step Main purification step Final purification step
Detergent
References
Deoxycholate
a
Laurylmaltoside Triton X-100 Tween 80, Triton X-100 Laurylmaltoside
b c d b
a T. Ozawa, M. Okumura, and K. Yagi, Biochem. Biophys. Res. Commun. 65, 1102 (1975). b D. A. Thompson and S. Ferguson-Miller, Biochemistry 22, 3178 (1983). c H. Weiss and H. J. Kolb, Eur. J. Biochem. 99, 139 (1979). d R. J. Rascati and P. Parsons, J. Biol. Chem. 254, 1586 (1979).
ture for 2 hr in 5 ml of 0.1 M NaHCO3, pH 8, containing 0.5 M NaCI and 4 mg of horse heart cytochrome c (1 mg per milliliter of swollen gel). The nonbound cytochrome c is removed by washing the gel with 10 ml of coupling buffer, and the remaining active groups are reacted at 4° for 1 hr with 50 ml of 1 M ethanolamine-HCl, pH 8. The gel is then washed at 4 ° in turn with 1 M NaC1, 0.1 M sodium acetate, pH 4, 1 M NaCI, and 0.1 M sodium borate, pH 8, and subsequently with the buffer which will be used for the affinity chromatography procedure, e.g., 50 mM Tris-HCl, pH 7.4. Applications These are summarized in Table I. A detailed procedure for the purification of cytochrome-c oxidase from Neurospora crassa has been published in an earlier volume in this series: and the procedures for the purification of the enzyme from other sources can be found in the original literature as indicated in Table I. Comments The above-described procedure is not applicable to all starting materials and with all detergents. For example, it was reported that the bovine heart and yeast enzymes cannot be purified in Triton X-100 by using this method. 7 It wa~ assumed that Triton X-100 might not be able to disperse 7 H. Weiss and H. J. Kolb, Eur. J. Biochem. 99, 139 (1979).
[6]
AFFINITY CHROMATOGRAPHY PURIFICATION OF CYTOCHROME OXIDASE
67
the enzyme completely. On the other hand, it was shown that bovine heart cytochrome-c oxidase dispersed in Triton X-100 can be purified by using the affinity gel described below. It has to be stressed that cytochrome c may be attached to the CNBr-activated gel through some of its lysine residues which are known to be crucial for the interaction with the oxidase. This drawback can apparently be overcome, at least partially, by inactivating some of the CNBr-activated groups of the gel, as mentioned above, in order to avoid multipoint attachment of cytochrome c. Indeed the gel is useful in some applications.
Yeast Cytochrome c Linked to Activated Thiol-Sepharose 4B Materials
For preparation of 30 ml of gel: Freeze-dried activated thiol-Sepharose 4B (8 g) (Pharmacia Fine Chemicals AB, Uppsala, Sweden) 75 ml of 50 mM Tris-HC1, pH 7.2 Cytochrome c (90 mg) from Saccharomyces cerevisiae (Sigma Chemical Company, St. Louis, MO 63178; type VIII) 50 ml of 50 mM sodium acetate, pH 4.5, containing 1.5 mM 2-mercaptoethanol 1 liter of 50 mM Tris-HCl, pH 7.2, containing 1 M NaCI, 1% Triton X-100, and 1 mM potassium hexacyanoferrate(III) Preparation s
Activated thiol-Sepharose 4B (8 g) is swollen and washed at room temperature for 15 min with 50 mM Tris-HCl, pH 7.2, yielding about 30 ml of swollen gel. Alternatively, the gel can be prepared from Sepharose 4B according to a method published in a previous volume in this series. 9 The gel is suspended in 50 mM Tris-HCl, pH 7.2, and 3 mg of S. cereoisiae cytochrome c per milliliter of swollen gel are added in the same buffer. The final volume should be about 50 ml. The suspension is shaken gently overnight at 4°, after which time practically all the cytochrome c is bound to the gel. The almost colorless buffer is removed and the gel is incubated for 30 min in 50 ml of 50 mM sodium acetate, pH 4.5, containing 1.5 mM 2-mercaptoethanol, in order to inactivate residual active groups. Under these conditions no covalently bound cytochrome c is removed 8 A. Azzi, K. Bill, and C. Broger, Proc. Natl. Acad. Sci. U.S.A. 79, 2447 (1982). 9 K. Brocklehurst, J. Carlsson, M. P. J. Kierstan, and E. M. Crook, this series, Vol. 34, p. 531.
68
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[6]
HOOO-CI-I
9 )rz
F16. 1. (A) Schematic drawing of yeast cytochrome c linked to activated thiol-Sepharose 4B. The hatched square in cytochrome c represents the heme plane and the numbers indicate the lysine residues, which are important in the interaction of this protein with cytochrome-c oxidase. Notice that cytochrome c is attached through a cysteine which is located on the opposite side of the molecule. (B) Schematic drawing of yeast cytochrome c linked to Affi-Gel 102 through the heterobifunctional reagent SMPB. The spacer between the gel and cytochrome c is longer as compared to the gel above and not sensitive to reducing agents.
from the gel. The gel is then washed with 1 liter of 50 mM Tris-HCl, pH 7.2, containing 1 M NaCI, 1% Triton X-100 (the detergent to be used for the purification procedure described below), and 1 mM potassium hexacyanoferrate(III) in order to remove not covalently bound cytochrome c and to reoxidize the gel fully. The amount of cytochrome c remaining bound to the gel is about 125 nmol/ml. A schematic drawing of the gel is shown in Fig. 1A.
[~]
AFFINITY CHROMATOGRAPHY PURIFICATION OF CYTOCHROME OXIDASE
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As an example for the use of the affinity chromatography technique, the purification of cytochrome-c oxidase from bovine heart mitochondria is presented in detail. Materials
The following are used for preparation of 30 ml of affinity gel: Bovine heart mitochondria isolated according to a standard method, such as described in Ref. 10, about 1 g of protein I00 ml of I0 mM Tris-HCl, pH 7.2, containing 150 mM KCI 1 liter of 10 mM Tris-HCl, pH 7.2 500 ml of 50 mM Tris-HC1, pH 7.2 Triton X-100 stock solution, 25% in water 200 ml of 50 mM Tris-HCl, pH 7.2, containing 1% Triton X-100 200 ml of 50 mM Tris-HCl, pH 7.2, containing 0.1% Triton X-100 100 ml of 50 mM Tris-HCl, pH 7.2, containing 0.1% Triton and 50 mM NaC1 The entire procedure is carried out at 4°. The mitochondria are suspended in 50 ml of 150 mM KCI buffered with 10 mM Tris-HCl, pH 7.2, and depleted of their outer membrane and cytochrome c by dilution into 10 volumes of 10 mM Tris-HCl, pH 7.2, and subsequent centrifugation at 27,000 g for 20 min. The pellet of mitoplasts is suspended, diluted, and centrifuged again, and then taken up in 50 mM Tris-HC1, pH 7.2, at a protein concentration of 2 mg/ml. Triton X-100 is added to a final concentration of 1%. After incubation for about 30 min the suspension is centrifuged at 27,000 g for 1 hr. The supernatant has a protein concentration of about 1.1 mg/ml. About 400 ml of this solution are applied at a flow rate of 35 ml/hr to the affinity column (2 × 10 cm), equilibrated in 50 mM TrisHC1, pH 7.2, containing 1% Triton X-100. Only cytochrome-c oxidase and reductase bind to the gel; the other proteins are eluted without retardation. After loading, the column is washed with 50 mM Tris-HC1, pH 7.2, containing 0.1% Triton X-100 until the eluate is free of protein and hemes. Elution of the cytochrome-c oxidase is carried out by adding 50 mM NaC1 to the washing buffer or by applying a gradient of NaCI from 0 to 150 mM in the same buffer. The enzyme is eluted at a salt concentration of 50 mM or less, whereas cytochrome-c reductase remains bound to the column and may be eluted at higher salt concentration. The eluted enzyme can be concentrated by using an Amicon filter PM30. Ratios of 10 nmol heme a per milligram protein are reached with this preparation. The enzyme contains about 30-40 mol of phospholipid per mol of heme aa3. It to A. L. Smith, this series, Vol. 10, p. 81.
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TABLE II PURIFICATION OF CYTOCHROME-C OXIDASE FROM VARIOUS SOURCES BY AFFINITY CHROMATOGRAPHY ON YEAST CYTOCHROME c-AcTIVATED THIOL-SEPHAROSE 4B Source Bovine heart Bacillus subtilis Rhodopseudomonas sphaeroides Paracoccus denitrificans
Comment
Detergent
References
One-step purification One-step purification Main purification step
Triton X-100 Laurylmaltoside Triton X-100
a b c
Final purification step
Triton X-100
d
a A. Azzi, K. Bill, and C. Broger, Proc. Natl. Acad. Sci. U.S.A. 79, 2447 (1982). b W. de Vrij, A. Azzi, and W. N. Konings, Ear. J. Biochem. 131, 97 (1983). c R. B. Gennis, R. P. Casey, A. Azzi, and B. Ludwig, Ear. J. Biochem. 125, 189 (1982). M. Solioz, E. Carafoli, and B. Ludwig, J. Biol. Chem. 257, 1579 (1982).
consists of 12-13 polypeptides, as in the traditional preparations, as analyzed by polyacrylamide gel electrophoresis according to Kadenbach et al. 11 The spectral characteristics of the enzyme are identical to the best conventional preparations. Further Applications
Table II summarizes further applications of the method for the purification of cytochrome-c oxidases from various sources. The general procedures are similar to the one described in detail above. Some technical details can be found in the original literature as indicated in Table II. Comments
It is important to block activated thiol groups after coupling cytochrome c to the gel in order to prevent cysteine-containing proteins to bind covalently to the resin. It was observed that under some circumstances a gel with lower substitution of cytochrome c has a relatively higher capacity for binding of cytochrome-c oxidase (Bernd Ludwig, personal communication). This might be due to less steric hindrance. The method is very useful also for microscale purifications and can be used for the preparation of other proteins which interact specifically with cytochrome c, as cytochrome-c reductase 8 or photosynthetic reaction ~i B. Kadenbach, J. Jarausch, R. Hartmann, and P. Merle, Anal. Biochem. 129, 517 (1983).
[6]
AFFINITY CHROMATOGRAPHY PURIFICATION OF CYTOCHROME OXIDASE
71
centers, which in fact have been copurified with cytochrome-c oxidase from Rhodopseudomonas sphaeroides.12 The preparation of the affinity gel just described is extremely simple and its applicability is wide; it cannot be used, however, under reducing conditions because the S-S bridge between cytochrome c and the column would be broken. Recently, a new type of gel has been proposed 13which not only is stable under reducing conditions, but also has a higher capacity for cytochrome-c oxidase, probably as a consequence of less steric hindrance due to a longer spacer. The preparation of this gel is also simple and uses commercially available chemicals. It is described below.
Yeast Cytochrome c Linked to Affi-Gel 102-SMPB
Materials For preparation of 15 ml of gel: 15 ml of Affi-Gel 102 (Bio-Rad, Richmond, CA 94804) 500 ml of 25 mM sodium phosphate, pH 7.4 75 ml of anhydrous dioxane 20 mg of succinimidyl-4-(p-maleimidophenyl)butyrate (SMPB) (Pierce Chemical Company, Rockford, IL 61105) 12 mg of cytochrome c from S. cerevisiae (Sigma Chemical Company, St. Louis, MO 63178; type VIII) 20 ml of swollen Sephadex G-25 (Pharmacia Fine Chemicals AB, Uppsala, Sweden) 200 ml of 25 mM sodium phosphate, pH 7.4, containing 1 M NaC1
Preparation 13 Swollen Affi-Gel 102 (15 ml) is washed on a sintered glass funnel with 25 mM sodium phosphate, pH 7.4. After being drained from buffer, the gel is washed three times with about 10 ml of anhydrous dioxane and finally suspended in 5 ml of dioxane. SMPB (20 rag) is dissolved in 2 ml of dioxane and added to the gel. The suspension is stirred under nitrogen for 3 hr in the dark. The gel is washed with dioxane to remove unbound SMPB and subsequently with several small portions of 25 mM sodium phosphate, pH 7.4. Finally, it is suspended in l0 ml of the buffer. Yeast cytochrome c (12 mg) is dissolved in the same buffer, reduced by the addition of sodium dithionite, and passed through a Sephadex G-25 column (2 × 20 cm) equilibrated in the same buffer. About 900 nmol of the 12 R. B. Gennis, R. P. Casey, A. Azzi, and B. Ludwig, Eur. J. Biochem. 125, 189 (1982). 13 K. Bill and A. Azzi, Biochem. Biophys. Res. Commun. 120, 124 (1984).
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reduced cytochrome c are added to the gel, and the suspension is stirred for 12 hr at 4 ° in the dark. The gel is washed free of nonbound cytochrome c with 25 mM sodium phosphate, pH 7.4, containing 1 M sodium chloride. More than 80% of the added cytochrome c remains bound to the resin. A schematic drawing of the gel is shown in Fig. lB.
Comments This gel has originally been used for measuring the binding affinities of cytochrome-c oxidase and reductase for oxidized and reduced cytochrome c. It will result in the useful purification of cytochrome-c oxidases from various sources not only because of its high capacity, but also because it can be used under reducing conditions where the binding affinity for cytochrome-c oxidase is higher.
[7] R e s o l u t i o n o f C y t o c h r o m e - c O x i d a s e By
ANGELO
AzzI,
CLEMENS BROGER, K U R T B I L L ,
and
MICHAEL J. CORBLEY
Cytochrome-c oxidase from bovine heart mitochondria is a multisubunit complex consisting of 13 polypeptides ~ and four prosthetic groups, two hemes and two coppers. 2 There have been mainly two approaches to study the role of the individual subunits in the function of the enzyme and the location of the prosthetic groups. Bacterial cytochrome-c oxidases 3 have been found to consist of a smaller number of subunits than mammalian enzymes, and in some cases it has been shown that they possess immuno-cross-reactivity with the largest subunits of the mitochondrial enzymes. In a second approach, bovine heart cytochrome-c oxidase has been split into subcomplexes and their polypeptide content and prosthetic groups have been analyzed. Alternatively, cytochrome-c oxidase has been depleted specifically of certain subunits and the function of the depleted complex has been studied. The purification of bacterial cytochrome-c oxidases by using affinity chromatography is described in ani B. Kadenbach, J. Jarausch, R. Hartmann, and P. Merle, Anal. Biochem. 129, 517 (1983). M. Wikstr0m, K. Krab, and M. Saraste, in "Cytochrome Oxidase, A Synthesis," p. 55. Academic Press, 1981. 3 B. Ludwig, Biochim. Biophys. Acta 594, 177 (1980).
METHODS IN ENZYMOLOGY, VOL. 126
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