222
CYTOCHROMES
[24]
[24] Purification and Subunit Composition of Cytochrome C 1 from Bakers' Yeast Saccharomyces cere visiae By ELLIOTT M. Ross and GOTTFRIED SCHATZ
Although bovine cardiac c y t o c h r o m e ci has been purified in several laboratories,l-3 little is known about c y t o c h r o m e c, from nonmammalian sources. The purification of yeast c y t o c h r o m e c, has allowed the study of its synthesis and incorporation into the inner mitochondrial membrane and initial studies of its structure with relationship to the rest of the c y t o c h r o m e b-c1 complex. 4'~ Purification of C y t o c h r o m e c, '~
All procedures are performed at 0-4 °. Cholate, deoxycholate, and phenylmethylsulfonylfluoride used during development of these procedures were obtained from Sigma Chemical Co.; diisopropylfluorophosphate from Aldrich Chemical Co; and ammonium sulfate (Enzyme Grade) from Schwartz-Mann. Reagents from other sources were not tried. Preparation o f Submitochondrial Particles. Yeast mitochondria are routinely prepared by the large-scale method of Mason et al. 6 modified to include 0.5 mM phenylmethylsulfonylfluoride in all solutions. This concentration of the protease inhibitor is also included in all solutions used in the purification of c y t o c h r o m e c,. Sodium phosphate buffer, pH 7.5, 0.5 M, is then added to the mitochondria to a final concentration of 0.1 M, and the mitochondrial suspension is sonicated twice for 30 sec at the highest setting of a Heat System Model W185 Sonifier (about 75 W output). The suspension is centrifuged for 150 min at 28,000 rpm in a Spinco No. 30 rotor, and the particles are resuspended to 30 mg ml - ' in 0.1 M sodium phosphate-0.5 mM EDTA, pH 7.5 (PE buffer). Step 1. Solubilization and A m m o n i u m Sulfate Fractionation. A 20% (w/v) solution of sodium cholate, pH 7.8, is added slowly to the suspen-
1C. A. Yu, L. Yu, and T. E. King, J. Biol. Chem. 247, 1012 (1972). 2 B. Trumpower and A. Katki, Biochemistry 14, 3635 (1975). 3 j. S. Rieske and H. Tisdale, this series, Vol. 10, p. 349. 4 E. Ross and G. Schatz, J. Biol. Chem. 251, 1991 (1976). 5 E. Ross and G. Schatz, J. Biol. Chem. 251, 1997 (1976). 6 T. L. Mason, R. O. Poyton, D. C. Wharton, and G. Schatz, J. Biol. Chem. 248, 1346 (1973).
[24]
PURIFICATION OF CYTOCHROME C1
223
sion of submitochondrial particles to a final cholate concentration of 3.6%. Solid (NH4)zSO4 (144 mg ml -~ suspension) is then added with constant stirring. After stirring for 60 min, an additional 60 mg of (NH4)2SO4 is then added for each milliliter of the original cholate-containing particle suspension, and, after 30 min, the mixture is centrifuged for 40 min at 28,000 rpm in a Spinco No. 30 rotor. To the supernatant are added 63 mg of (NH4)2SO4 per milliliter of supernatant; after 50 min of stirring, a green precipitate containing cytochrome aaa is removed by centrifugation as before.
Step 2. Precipitation of the Cytochrome bci Complex. The orange supernatant from step 1 is dialyzed against PE buffer containing 0.25% sodium cholate and concentrated at least 2-fold either by ultrafiltration or by dialysis against solid sucrose. The dialyzed mixture is centrifuged for 60 min at 48,000 rpm in a Spinco No. 50 Ti rotor. The deep red pellet, which contains cytochromes b and c~, is resuspended in PE buffer to a protein concentration of 10 mg ml-a; 2-mercaptoethanol is added to 0.1% (v/v), and the suspension is titrated to pH 8.6 with 1 N NaOH. After 612 hr, the mixture is centrifuged as above, and the pellet is resuspended in PE buffer to the same volume. Step 3. Separation of Cytochromes b and Ca. Cytochromes b and cl are separated according to a modification of the procedure developed by Yu et al. ~ for purifying beef cytochrome cl. The following are added to the stirred suspension in the order indicated: 20% sodium cholate to 2% final concentration, 10% sodium deoxycholate to 0.5%, 2-mercaptoethanol to 6%, and (NH4)zSO4 to 144 mg m1-1 total volume after the addition of detergents and 2-mercaptoethanol. After stirring for 90 rain, the precipitate is removed by centrifugation for 30 rain at 35,000 rpm in a Spinco No. 40 rotor. The pellet contains spectrally pure cytochrome b, which, however, contains only 2-3 nmol of heme b mg -~. The pink supernatant contains spectrally pure cytochrome c~; it is dialyzed overnight against 10 mM sodium phosphate-0.5 mM EDTA-0.1% DEAE-purified cholate z0.01% 2-mercaptoethanol-0.1 M NaC1, pH 7.5 (Pi-EDTA-cholate-mercaptoethanol-NaCl buffer). A white precipitate usually forms; it is removed by low-speed centrifugation. The crude cytochrome cl obtained in this step is water soluble, although the continued presence of deter7 Sodium cholate used during DEAE-cellulose c h r o m a t o g r a p h y should be purified by passage over DEAE-cellulose as a 2% solution, p H 7.8. Cholic acid in the eluate is precipitated by the addition of HCI, filtered, and w a s h e d with cold 1 m M HC1. It dissolves in N a O H to form a colorless 20% (w/v) solution, p H 7.5. Cholate and deoxycholate used earlier in the purification were not specially purified.
224
CYTOCHROMES
[24]
gents is necessary to maintain the solubility of impurities during chromatography. Step 4. DEAE-Cellulose Chromatography. The clear pink supernatant from the preceding step is applied to a column of DEAE-cellulose 8 that has been equilibrated with P i - E D T A - c h o l a t e - m e r c a p t o e t h a n o l - N a C l buffer detailed in the preceding step. The column is washed with one bed volume of the same buffer, and c y t o c h r o m e Cl is eluted with a linear NaCI gradient ranging from 0.1 to 0.7 M NaC1 in the same buffer. A 20 cm × 1.5 cm DEAE-cellulose column is sufficient for 30-150 mg of applied protein. The volume o f the gradient should be about six times the bed volume. Pure c y t o c h r o m e cl elutes at approximately 0.45 M NaCI. N o t e on the Choice o f Yeast. As previously noted for yeast cytochrome c oxidase, 6"9the purification procedures and the purity of isolated yeast c y t o c h r o m e cl vary with the yeast strain and with the conditions of cell growth. The procedures described above were developed for commercially grown Fleischmann's pressed yeast and may require modification for use with other strains. With Fleischmann's yeast, about 60% of the c y t o c h r o m e cl present in the initial cholate extract precipitates upon removal of cholate (step 2), the remainder being left in the supernatant. All c y t o c h r o m e b is found in the pellet. When either commercial Red Star yeast or the laboratory strain D273-10B (ATCC 24657; ctPETp+) TM was used as starting material, however, less than 20% of the c y t o c h r o m e cl could be precipitated at this point. Evidently a c y t o c h r o m e bc1 complex is the species precipitated in this step, and the ratio of c y t o c h r o m e Cl in the pellet to that in the supernatant reflects the stability of the complex to the detergent and salt used in the preceding steps. The lability of the c y t o c h r o m e bc, complex in other strains is consistent with the fact that Katan et al. ~ required the stabilizing effect of antimycin A 12 in order to prepare a c y t o c h r o m e bc~ complex from their strain of yeast. A less stable c y t o c h r o m e bcl complex would also explain the differences between this procedure for purifying yeast c y t o c h r o m e c~ and that mentioned by Sekuzu et al.,~a as well as the fact that we were unsuccessful
s We have routinely used Whatman DE-52 microgranular DEAE-cellulose. Other products have not been tested. 9 G. D. Eytan and G. Schatz, J. Biol. Chem. 250, 767 (1975). 10F. Sherman, in "Mrcanismes de Rrgulation des Activitrs Cellulaires chez les Microorganismes," p. 465. CNRS, Paris, 1965. 11M. B. Katan, L. Pool, and G. S. P. Groot, Ettr. J. Biochem. 65, 95 (1976). 12j. S. Rieske, H. Baum, C. D. Stoner, and S. H. Lipton, J. Biol. Chem. 242, 4854 (1971). la 1. Sekuzu, H. Mizushima, and K. Okunuki, Biochim. Biophys. Acta 85, 516 (1964).
[24]
P U R I F I C A T I O N OF C Y T O C H R O M E C 1
225
in applying the procedure of Sekuzu et al. to submitochondrial particles from Fleischman's yeast. The purity of a given cytochrome cl preparation is also dependent on the quality of the submitochondrial particles used as starting material, which is itself dependent on the batch of yeast from which they are prepared. The cytochrome c~ content of the particles must be at least 0.15 nmol of heme c~ mg -~ protein in order to reach a final purity of greater than 25 nmol mg -~. Some batches of yeast, which can be identified by a gray color and unpleasant odor, yielded particles with a heme cl content of 0.07-0.10 nmol mg-k These were unsuitable for the purification of cytochrome c~. Properties
Purity. This procedure for the purification of yeast cytochrome cl yields a product with a heme cl content equal to or greater than that obtained with the analogous cytochrome from bovine heart L2 (cf. the table). The highest heme content determined with our preparation, 32 nmol mg -1, implies a molecular weight of 31,000; this is the actual molecular weight determined for the cytochrome cl heme protein (see below). PURIFICATION OF CYTOCHROME C1 FROM Saccharomyces eerevisiae "'~
Fraction Submitochondrial particles Tept 1:(NH4)zSO4 fractionation Step 2: Cythchrome be, precipitate Step 3: crude cytochrome c, Step 4: DEAE-cellulose chromatography
Total protein (mg)
Total heine c, (nmol)
Heme ci/ protein (nmol/mg)
Purification (fold)
17,800
2190
0.16
(1)
1,300
950
0.52
3.3
43
325
572
2.50
16.0
26
43
412
9.70
61.0
19
13
341
169.0
16
27.0
Yield (~?i) (100)
" From E. Ross and G. Schatz, J. Biol. Chem. 251, 1991 (1976). t, The fraction obtained in step 4 represents the pooled fractions from DEAE chromatography which had a heme content of at least 26 nmol of heine cl mg protein -1. This represents about 85% of the cytochrome c~ recovered from the column. The remainder is usually rechromatographed. The heme content of the peak fraction from the column was 29 nmol of heme cl mg protein -1.
226
CYTOCHROMES
[24]
0.4
555i4 0.5 523
0 ]2~ 0.1
\
o400 420 440 460 480 500 520 540 560 580 6o0 Wevelength (nm) FIG. l. Absolute room-temperature spectra of yeast cytochrome c~. - - , reduced with Na,~S204; - - - , oxidized with Na2S208. Protein concentration was 0.11 mg m1-1 in the lower two curves and 0.54 mg m1-1 in the upper curve. All samples were in 0.1 M NaPi-0.5 mM EDTA, pH 7.5. The light path was 10 ram. The blank contained the buffer mentioned above. Reproduced from E. Ross and G. Schatz, J. Biol. Chem. 251, 1991 (1976).
Spectrum and Redox Properties. Cytochrome cl purified by this procedure is spectrally pure, and its absorbance characteristics are not grossly different from those observed in submitochondrial particles (Fig. 1). At 20 °, oxidized cytochrome cl has a Soret absorbance maximum at 416 nm, and some absorbance in the 510-560 nm region, whereas the reduced cytochrome shows peaks at 418 nm, 523.0 nm, and 553.4 nm. At -196 °, the a maximum is sharpened and shifted to 552.7 nm, with a shoulder at 548.5 nm. The fl-absorbance is resolved into a family of peaks, the strongest being at 510.8 nm, 520.6 nm, and 529.0 nm. These spectra resemble those of beef cytochrome c1. Yeast cytochrome c1 is reduced by dithionite or ascorbate and oxidized by ferricyanide, persulfate, or hydrogen peroxide. It is isolated in the reduced form; the reduced form does not react with oxygen, nor does it bind carbon monoxide. This suggests that the environment of the heme group is not grossly altered relative to the membrane-bound cytochrome. Polypeptide Composition. Upon electrophoresis in polyacrylamide gels containing dodecyl sulfate, purified cytochrome Cl displays a major band of molecular weight 31,000 and variable amounts of a minor band
[24]
PURIFICATION
OF
CYTOCHROME
C1
227
31,0OO
0;
tO
I
I
I
31,000
I
I
29,000
I
I
0.5 I1.0 Relative Migration
Fic. 2. Dodecyl sulfate-polyacrylamide gel electrophoresis of yeast cytochrome c~.
Two cytochrome cl fractions were dissociated, subjected to electrophoresis, stained with Coomassie Brilliant Blue, and scanned at 560 nm. The arrow at the right marks the position of the bromphenol blue dye front. The calibration bars represent 0.1 absorbance unit. Top: A typical fraction containing both the 31,000-dalton heme protein and the 18,500dalton protein. The shoulder on the 31,000-dalton peak is of 29,000 daltons. Bottom: A fraction essentially lacking the 18,500-daltons protein. The amount of protein applied was 15 /zg (top) and 8.8 /xg (bottom). From E. Ross and G. Schatz, J. Biol. Chem. 251, 1991 (1976).
of molecular weight 18,500 (Fig. 2). All the covalently bound heme is associated with the 31,000-dalton protein (Fig. 3). The presence of a 31,000 heme protein and a smaller (reports of exact molecular weight vary) nonheme protein has also been noted in preparations of bovine c y t o c h r o m e c l , 1"2"14 and Yu e t al. ~ have claimed that the smaller protein is a subunit of the enzyme. We always observe less than 1 mol of 18,500dalton peptide per mole of heme protein, 5 as have T r u m p o w e r and Katki 2 using the bovine c y t o c h r o m e . In some preparations, the small protein is missing altogether (Fig. 1). The small protein is also not found in cytochrome c~ isolated from mitochondrial extracts by immunoprecipitation with antisera directed against the heme protein. 6 It thus seems unlikely that this smaller protein is a true subunit of c y t o c h r o m e c~. Protease
Lability.
Yeast c y t o c h r o m e cl hemeprotein is extremely
;4 p. Gellerfors and B. D. Nelson, Eur. J. Biochem. 52, 433 (1975).
228
CYTOCHROMES
[24]
29,000 50{
I
29,000 I
F
i 01.5 I i10 Relative Migrotion
FIG. 3. Detection of the heme absorbance of cytochrome cl on polyacrylamide gels. Cytochrome cl (57/.tg) was dissociated and subjected to electrophoresis as in Fig. 2. Before staining, the gel was scanned at 409 nm (bottom), the absorbance maximum of dodecyl sulfate-denatured cytochrome cl. The gel was then stained with Coomassie Brilliant Blue and scanned again at 560 nm (top). The heme protein in this sample had been shortened to 29,000 daltons during purifcation. The calibration bar represents 0.1 absorbance unit at 409 nm and 0.3 unit at 560 nm. In this particular experiment, only little cytochrome cl was obtained as the " n a t i v e " 31,000-dalton species, despite the presence of protease inhibitors during purification. From E. Ross and G. Schatz, J. Biol. Chem. 251, 1991 (1976).
labile to proteolysis by endogenous yeast proteases, and purified cytochrome cl preparations often contain at least small amounts of 29,000and 27,000-dalton proteolysis products of the heme proteins. ~ Proteolysis is minimized by the use of the protease inhibitor phenylmethylsulfonylfluoride during the preparation, as suggested above, and is further minimized by the simultaneous addition of diisopropylfluorophosphate. 15 Since diisopropylfluorophosphate is extremely toxic, we have preferred to use only phenylmethylsulfonylfluoride and low temperature (as close 15 Anyone planning to work with diisopropylfluorophosphate should familiarize himself with the toxic properties of the compound (cf., e.g., L. S. Goodman and A. Gilman, "The Pharmacological Basis of Therapeutics," 5th ed., p. 456. Macmillan, New York, 1975). Initial dilution should be done in a well-vented fume hood, using gloves. Phenylmethylsulfonylfluoride, while poisonous, is less dangerous than diisopropylfluorophosphate because of its lower volatility and slower reaction with acetylcholinesterase [D. E. Fahrney and A. M. Gold, J. A m . Chem. Soc. 85, 997 (1963)].
[25]
PURIFICATION OF BACTERIAL CYTOCHROME C
229
as possible to 0 °) for large-scale preparations. If no precautions are taken to avoid proteolysis, cytochrome cl heme protein is isolated as 27,000and 25,000-dalton proteolysis products, and none of the native 31,000dalton protein is recovered, a6 The bovine Cl heme protein is also labile to proteolysis, and proteolytic cleavage during purification may account for reports of the molecular weight as 29,000.1'2'J4
Stability. Yeast cytochrome c~ is stable at 0-4 ° in neutral buffer for several months, as determined by lack of spectral change and lack of autoxidizability. J" E. Ross, E. Ebner, R. O. Poyton, T. L. Mason, B. Ono, and G. Schatz, in "'The Biogenesis of Mitochondria" (A. Kroon and C. Saccone, eds.), p. 477. Academic Press, New York, 1974.
[25] P u r i f i c a t i o n
of Bacterial Cytochrome Focusing
By
c by Isoelectric
L U C I L E SMITH
Cytochrome c can be washed out of intact cells of Paracoccus (formerly Micrococcus) denitrificans and purified by a combination of ammonium sulfate precipitation and chromatography on DEAE-cellulose and Sephadex.1 Further purification by isoelectric focusing removes several minor components, leaving the main fraction easily crystallizable. We have used a modification of isoelectric focusing in a pH gradient that has evolved during several years of experience by Mr. G. McLain, with purifying cytochrome c from as divergent sources as mammalian heart and aerobic bacteria. Our efforts followed Flatmark's observation 2 that proteins with small differences in charge could be separated from the predominant form of cytochrome c by this method. Flatmark demonstrate& that the minor forms removed from beef cytochrome c were singly or multiply deamidated derivatives. He did not devise conditions for large-scale separation of the different forms. In addition, we found that isoelectric focusing can remove ions that bind to the highly charged 1 p. B. Scholes, G. McLain, and L. Smith, Biochemistry 10, 2072 (1971). There is a misprint in this paper: p. 2073, line 12, 0.15 M KC1 should be 0.5 M. We have since observed that the molecular weight of the bacterial cytochrome c appears to be abnormally high on Sephadex chromatography. 2 T. Flatmark and O. Vesterberg, Acta Chem. Scand. 20, 1497 (1966). 3 T. Flatmark, Acta Chem. Scand. 20, 1487 (1966).
CYTOCHROMES
[24]
[24] Purification and Subunit Composition of Cytochrome C 1 from Bakers' Yeast Saccharomyces cere visiae By ELLIOTT M. Ross and GOTTFRIED SCHATZ
Although bovine cardiac c y t o c h r o m e ci has been purified in several laboratories,l-3 little is known about c y t o c h r o m e c, from nonmammalian sources. The purification of yeast c y t o c h r o m e c, has allowed the study of its synthesis and incorporation into the inner mitochondrial membrane and initial studies of its structure with relationship to the rest of the c y t o c h r o m e b-c1 complex. 4'~ Purification of C y t o c h r o m e c, '~
All procedures are performed at 0-4 °. Cholate, deoxycholate, and phenylmethylsulfonylfluoride used during development of these procedures were obtained from Sigma Chemical Co.; diisopropylfluorophosphate from Aldrich Chemical Co; and ammonium sulfate (Enzyme Grade) from Schwartz-Mann. Reagents from other sources were not tried. Preparation o f Submitochondrial Particles. Yeast mitochondria are routinely prepared by the large-scale method of Mason et al. 6 modified to include 0.5 mM phenylmethylsulfonylfluoride in all solutions. This concentration of the protease inhibitor is also included in all solutions used in the purification of c y t o c h r o m e c,. Sodium phosphate buffer, pH 7.5, 0.5 M, is then added to the mitochondria to a final concentration of 0.1 M, and the mitochondrial suspension is sonicated twice for 30 sec at the highest setting of a Heat System Model W185 Sonifier (about 75 W output). The suspension is centrifuged for 150 min at 28,000 rpm in a Spinco No. 30 rotor, and the particles are resuspended to 30 mg ml - ' in 0.1 M sodium phosphate-0.5 mM EDTA, pH 7.5 (PE buffer). Step 1. Solubilization and A m m o n i u m Sulfate Fractionation. A 20% (w/v) solution of sodium cholate, pH 7.8, is added slowly to the suspen-
1C. A. Yu, L. Yu, and T. E. King, J. Biol. Chem. 247, 1012 (1972). 2 B. Trumpower and A. Katki, Biochemistry 14, 3635 (1975). 3 j. S. Rieske and H. Tisdale, this series, Vol. 10, p. 349. 4 E. Ross and G. Schatz, J. Biol. Chem. 251, 1991 (1976). 5 E. Ross and G. Schatz, J. Biol. Chem. 251, 1997 (1976). 6 T. L. Mason, R. O. Poyton, D. C. Wharton, and G. Schatz, J. Biol. Chem. 248, 1346 (1973).
[24]
PURIFICATION OF CYTOCHROME C1
223
sion of submitochondrial particles to a final cholate concentration of 3.6%. Solid (NH4)zSO4 (144 mg ml -~ suspension) is then added with constant stirring. After stirring for 60 min, an additional 60 mg of (NH4)2SO4 is then added for each milliliter of the original cholate-containing particle suspension, and, after 30 min, the mixture is centrifuged for 40 min at 28,000 rpm in a Spinco No. 30 rotor. To the supernatant are added 63 mg of (NH4)2SO4 per milliliter of supernatant; after 50 min of stirring, a green precipitate containing cytochrome aaa is removed by centrifugation as before.
Step 2. Precipitation of the Cytochrome bci Complex. The orange supernatant from step 1 is dialyzed against PE buffer containing 0.25% sodium cholate and concentrated at least 2-fold either by ultrafiltration or by dialysis against solid sucrose. The dialyzed mixture is centrifuged for 60 min at 48,000 rpm in a Spinco No. 50 Ti rotor. The deep red pellet, which contains cytochromes b and c~, is resuspended in PE buffer to a protein concentration of 10 mg ml-a; 2-mercaptoethanol is added to 0.1% (v/v), and the suspension is titrated to pH 8.6 with 1 N NaOH. After 612 hr, the mixture is centrifuged as above, and the pellet is resuspended in PE buffer to the same volume. Step 3. Separation of Cytochromes b and Ca. Cytochromes b and cl are separated according to a modification of the procedure developed by Yu et al. ~ for purifying beef cytochrome cl. The following are added to the stirred suspension in the order indicated: 20% sodium cholate to 2% final concentration, 10% sodium deoxycholate to 0.5%, 2-mercaptoethanol to 6%, and (NH4)zSO4 to 144 mg m1-1 total volume after the addition of detergents and 2-mercaptoethanol. After stirring for 90 rain, the precipitate is removed by centrifugation for 30 rain at 35,000 rpm in a Spinco No. 40 rotor. The pellet contains spectrally pure cytochrome b, which, however, contains only 2-3 nmol of heme b mg -~. The pink supernatant contains spectrally pure cytochrome c~; it is dialyzed overnight against 10 mM sodium phosphate-0.5 mM EDTA-0.1% DEAE-purified cholate z0.01% 2-mercaptoethanol-0.1 M NaC1, pH 7.5 (Pi-EDTA-cholate-mercaptoethanol-NaCl buffer). A white precipitate usually forms; it is removed by low-speed centrifugation. The crude cytochrome cl obtained in this step is water soluble, although the continued presence of deter7 Sodium cholate used during DEAE-cellulose c h r o m a t o g r a p h y should be purified by passage over DEAE-cellulose as a 2% solution, p H 7.8. Cholic acid in the eluate is precipitated by the addition of HCI, filtered, and w a s h e d with cold 1 m M HC1. It dissolves in N a O H to form a colorless 20% (w/v) solution, p H 7.5. Cholate and deoxycholate used earlier in the purification were not specially purified.
224
CYTOCHROMES
[24]
gents is necessary to maintain the solubility of impurities during chromatography. Step 4. DEAE-Cellulose Chromatography. The clear pink supernatant from the preceding step is applied to a column of DEAE-cellulose 8 that has been equilibrated with P i - E D T A - c h o l a t e - m e r c a p t o e t h a n o l - N a C l buffer detailed in the preceding step. The column is washed with one bed volume of the same buffer, and c y t o c h r o m e Cl is eluted with a linear NaCI gradient ranging from 0.1 to 0.7 M NaC1 in the same buffer. A 20 cm × 1.5 cm DEAE-cellulose column is sufficient for 30-150 mg of applied protein. The volume o f the gradient should be about six times the bed volume. Pure c y t o c h r o m e cl elutes at approximately 0.45 M NaCI. N o t e on the Choice o f Yeast. As previously noted for yeast cytochrome c oxidase, 6"9the purification procedures and the purity of isolated yeast c y t o c h r o m e cl vary with the yeast strain and with the conditions of cell growth. The procedures described above were developed for commercially grown Fleischmann's pressed yeast and may require modification for use with other strains. With Fleischmann's yeast, about 60% of the c y t o c h r o m e cl present in the initial cholate extract precipitates upon removal of cholate (step 2), the remainder being left in the supernatant. All c y t o c h r o m e b is found in the pellet. When either commercial Red Star yeast or the laboratory strain D273-10B (ATCC 24657; ctPETp+) TM was used as starting material, however, less than 20% of the c y t o c h r o m e cl could be precipitated at this point. Evidently a c y t o c h r o m e bc1 complex is the species precipitated in this step, and the ratio of c y t o c h r o m e Cl in the pellet to that in the supernatant reflects the stability of the complex to the detergent and salt used in the preceding steps. The lability of the c y t o c h r o m e bc, complex in other strains is consistent with the fact that Katan et al. ~ required the stabilizing effect of antimycin A 12 in order to prepare a c y t o c h r o m e bc~ complex from their strain of yeast. A less stable c y t o c h r o m e bcl complex would also explain the differences between this procedure for purifying yeast c y t o c h r o m e c~ and that mentioned by Sekuzu et al.,~a as well as the fact that we were unsuccessful
s We have routinely used Whatman DE-52 microgranular DEAE-cellulose. Other products have not been tested. 9 G. D. Eytan and G. Schatz, J. Biol. Chem. 250, 767 (1975). 10F. Sherman, in "Mrcanismes de Rrgulation des Activitrs Cellulaires chez les Microorganismes," p. 465. CNRS, Paris, 1965. 11M. B. Katan, L. Pool, and G. S. P. Groot, Ettr. J. Biochem. 65, 95 (1976). 12j. S. Rieske, H. Baum, C. D. Stoner, and S. H. Lipton, J. Biol. Chem. 242, 4854 (1971). la 1. Sekuzu, H. Mizushima, and K. Okunuki, Biochim. Biophys. Acta 85, 516 (1964).
[24]
P U R I F I C A T I O N OF C Y T O C H R O M E C 1
225
in applying the procedure of Sekuzu et al. to submitochondrial particles from Fleischman's yeast. The purity of a given cytochrome cl preparation is also dependent on the quality of the submitochondrial particles used as starting material, which is itself dependent on the batch of yeast from which they are prepared. The cytochrome c~ content of the particles must be at least 0.15 nmol of heme c~ mg -~ protein in order to reach a final purity of greater than 25 nmol mg -~. Some batches of yeast, which can be identified by a gray color and unpleasant odor, yielded particles with a heme cl content of 0.07-0.10 nmol mg-k These were unsuitable for the purification of cytochrome c~. Properties
Purity. This procedure for the purification of yeast cytochrome cl yields a product with a heme cl content equal to or greater than that obtained with the analogous cytochrome from bovine heart L2 (cf. the table). The highest heme content determined with our preparation, 32 nmol mg -1, implies a molecular weight of 31,000; this is the actual molecular weight determined for the cytochrome cl heme protein (see below). PURIFICATION OF CYTOCHROME C1 FROM Saccharomyces eerevisiae "'~
Fraction Submitochondrial particles Tept 1:(NH4)zSO4 fractionation Step 2: Cythchrome be, precipitate Step 3: crude cytochrome c, Step 4: DEAE-cellulose chromatography
Total protein (mg)
Total heine c, (nmol)
Heme ci/ protein (nmol/mg)
Purification (fold)
17,800
2190
0.16
(1)
1,300
950
0.52
3.3
43
325
572
2.50
16.0
26
43
412
9.70
61.0
19
13
341
169.0
16
27.0
Yield (~?i) (100)
" From E. Ross and G. Schatz, J. Biol. Chem. 251, 1991 (1976). t, The fraction obtained in step 4 represents the pooled fractions from DEAE chromatography which had a heme content of at least 26 nmol of heine cl mg protein -1. This represents about 85% of the cytochrome c~ recovered from the column. The remainder is usually rechromatographed. The heme content of the peak fraction from the column was 29 nmol of heme cl mg protein -1.
226
CYTOCHROMES
[24]
0.4
555i4 0.5 523
0 ]2~ 0.1
\
o400 420 440 460 480 500 520 540 560 580 6o0 Wevelength (nm) FIG. l. Absolute room-temperature spectra of yeast cytochrome c~. - - , reduced with Na,~S204; - - - , oxidized with Na2S208. Protein concentration was 0.11 mg m1-1 in the lower two curves and 0.54 mg m1-1 in the upper curve. All samples were in 0.1 M NaPi-0.5 mM EDTA, pH 7.5. The light path was 10 ram. The blank contained the buffer mentioned above. Reproduced from E. Ross and G. Schatz, J. Biol. Chem. 251, 1991 (1976).
Spectrum and Redox Properties. Cytochrome cl purified by this procedure is spectrally pure, and its absorbance characteristics are not grossly different from those observed in submitochondrial particles (Fig. 1). At 20 °, oxidized cytochrome cl has a Soret absorbance maximum at 416 nm, and some absorbance in the 510-560 nm region, whereas the reduced cytochrome shows peaks at 418 nm, 523.0 nm, and 553.4 nm. At -196 °, the a maximum is sharpened and shifted to 552.7 nm, with a shoulder at 548.5 nm. The fl-absorbance is resolved into a family of peaks, the strongest being at 510.8 nm, 520.6 nm, and 529.0 nm. These spectra resemble those of beef cytochrome c1. Yeast cytochrome c1 is reduced by dithionite or ascorbate and oxidized by ferricyanide, persulfate, or hydrogen peroxide. It is isolated in the reduced form; the reduced form does not react with oxygen, nor does it bind carbon monoxide. This suggests that the environment of the heme group is not grossly altered relative to the membrane-bound cytochrome. Polypeptide Composition. Upon electrophoresis in polyacrylamide gels containing dodecyl sulfate, purified cytochrome Cl displays a major band of molecular weight 31,000 and variable amounts of a minor band
[24]
PURIFICATION
OF
CYTOCHROME
C1
227
31,0OO
0;
tO
I
I
I
31,000
I
I
29,000
I
I
0.5 I1.0 Relative Migration
Fic. 2. Dodecyl sulfate-polyacrylamide gel electrophoresis of yeast cytochrome c~.
Two cytochrome cl fractions were dissociated, subjected to electrophoresis, stained with Coomassie Brilliant Blue, and scanned at 560 nm. The arrow at the right marks the position of the bromphenol blue dye front. The calibration bars represent 0.1 absorbance unit. Top: A typical fraction containing both the 31,000-dalton heme protein and the 18,500dalton protein. The shoulder on the 31,000-dalton peak is of 29,000 daltons. Bottom: A fraction essentially lacking the 18,500-daltons protein. The amount of protein applied was 15 /zg (top) and 8.8 /xg (bottom). From E. Ross and G. Schatz, J. Biol. Chem. 251, 1991 (1976).
of molecular weight 18,500 (Fig. 2). All the covalently bound heme is associated with the 31,000-dalton protein (Fig. 3). The presence of a 31,000 heme protein and a smaller (reports of exact molecular weight vary) nonheme protein has also been noted in preparations of bovine c y t o c h r o m e c l , 1"2"14 and Yu e t al. ~ have claimed that the smaller protein is a subunit of the enzyme. We always observe less than 1 mol of 18,500dalton peptide per mole of heme protein, 5 as have T r u m p o w e r and Katki 2 using the bovine c y t o c h r o m e . In some preparations, the small protein is missing altogether (Fig. 1). The small protein is also not found in cytochrome c~ isolated from mitochondrial extracts by immunoprecipitation with antisera directed against the heme protein. 6 It thus seems unlikely that this smaller protein is a true subunit of c y t o c h r o m e c~. Protease
Lability.
Yeast c y t o c h r o m e cl hemeprotein is extremely
;4 p. Gellerfors and B. D. Nelson, Eur. J. Biochem. 52, 433 (1975).
228
CYTOCHROMES
[24]
29,000 50{
I
29,000 I
F
i 01.5 I i10 Relative Migrotion
FIG. 3. Detection of the heme absorbance of cytochrome cl on polyacrylamide gels. Cytochrome cl (57/.tg) was dissociated and subjected to electrophoresis as in Fig. 2. Before staining, the gel was scanned at 409 nm (bottom), the absorbance maximum of dodecyl sulfate-denatured cytochrome cl. The gel was then stained with Coomassie Brilliant Blue and scanned again at 560 nm (top). The heme protein in this sample had been shortened to 29,000 daltons during purifcation. The calibration bar represents 0.1 absorbance unit at 409 nm and 0.3 unit at 560 nm. In this particular experiment, only little cytochrome cl was obtained as the " n a t i v e " 31,000-dalton species, despite the presence of protease inhibitors during purification. From E. Ross and G. Schatz, J. Biol. Chem. 251, 1991 (1976).
labile to proteolysis by endogenous yeast proteases, and purified cytochrome cl preparations often contain at least small amounts of 29,000and 27,000-dalton proteolysis products of the heme proteins. ~ Proteolysis is minimized by the use of the protease inhibitor phenylmethylsulfonylfluoride during the preparation, as suggested above, and is further minimized by the simultaneous addition of diisopropylfluorophosphate. 15 Since diisopropylfluorophosphate is extremely toxic, we have preferred to use only phenylmethylsulfonylfluoride and low temperature (as close 15 Anyone planning to work with diisopropylfluorophosphate should familiarize himself with the toxic properties of the compound (cf., e.g., L. S. Goodman and A. Gilman, "The Pharmacological Basis of Therapeutics," 5th ed., p. 456. Macmillan, New York, 1975). Initial dilution should be done in a well-vented fume hood, using gloves. Phenylmethylsulfonylfluoride, while poisonous, is less dangerous than diisopropylfluorophosphate because of its lower volatility and slower reaction with acetylcholinesterase [D. E. Fahrney and A. M. Gold, J. A m . Chem. Soc. 85, 997 (1963)].
[25]
PURIFICATION OF BACTERIAL CYTOCHROME C
229
as possible to 0 °) for large-scale preparations. If no precautions are taken to avoid proteolysis, cytochrome cl heme protein is isolated as 27,000and 25,000-dalton proteolysis products, and none of the native 31,000dalton protein is recovered, a6 The bovine Cl heme protein is also labile to proteolysis, and proteolytic cleavage during purification may account for reports of the molecular weight as 29,000.1'2'J4
Stability. Yeast cytochrome c~ is stable at 0-4 ° in neutral buffer for several months, as determined by lack of spectral change and lack of autoxidizability. J" E. Ross, E. Ebner, R. O. Poyton, T. L. Mason, B. Ono, and G. Schatz, in "'The Biogenesis of Mitochondria" (A. Kroon and C. Saccone, eds.), p. 477. Academic Press, New York, 1974.
[25] P u r i f i c a t i o n
of Bacterial Cytochrome Focusing
By
c by Isoelectric
L U C I L E SMITH
Cytochrome c can be washed out of intact cells of Paracoccus (formerly Micrococcus) denitrificans and purified by a combination of ammonium sulfate precipitation and chromatography on DEAE-cellulose and Sephadex.1 Further purification by isoelectric focusing removes several minor components, leaving the main fraction easily crystallizable. We have used a modification of isoelectric focusing in a pH gradient that has evolved during several years of experience by Mr. G. McLain, with purifying cytochrome c from as divergent sources as mammalian heart and aerobic bacteria. Our efforts followed Flatmark's observation 2 that proteins with small differences in charge could be separated from the predominant form of cytochrome c by this method. Flatmark demonstrate& that the minor forms removed from beef cytochrome c were singly or multiply deamidated derivatives. He did not devise conditions for large-scale separation of the different forms. In addition, we found that isoelectric focusing can remove ions that bind to the highly charged 1 p. B. Scholes, G. McLain, and L. Smith, Biochemistry 10, 2072 (1971). There is a misprint in this paper: p. 2073, line 12, 0.15 M KC1 should be 0.5 M. We have since observed that the molecular weight of the bacterial cytochrome c appears to be abnormally high on Sephadex chromatography. 2 T. Flatmark and O. Vesterberg, Acta Chem. Scand. 20, 1497 (1966). 3 T. Flatmark, Acta Chem. Scand. 20, 1487 (1966).
