[12]
CYTOCHROME
OXIDASEOF Saccharomyces cerevisiae
73
TABLE Vl APPARENT MOLECULAR WEIGHTS OF THE SUBUNITS OF CYTOCHROME OX[DASE FROM VARIOUS ORGANISMS
Organism
Ne,rospora crassa Polytoma mirmn Loc,sta migratoria Xenolms m,elleri Ratt,s norwe~,,ic,s
Apparent molecular weight × l0 :~ 41 40 38 42 40
28.5 30 24 24 22
21 18 19 22 20
16 15 14.5 16 15
14 15 12.5 14 12
11.5 I 1.5 10 12 10
10
9.5 10.5 8 9 8
molecular weight per 2 tool of heme, namely 140,000, which is derived from the specific heine content of purified cytochrome oxidase. Amino Acid Composition. With regard to the amino acid composition, cytochrome oxidase seems to be composed of two types of subunits: on the one hand, subunits 1, 2, and 3, which have a high content of unpolar amino acids, and, on the other hand, subunits 4-8, the amino acid compositions of which are more polar (Table IV). ~ As described in this series, Vol. 56 [5], the subunits 1-3 are translated on mitochondrial ribosomes and are most probably coded for on mitochondrial DNA whereas subunits 4-8 are provided by extramintochondrial protein synthesis. Cytochrome Oxidase Purified from Other Sources. By means of the chromatographic procedure described above, cytochrome oxidase has, in addition, been purified from mitochondria of Polytoma mirum, 4 Locusta rnigratoria, ~Xenopus muelleri, 4 and Rattus norwegicus. 4 All preparations showed similar enzymic activity and absorption spectra. The wavelength positions of their main absorption bands are shown in Table V. Upon dodecyl sulfate gel electrophoresis, all preparations showed a similar subunit pattern (Table VI). [12] C y t o c h r o m e
O x i d a s e o f S a c c h a r o m y c e s cerevisiae
B y MERYL S. RUBIN a n d ALEXANDER TZAGOLOFF Ferrocytochrome c + 2H* + ½ 02 -~ ferricytochrome c + H20
Assay Method Principle. The rate of oxidation of reduced cytochrome c is measured spectrophotometrically. 1 L. Smith, in "Methods in Biochemical Analysis," Vol. If, p. 427. Wiley (Interscience), New York, 1955.
74
ELECTRON TRANSFER COMPLEXES
[12]
Reagents 1. Deoxycholate, 10% (w/v), neutralized with KOH to pH 8.2 2. Asolectin, 20 mg/ml in 10 mM Tris-acetate, pH 7.5, 1 mM EDTA 3. Cytochrome c (Sigma Chemical Co., horse heart, type III), 1% in 50 mM Tris-chloride, pH 8.0, reduced with sodium dithionite Procedure. To a 1-ml cuvette with a 10-mm light path was added 0.93 ml of 50 mM potassium phosphate buffer, pH 7.1, and 0.07 ml of 1% reduced cytochrome c. The same amount of cytochrome c oxidized with potassium ferricyanide was added to another cuvette, which served as the blank. An appropriate amount of enzyme (1-250 ~g of protein) was mixed with 10 /~1 of 10% deoxycholate and 100 /~1 of the Asolectin solution. The reaction was started by adding 5-10 txl of the enzymeAsolectin mixture to the cuvette, and the decrease in absorbancy at 550 nm was followed against the blank. Units. The first-order velocity constant was used to calculate the specific activity as described by Smith.1 A unit of cytochrome oxidase activity is defined as the amount of enzyme that oxidizes 1 /zmol of ferrocytochrome c per minute. Purification of Cytochrome Oxidase 2 Yeast submitochondrial particles were prepared as described by Trembath and Tzagoloff3 and suspended in 0.25 M sucrose, 10 mM Trisacetate, pH 7.5, at 25 mg protein per milliliter. The purification of the enzyme was carried out at 4 °, except where specified. Deoxycholic and cholic acid were decolorized with Norit A and recrystallized from 50% ethanol. A 10% (w/v) solution of deoxycholate was prepared by neutralizing the acid with 6 N KOH to pH 8.2. Cholate (20%, w/v) was prepared in an analogous fashion.
Step 1. Fractional Extraction of Submitochondrial Particles. To 250 ml of submitochondrial particle suspension (6250 mg of protein) was added 18.7 g of KC1, and the pH was adjusted to 7.6 with 2 M Tris base. Deoxycholate, 18.75 ml, was added (0.3 mg deoxycholate per milligram of protein), and the suspension was centrifuged at 105,000 gav for 30 min. The clear yellow supernatant was discarded, and the brownish oil and pellet were resuspended in 0.25 M sucrose, 10 mM Tris-acetate, pH 7.5, at a protein concentration of 20 mg/ml. Step 2. Solubilization of Cytochrome Oxidase. To the suspension z M. S. Rubin and A. Tzagoloff, J. Biol. Chem. 248, 4269 (1973). M. K. T r e m b a t h and A. Tzagoloff, this series, Vol. 55 [20].
[12]
C Y T O C H R O M E O X I D A S E OF
Saccharomyces cerevisiae
75
obtained from step 1 was added solid KCI (7.5 g/100 ml) and deoxycholate (0.5 mg per milligram of protein). The suspension was centrifuged at 105,000 gay for 20 min, and the clear light-green supernatant was collected.
Step 3. First Ammonium Sulfate Fractionation. To' the extract from step 2 was added 0.1 volume of 20% cholate, 1 volume of cold water, and sufficient saturated ammonium sulfate (saturated at 4 ° and neutralized with NH4OH) to make the solution 30% saturated in ammonium sulfate (42.9 ml per 100 ml of solution). The supernatant obtained after centrifugation at 79,000 gav for 10 min was adjusted to 40% saturation by the addition of 16.7 ml of saturated ammonium sulfate per 100 ml and again centrifuged at 79,000 gav for 10 min. The green precipitate was dissolved in 50 ml of a solution containing 1% deoxycholate, 1% cholate, and 10 mM Tris-acetate, pH 7.5. Step 4. Second Ammonium Sulfate Fractionation. To the crude enzyme from step 3 was added 13.3 ml of saturated ammonium sulfate (21% saturation). The precipitate was removed by centrifugation at 105,000 gav for 10 min, and the supernatant was adjusted to 23% saturation in ammonium sulfate. The precipitate formed was again removed by centrifugation at 105,000 gav, and the clear green supernatant was adjusted to 26% saturation in ammonium sulfate. This last ammonium sulfate cut precipitated the enzyme, which after centrifugation at 105,000 g~v was dissolved in 50 ml of 0.1% (w/v) Triton X-100, 10 mM Tris-acetate, pH 7.5. Step 5. Anion-Exchange Chromatography. The enzyme from step 4 was applied to a 2 x 7 cm column of DEAE-cellulose (Cellex D, BioRad) equilibrated with 0.1% Triton X-100 in 10 mM Tris-acetate, pH 7.5. The column was washed with 20 ml of 0.1% Triton X-100 in 10 mM Trisacetate, followed by 150 ml of 0.1% Triton X-100 in 10 mM Tris-acetate, pH 7.5 containing 75 to,S/ KC1. Cytochrome oxidase was then eluted from the column with 0.1% Triton X-100 in 10 mM Tris-acetate, pH 7.5, containing 300 mM KCI. Only the green portion of the eluate was collected. Step 6. Ammonium Sulfate Fractionation at Elevated Temperature. The volume of the DEAE eluate was adjusted to 16.5 ml with the eluting buffer, and 4.5 ml of 20% cholate and 9.0 ml of saturated ammonium sulfate was added (3 and 30% final concentration, respectively). The mixture was incubated at 30 ° for 30 min and at 37 ° for 10 min. The copious precipitate was removed by centrifugation at 10,000 g~v for 10 min. To the supernatant was added 10% Triton X-100 to a final concentration of 0.5%, and saturated ammonium sulfate to 30% saturation. The
76
ELECTRON TRANSFER COMPLEXES
[12]
suspension was centrifuged at 105,000 gav for 10 min, and the green pellet was dissolved in 10 mM Tris-acetate, pH 7.5, at a protein concentration of 3-7 mg/ml. Properties
Purity. The enzyme contained 14-15 nmol of heme a per milligram of protein and 1.4-1.5 as much copper. No contamination by cytochrome b or c was evident in the absorption spectra. The specific activity ranged from 100 to 170/zmol of cytochrome c oxidized per minute per milligram of protein when measured at 23 °. The activity was completely dependent on exogenously added phospholipids. The phospholipid content--assuming that all the phosphorus measured, 37-60 nmol per milligram of protein, corresponds to phospholipid--averages 3.8% by weight. No carbohydrate was detectable by periodic acid-Schiff staining of gels of the enzyme. Stability. The purified yeast cytochrome oxidase retained enzymic activity after prolonged storage at - 7 0 °. Overnight storage at 4 ° led to 50% loss of activity. Subunit Composition. The protein composition of cytochrome oxidase was assessed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, a The enzyme is composed of 7 nonidentical subunits whose molecular weights as determined in 10% polyacrylamide are: subunit 1 = 40,000, subunit 2 = 27,300, subunit 3 = 25,000, subunit 4 = 13,800, subunit 5 = 13,000, subunit 6 = 10,200, and subunit 7 = 9500. A stained gel of the enzyme depolymerized in 1% SDS and separated on a 7.5% polyacrylamide gel is shown in Fig. 1. Purification of Subunits Yeast cytochrome oxidase has been subfractionated to yield pure subunits 2, 4, 5, 6, and 7 and a hemoprotein fraction, which probably corresponds to aggregated subunit 1. The entire procedure is carried out at room temperature.
Step 1. Extraction of Subunits 4 and 6. To 20 ml of yeast cytochrome oxidase at a protein concentration of 4 mg/ml was added 180 ml of acetone. The mixture was stirred for l0 rain and centrifuged at 3,000 gay for l0 min. The supernatant was discarded, and the pellet was suspended and homogenized in 20 ml of 2 mM NaPO4, pH 7.5. The clear extract obtained after centrifugation at 12,000 gav for l0 rain contained pure subunits 4 and 6. 4 K. W e b e r and M. Osborn, J. Biol. Chem. 244, 4406 (1969).
[12]
CYTOCHROME OXIDASE OF Saccharomyces cerevisiae
77
---I --2 "-3 --4 --5
FIG. 1. Subunits of yeast cytochrome oxidase. The purified enzyme was depolymerized in 1% sodium dodecyl sulfate and separated on a 7.5% polyacrylamide gel according to the method of K. Weber and M. Osborn [J. Biol. Chem. 244, 4406 (1%9)].
Step 2. Separation of Subunits 4 and 6 on Hydroxyapatite. The extract f r o m step 1 was applied to a 1.3 x 4 cm column of hydroxyapatite (BioGel, H T P ) equilibrated in 2 m M NaPO4, p H 7.5, and the column was washed with (1) 10 ml of 2 m M NaPO4, p H 7.5, (2) 10 ml of 20 m M NaPO4, p H 7.5, and (3) 10 ml of 100 mM NaPO4, p H 7.5. Subunit 6 eluted with 20 m M p h o s p h a t e buffer and subunit 4 with 100 m M phosphate buffer. Step 3. Fractional Solubilization with Guanidine Thiocyanate. The extracted pellet f r o m step 1 was dissolved in 10 ml of 6 M guanidine thiocyanate (guanidine thiocyanate was purchased from I C N - K & K , Inc.;
78
[12]
ELECTRON TRANSFER COMPLEXES
a 6 M solution in water was decolorized with Norit A). The clear greet solution was diluted with 30 ml of water and centrifuged at 12,000 gay for 10 min. The green pellet was dissolved in 10 ml of 6 M guanidine thiocyanate.
Step 4. Purification of Subunit 5. The supernatant from step 3 was dialyzed twice against 2 liters of water for 16 hr total. The precipitate formed was collected by centrifugation at 3000 gay for 10 min and dissolved in 6 ml of 8 M urea, 10 mM Tris-acetate, pH 7.5. The protein solution was applied to a 1 x 5 cm column of DEAE-Sephadex equilibrated in 8 M urea, 10 mM Tris-acetate, pH 7.5. The column was washed with 20 ml of 8 M urea, 10 mM Tris-acetate, pH 7.5. The sample and wash containing subunit 4 were dialyzed against water to remove the urea. Step 5. Separation of the Hemoprotein from Subunit 7. The guanidine thiocyanate solution from step 3 was layered on a 2.5 x 90 cm column of Sephadex G-200 equilibrated with 4 M guanidine thiocyanate and developed with the same solvent. Three peaks with 280 nm absorbing material were detected (Fig. 2). Peak I eluted in the void volume and contained all the heme a and most of the protein applied to the column. Peak II contained pure subunit 7. Peak III did not contain any protein. The pooled fractions of peak I (only the first two-thirds of this peak were pooled) and peak II were dialyzed against 2 liters of water for 16 hr. The precipitated proteins were centrifuged and suspended in a small volume of buffer.
I
[
I
I
I
I
I .........
IO E O
Qo 0 8 ¢4 W
o
0.6
Z
a::
0.4
0 03 (313
CYTOCHROME
OXIDASEOF Saccharomyces cerevisiae
73
TABLE Vl APPARENT MOLECULAR WEIGHTS OF THE SUBUNITS OF CYTOCHROME OX[DASE FROM VARIOUS ORGANISMS
Organism
Ne,rospora crassa Polytoma mirmn Loc,sta migratoria Xenolms m,elleri Ratt,s norwe~,,ic,s
Apparent molecular weight × l0 :~ 41 40 38 42 40
28.5 30 24 24 22
21 18 19 22 20
16 15 14.5 16 15
14 15 12.5 14 12
11.5 I 1.5 10 12 10
10
9.5 10.5 8 9 8
molecular weight per 2 tool of heme, namely 140,000, which is derived from the specific heine content of purified cytochrome oxidase. Amino Acid Composition. With regard to the amino acid composition, cytochrome oxidase seems to be composed of two types of subunits: on the one hand, subunits 1, 2, and 3, which have a high content of unpolar amino acids, and, on the other hand, subunits 4-8, the amino acid compositions of which are more polar (Table IV). ~ As described in this series, Vol. 56 [5], the subunits 1-3 are translated on mitochondrial ribosomes and are most probably coded for on mitochondrial DNA whereas subunits 4-8 are provided by extramintochondrial protein synthesis. Cytochrome Oxidase Purified from Other Sources. By means of the chromatographic procedure described above, cytochrome oxidase has, in addition, been purified from mitochondria of Polytoma mirum, 4 Locusta rnigratoria, ~Xenopus muelleri, 4 and Rattus norwegicus. 4 All preparations showed similar enzymic activity and absorption spectra. The wavelength positions of their main absorption bands are shown in Table V. Upon dodecyl sulfate gel electrophoresis, all preparations showed a similar subunit pattern (Table VI). [12] C y t o c h r o m e
O x i d a s e o f S a c c h a r o m y c e s cerevisiae
B y MERYL S. RUBIN a n d ALEXANDER TZAGOLOFF Ferrocytochrome c + 2H* + ½ 02 -~ ferricytochrome c + H20
Assay Method Principle. The rate of oxidation of reduced cytochrome c is measured spectrophotometrically. 1 L. Smith, in "Methods in Biochemical Analysis," Vol. If, p. 427. Wiley (Interscience), New York, 1955.
74
ELECTRON TRANSFER COMPLEXES
[12]
Reagents 1. Deoxycholate, 10% (w/v), neutralized with KOH to pH 8.2 2. Asolectin, 20 mg/ml in 10 mM Tris-acetate, pH 7.5, 1 mM EDTA 3. Cytochrome c (Sigma Chemical Co., horse heart, type III), 1% in 50 mM Tris-chloride, pH 8.0, reduced with sodium dithionite Procedure. To a 1-ml cuvette with a 10-mm light path was added 0.93 ml of 50 mM potassium phosphate buffer, pH 7.1, and 0.07 ml of 1% reduced cytochrome c. The same amount of cytochrome c oxidized with potassium ferricyanide was added to another cuvette, which served as the blank. An appropriate amount of enzyme (1-250 ~g of protein) was mixed with 10 /~1 of 10% deoxycholate and 100 /~1 of the Asolectin solution. The reaction was started by adding 5-10 txl of the enzymeAsolectin mixture to the cuvette, and the decrease in absorbancy at 550 nm was followed against the blank. Units. The first-order velocity constant was used to calculate the specific activity as described by Smith.1 A unit of cytochrome oxidase activity is defined as the amount of enzyme that oxidizes 1 /zmol of ferrocytochrome c per minute. Purification of Cytochrome Oxidase 2 Yeast submitochondrial particles were prepared as described by Trembath and Tzagoloff3 and suspended in 0.25 M sucrose, 10 mM Trisacetate, pH 7.5, at 25 mg protein per milliliter. The purification of the enzyme was carried out at 4 °, except where specified. Deoxycholic and cholic acid were decolorized with Norit A and recrystallized from 50% ethanol. A 10% (w/v) solution of deoxycholate was prepared by neutralizing the acid with 6 N KOH to pH 8.2. Cholate (20%, w/v) was prepared in an analogous fashion.
Step 1. Fractional Extraction of Submitochondrial Particles. To 250 ml of submitochondrial particle suspension (6250 mg of protein) was added 18.7 g of KC1, and the pH was adjusted to 7.6 with 2 M Tris base. Deoxycholate, 18.75 ml, was added (0.3 mg deoxycholate per milligram of protein), and the suspension was centrifuged at 105,000 gav for 30 min. The clear yellow supernatant was discarded, and the brownish oil and pellet were resuspended in 0.25 M sucrose, 10 mM Tris-acetate, pH 7.5, at a protein concentration of 20 mg/ml. Step 2. Solubilization of Cytochrome Oxidase. To the suspension z M. S. Rubin and A. Tzagoloff, J. Biol. Chem. 248, 4269 (1973). M. K. T r e m b a t h and A. Tzagoloff, this series, Vol. 55 [20].
[12]
C Y T O C H R O M E O X I D A S E OF
Saccharomyces cerevisiae
75
obtained from step 1 was added solid KCI (7.5 g/100 ml) and deoxycholate (0.5 mg per milligram of protein). The suspension was centrifuged at 105,000 gay for 20 min, and the clear light-green supernatant was collected.
Step 3. First Ammonium Sulfate Fractionation. To' the extract from step 2 was added 0.1 volume of 20% cholate, 1 volume of cold water, and sufficient saturated ammonium sulfate (saturated at 4 ° and neutralized with NH4OH) to make the solution 30% saturated in ammonium sulfate (42.9 ml per 100 ml of solution). The supernatant obtained after centrifugation at 79,000 gav for 10 min was adjusted to 40% saturation by the addition of 16.7 ml of saturated ammonium sulfate per 100 ml and again centrifuged at 79,000 gav for 10 min. The green precipitate was dissolved in 50 ml of a solution containing 1% deoxycholate, 1% cholate, and 10 mM Tris-acetate, pH 7.5. Step 4. Second Ammonium Sulfate Fractionation. To the crude enzyme from step 3 was added 13.3 ml of saturated ammonium sulfate (21% saturation). The precipitate was removed by centrifugation at 105,000 gav for 10 min, and the supernatant was adjusted to 23% saturation in ammonium sulfate. The precipitate formed was again removed by centrifugation at 105,000 gav, and the clear green supernatant was adjusted to 26% saturation in ammonium sulfate. This last ammonium sulfate cut precipitated the enzyme, which after centrifugation at 105,000 g~v was dissolved in 50 ml of 0.1% (w/v) Triton X-100, 10 mM Tris-acetate, pH 7.5. Step 5. Anion-Exchange Chromatography. The enzyme from step 4 was applied to a 2 x 7 cm column of DEAE-cellulose (Cellex D, BioRad) equilibrated with 0.1% Triton X-100 in 10 mM Tris-acetate, pH 7.5. The column was washed with 20 ml of 0.1% Triton X-100 in 10 mM Trisacetate, followed by 150 ml of 0.1% Triton X-100 in 10 mM Tris-acetate, pH 7.5 containing 75 to,S/ KC1. Cytochrome oxidase was then eluted from the column with 0.1% Triton X-100 in 10 mM Tris-acetate, pH 7.5, containing 300 mM KCI. Only the green portion of the eluate was collected. Step 6. Ammonium Sulfate Fractionation at Elevated Temperature. The volume of the DEAE eluate was adjusted to 16.5 ml with the eluting buffer, and 4.5 ml of 20% cholate and 9.0 ml of saturated ammonium sulfate was added (3 and 30% final concentration, respectively). The mixture was incubated at 30 ° for 30 min and at 37 ° for 10 min. The copious precipitate was removed by centrifugation at 10,000 g~v for 10 min. To the supernatant was added 10% Triton X-100 to a final concentration of 0.5%, and saturated ammonium sulfate to 30% saturation. The
76
ELECTRON TRANSFER COMPLEXES
[12]
suspension was centrifuged at 105,000 gav for 10 min, and the green pellet was dissolved in 10 mM Tris-acetate, pH 7.5, at a protein concentration of 3-7 mg/ml. Properties
Purity. The enzyme contained 14-15 nmol of heme a per milligram of protein and 1.4-1.5 as much copper. No contamination by cytochrome b or c was evident in the absorption spectra. The specific activity ranged from 100 to 170/zmol of cytochrome c oxidized per minute per milligram of protein when measured at 23 °. The activity was completely dependent on exogenously added phospholipids. The phospholipid content--assuming that all the phosphorus measured, 37-60 nmol per milligram of protein, corresponds to phospholipid--averages 3.8% by weight. No carbohydrate was detectable by periodic acid-Schiff staining of gels of the enzyme. Stability. The purified yeast cytochrome oxidase retained enzymic activity after prolonged storage at - 7 0 °. Overnight storage at 4 ° led to 50% loss of activity. Subunit Composition. The protein composition of cytochrome oxidase was assessed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, a The enzyme is composed of 7 nonidentical subunits whose molecular weights as determined in 10% polyacrylamide are: subunit 1 = 40,000, subunit 2 = 27,300, subunit 3 = 25,000, subunit 4 = 13,800, subunit 5 = 13,000, subunit 6 = 10,200, and subunit 7 = 9500. A stained gel of the enzyme depolymerized in 1% SDS and separated on a 7.5% polyacrylamide gel is shown in Fig. 1. Purification of Subunits Yeast cytochrome oxidase has been subfractionated to yield pure subunits 2, 4, 5, 6, and 7 and a hemoprotein fraction, which probably corresponds to aggregated subunit 1. The entire procedure is carried out at room temperature.
Step 1. Extraction of Subunits 4 and 6. To 20 ml of yeast cytochrome oxidase at a protein concentration of 4 mg/ml was added 180 ml of acetone. The mixture was stirred for l0 rain and centrifuged at 3,000 gay for l0 min. The supernatant was discarded, and the pellet was suspended and homogenized in 20 ml of 2 mM NaPO4, pH 7.5. The clear extract obtained after centrifugation at 12,000 gav for l0 rain contained pure subunits 4 and 6. 4 K. W e b e r and M. Osborn, J. Biol. Chem. 244, 4406 (1969).
[12]
CYTOCHROME OXIDASE OF Saccharomyces cerevisiae
77
---I --2 "-3 --4 --5
FIG. 1. Subunits of yeast cytochrome oxidase. The purified enzyme was depolymerized in 1% sodium dodecyl sulfate and separated on a 7.5% polyacrylamide gel according to the method of K. Weber and M. Osborn [J. Biol. Chem. 244, 4406 (1%9)].
Step 2. Separation of Subunits 4 and 6 on Hydroxyapatite. The extract f r o m step 1 was applied to a 1.3 x 4 cm column of hydroxyapatite (BioGel, H T P ) equilibrated in 2 m M NaPO4, p H 7.5, and the column was washed with (1) 10 ml of 2 m M NaPO4, p H 7.5, (2) 10 ml of 20 m M NaPO4, p H 7.5, and (3) 10 ml of 100 mM NaPO4, p H 7.5. Subunit 6 eluted with 20 m M p h o s p h a t e buffer and subunit 4 with 100 m M phosphate buffer. Step 3. Fractional Solubilization with Guanidine Thiocyanate. The extracted pellet f r o m step 1 was dissolved in 10 ml of 6 M guanidine thiocyanate (guanidine thiocyanate was purchased from I C N - K & K , Inc.;
78
[12]
ELECTRON TRANSFER COMPLEXES
a 6 M solution in water was decolorized with Norit A). The clear greet solution was diluted with 30 ml of water and centrifuged at 12,000 gay for 10 min. The green pellet was dissolved in 10 ml of 6 M guanidine thiocyanate.
Step 4. Purification of Subunit 5. The supernatant from step 3 was dialyzed twice against 2 liters of water for 16 hr total. The precipitate formed was collected by centrifugation at 3000 gay for 10 min and dissolved in 6 ml of 8 M urea, 10 mM Tris-acetate, pH 7.5. The protein solution was applied to a 1 x 5 cm column of DEAE-Sephadex equilibrated in 8 M urea, 10 mM Tris-acetate, pH 7.5. The column was washed with 20 ml of 8 M urea, 10 mM Tris-acetate, pH 7.5. The sample and wash containing subunit 4 were dialyzed against water to remove the urea. Step 5. Separation of the Hemoprotein from Subunit 7. The guanidine thiocyanate solution from step 3 was layered on a 2.5 x 90 cm column of Sephadex G-200 equilibrated with 4 M guanidine thiocyanate and developed with the same solvent. Three peaks with 280 nm absorbing material were detected (Fig. 2). Peak I eluted in the void volume and contained all the heme a and most of the protein applied to the column. Peak II contained pure subunit 7. Peak III did not contain any protein. The pooled fractions of peak I (only the first two-thirds of this peak were pooled) and peak II were dialyzed against 2 liters of water for 16 hr. The precipitated proteins were centrifuged and suspended in a small volume of buffer.
I
[
I
I
I
I
I .........
IO E O
Qo 0 8 ¢4 W
o
0.6
Z
a::
0.4
0 03 (313
