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[25] Purification of bacterial cytochrome c by isoelectric focusing
[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).
230
CYTOCHROMES
[2 5]
protein and inhibit its reaction with cytochrome c oxidase 4 or with cytochrome c-depleted mitochondria.5 Eukaryotic cytochromes c (isoelectric point around 10.5) are electrofocused on ampholytes of pH range 8 to 10, supplemented with arginine to extend the pH range. 2 With P. denitrificans cytochrome c (isoelectric point 4.5), ampholytes with a pH range of 4 to 6 (LKB) are employed. In our modification, the cytochrome is allowed to traverse a column from top to bottom to the stage of maximal separation of cytochrome bands, but not to equilibrium. Fortunately the different bands of cytochrome c are readily visible. Electrofocusing is carried out in LKB's 8100 column of ll0-ml capacity, using their recommended electrode solutions and heavy and light gradient solutions, 6 except for a double measure of the ampholyte because of the limited solubility of the bacterial cytochrome at its isoelectric point. Good results are obtained by applying the cytochrome c near the top of the column, but separated from the basic electrode solution. The heavy solution for the positive electrode is added to the bottom of the column, then the sucrose gradient-ampholyte mixture is brought up to 80% of the height of the column in the usual manner. The next 10 ml of the gradient solution are pumped into a weighed 10-ml volumetric flask, and its density is determined. Then 10 ml of cytochrome c solution containing around 50-75 mg and previously dialyzed against weak buffer (10 raM), pH 7, are carefully added. The cytochrome c should be reduced by addition of a minimal amount of solid Na2S204 or ascorbate to avoid having both oxidized and reduced forms, since, as shown by Flatmark, 2 the oxidized form becomes partially reduced during electrophoresis or electrofocusing. The 10 ml of cytochrome c solution must be of the same density, or slightly less, as the top sucrose-ampholyte mixture, so that it will float, yet remain below the final portion of the gradient solution when it is added. The density is adjusted by addition of sucrose, the discarded gradient solution, and, if necessary, water, as it is finally made up to 10 ml and added to the column. The next 5-7 ml of gradient solution are discarded, then the remainder is added to float on top of the cytochrome c sample. Finally, the upper electrode solution is pumped in, water is circulated around the column at 2°-5 °, the leads from a power supply (top - ) are connected, and the voltage is adjusted to 575-600 V. The current is allowed to run until several small red bands are well 4 L. Smith, M. Nava, and E. Margoliash, in "Oxidases and Related Redox Systems" (T. E. King, H. S. Mason, and M. Morrison, eds.), Vol. 2, p. 629. Univ. Park Press, Baltimore, Maryland, 1973. '~ E. Margoliash, Harvey Lect. 66, 177 (1972). 6 LKB 8100 Ampholine Electrofocusing Equipment Instruction Manual.
[25]
PURIFICATION OF BACTERIAL CYTOCHROME C
231
separated above the main band in the column. As many as 5 small bands can appear. This usually requires running the current for 20-25 hr, although sometimes a longer time is required. The electrofocusing is not carried to final equilibrium, but is stopped when the bands appear to be maximally separated. By running the cytochrome c from the top to the bottom of the column, the distance between two forms with a difference of only one charged group can be kept fairly great, even though the difference in isoelectric point is very small. The P. denitrificans cytochrome c must be electrofocused from the top of the column ( - electrode) to the bottom (+ electrode) so that the minor, less negatively charged forms are left behind the major band. The power supply is disconnected, then the separated cytochrome is slowly (0.3-0.5 ml per minute) drained into a fraction collector; the absorbance of each tube at 550 nm is plotted against the tube number. The tubes containing the major band are pooled and dialyzed exhaustively against the desired buffer to remove the sucrose-ampholyte mixture. Increasing the concentration of cytochrome c by use of ion-exchange resins should be avoided, since this requires the addition of binding ions, which may influence the activity of the cytochrome c. Instead, the concentration can be increased by covering the dialysis bag with Aquacide II (Calbiochem) or with a suitable size of Amicon pressure concentrating system, using the DM-5 membrane. The cytochrome c remains more than 90% reduced. If completely reduced cytochrome is required, it can be obtained by addition of a micro quantity of NaBH4, 7 followed by gentle shaking for a few minutes to remove the evolved H2. Once the minor components are removed, the cytochrome c can be readily crystallized by making the solution 1 M in KCI at room temperature, then adding solid ammonium sulfate gradually until crystals form. Crystals prepared by this method were employed to determine the tertiary structure. 8 The nature of the minor forms in the cytochrome c isolated from P. denitri/~cans is not known. The different forms found in beef cytochrome ¢ result from deamidation, which proceeds at increased rates at high pH, increased temperature, or increased ionic strength. 3 However the minor forms separated from P. denitrif~cans cytochrome c differ from the major form by being less negatively charged (migrate more slowly toward the positive electrode) and are thus different from the deamidated forms derived from beef cytochrome c. 7 L. Smith, H. C. Davies, M. Reichlin, and E. Margoliash, d. Biol. Chem. 248,237 (1973). R. Timkovich and R. E. Dickerson, J. Biol. Chem. 251, 4033 (1976).
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).
230
CYTOCHROMES
[2 5]
protein and inhibit its reaction with cytochrome c oxidase 4 or with cytochrome c-depleted mitochondria.5 Eukaryotic cytochromes c (isoelectric point around 10.5) are electrofocused on ampholytes of pH range 8 to 10, supplemented with arginine to extend the pH range. 2 With P. denitrificans cytochrome c (isoelectric point 4.5), ampholytes with a pH range of 4 to 6 (LKB) are employed. In our modification, the cytochrome is allowed to traverse a column from top to bottom to the stage of maximal separation of cytochrome bands, but not to equilibrium. Fortunately the different bands of cytochrome c are readily visible. Electrofocusing is carried out in LKB's 8100 column of ll0-ml capacity, using their recommended electrode solutions and heavy and light gradient solutions, 6 except for a double measure of the ampholyte because of the limited solubility of the bacterial cytochrome at its isoelectric point. Good results are obtained by applying the cytochrome c near the top of the column, but separated from the basic electrode solution. The heavy solution for the positive electrode is added to the bottom of the column, then the sucrose gradient-ampholyte mixture is brought up to 80% of the height of the column in the usual manner. The next 10 ml of the gradient solution are pumped into a weighed 10-ml volumetric flask, and its density is determined. Then 10 ml of cytochrome c solution containing around 50-75 mg and previously dialyzed against weak buffer (10 raM), pH 7, are carefully added. The cytochrome c should be reduced by addition of a minimal amount of solid Na2S204 or ascorbate to avoid having both oxidized and reduced forms, since, as shown by Flatmark, 2 the oxidized form becomes partially reduced during electrophoresis or electrofocusing. The 10 ml of cytochrome c solution must be of the same density, or slightly less, as the top sucrose-ampholyte mixture, so that it will float, yet remain below the final portion of the gradient solution when it is added. The density is adjusted by addition of sucrose, the discarded gradient solution, and, if necessary, water, as it is finally made up to 10 ml and added to the column. The next 5-7 ml of gradient solution are discarded, then the remainder is added to float on top of the cytochrome c sample. Finally, the upper electrode solution is pumped in, water is circulated around the column at 2°-5 °, the leads from a power supply (top - ) are connected, and the voltage is adjusted to 575-600 V. The current is allowed to run until several small red bands are well 4 L. Smith, M. Nava, and E. Margoliash, in "Oxidases and Related Redox Systems" (T. E. King, H. S. Mason, and M. Morrison, eds.), Vol. 2, p. 629. Univ. Park Press, Baltimore, Maryland, 1973. '~ E. Margoliash, Harvey Lect. 66, 177 (1972). 6 LKB 8100 Ampholine Electrofocusing Equipment Instruction Manual.
[25]
PURIFICATION OF BACTERIAL CYTOCHROME C
231
separated above the main band in the column. As many as 5 small bands can appear. This usually requires running the current for 20-25 hr, although sometimes a longer time is required. The electrofocusing is not carried to final equilibrium, but is stopped when the bands appear to be maximally separated. By running the cytochrome c from the top to the bottom of the column, the distance between two forms with a difference of only one charged group can be kept fairly great, even though the difference in isoelectric point is very small. The P. denitrificans cytochrome c must be electrofocused from the top of the column ( - electrode) to the bottom (+ electrode) so that the minor, less negatively charged forms are left behind the major band. The power supply is disconnected, then the separated cytochrome is slowly (0.3-0.5 ml per minute) drained into a fraction collector; the absorbance of each tube at 550 nm is plotted against the tube number. The tubes containing the major band are pooled and dialyzed exhaustively against the desired buffer to remove the sucrose-ampholyte mixture. Increasing the concentration of cytochrome c by use of ion-exchange resins should be avoided, since this requires the addition of binding ions, which may influence the activity of the cytochrome c. Instead, the concentration can be increased by covering the dialysis bag with Aquacide II (Calbiochem) or with a suitable size of Amicon pressure concentrating system, using the DM-5 membrane. The cytochrome c remains more than 90% reduced. If completely reduced cytochrome is required, it can be obtained by addition of a micro quantity of NaBH4, 7 followed by gentle shaking for a few minutes to remove the evolved H2. Once the minor components are removed, the cytochrome c can be readily crystallized by making the solution 1 M in KCI at room temperature, then adding solid ammonium sulfate gradually until crystals form. Crystals prepared by this method were employed to determine the tertiary structure. 8 The nature of the minor forms in the cytochrome c isolated from P. denitri/~cans is not known. The different forms found in beef cytochrome ¢ result from deamidation, which proceeds at increased rates at high pH, increased temperature, or increased ionic strength. 3 However the minor forms separated from P. denitrif~cans cytochrome c differ from the major form by being less negatively charged (migrate more slowly toward the positive electrode) and are thus different from the deamidated forms derived from beef cytochrome c. 7 L. Smith, H. C. Davies, M. Reichlin, and E. Margoliash, d. Biol. Chem. 248,237 (1973). R. Timkovich and R. E. Dickerson, J. Biol. Chem. 251, 4033 (1976).