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[134] Cytochrome c (ustilago)
[134]
CYTOCHROME c (USTILAGO)
755
25 °, m M K C N ) the cyanide cytochrome complex will contribute but little to the effect of cyanide on the respiration. With ferricytochrome c azide ion rapidly forms a compound with absorption bands at 540 m~ and 570 m~. 26 Because of the large dissociation constant (0.15 M at 25 °) also the azide effect on cytochrome c exerts little influence on the respiration. ~ B. L. Horecker and J. N. Stannard, J. Biol. Chem. 172, 589 (1948).
[134] C y t o c h r o m e
c (Ustilago)
By J. B. NEILANDS
Assay Method Principle. For isolation purposes a suitable approximation of the concentration of the hemoprotein m a y be obtained from the relative strength of the absorption bands of the reduced compound as seen with the hand spectroscope. Procedure. The material to be examined is suspended or dissolved in 0.1 M phosphate buffer and a "knife-point" of solid sodium hydrosulfite is added. The relative concentration m a y be estimated b y comparing the density of the 550-mr, band with t h a t of a similarly treated standard solution of beef heart cytochrome c. The concentration should be at t h a t level where the/~ band, in the region of 520 m~, is just barely visible. A Zeiss band spectroscope is suitable for the above observations.
Purification Procedure The procedure is essentially the same as t h a t described in the original publication. 1 Step 1. Cultivation of Ustilago sphaerogena. This aerobic organism is cultured under conditions which will provide maximum cell yields. The medium contains 1.0% glucose and 1.8% Difco yeast extract. ~ The culture is started in 100-ml. amounts of this medium in ten 500-ml. Erlenmeyer flasks. T h e inoculum is grown on a gentle rocker in a 25 ° room. Meanwhile a fermentation unit 3 is filled with 200 1. of the same medium, and the t a n k and contents are subjected to 15 lb. of steam pressure for 1j. B. Neilands, J. Biol. Chem. 197, 701 (1952). 2 Although Ustilago sphaerogena may be propagated on almost any type of natural material, maximum cytochrome formation has been obtained only on Difco yeast extract; see also the article by P. W. Grimm and P. Allen, Plant Physiol. 29, 369 (1954). s j. j. Stefaniak, F. B. Galley, C. S. Brown, and M. J. Johnson, Ind. Eng. Chem. 38, 666 (1946).
756
RESPIRATORY ENZYMES
[134]
1 hour. After heavy growth appears in the Erlenmeyer flasks" (about 2 days), the contents are pooled and aseptically transferred to the cooled fermentation unit. Aeration is maintained at the maximum rate that will not cause loss of medium as foam going through the exhaust. Small amounts of Dow Antifoam A may be added if foaming becomes too serious. The temperature of the fermentation is maintained at 25 + 1°. After 24 hours the pH of the medium is taken at 2-hour intervals, and when it has risen to 7.5 to 8.0 there is no further growth. At this point, about 36 hours after inoculation, the fermentation may be considered complete. Step 2. Extraction. The tank is opened and 7 kg. of Filter-Cel is thoroughly mixed with the broth. The suspension is pumped through a filter press, and the cake is washed with liberal quantities of tap water. Enough water is added to the filter cake to give a fluid suspension, and N NaOH is added to bring the pH to approximately 10. The mixture is stirred mechanically, NaOH being added from time to time to maintain the pH at about 10. After 4 hours of continuous stirring, the mixture is again pumped through the filter press, and this time the clear, brownish extract is saved. Step 3. Fractionation with Ammonium Sulfate. The pH of the extract is reduced to 7 with syrupy phosphoric acid, and 487 g. of ammonium sulfate is added per liter of solution (about 75 % saturation). The material is left at 5° for 3 to 4 hours, after which a white precipitate can be removed by filtration through fluted papers. The filtrate is completely saturated with ammonium sulfate and allowed to stand at 5 ° overnight. The next morning, the cytochrome, which has precipitated, is filtered off and dialyzed against cold 0.027 % ammonium hydroxide until free from sulfate ion. This preparation is lyophilized and stored at 5 °. The yield is about 0.6 g. Step 4. Chromatography on Amberlite IRC-50. Commercial Amberlite IRC-50 is pulverized with a mortar and pestle until the particle size is reduced to 200 to 400 mesh. 4 The resin is washed on the centrifuge in succession with 5% sulfuric acid, 5 % sodium hydroxide, distilled water, and 0.1 M phosphate buffer, pH 7.0. A chromatography tube, 2.0 X 20 cm., is closed with a one-hole rubber stopper; a layer of glass wool and a layer of asbestos are placed over the stopper in order to prevent the particles of Amberlite from escaping from the tube. The resin, suspended in phosphate buffer, is poured into the tube to form a column about 10 cm.-high. The column is then washed with phosphate buffer until the effluent has a negligible optical density at 280 m~. 4 This resin may now be obtained in a fine particle size (Amberlite XE-97) from R o h m and Haas Co., Philadelphia.
[134]
CYTOCHROME C (USTILAGO)
757
The sample, about 0.6 g., is dissolved in the least amount of buffer and placed on the column. On washing with the same buffer, 0.1 M phosphate at pH 7.0, the cytochrome remains rather strongly adsorbed while a brownish, noncytochrome fraction migrates rapidly down the column and into the effluent. After the optical density at 280 m~ in the effluent has fallen to a value of less than 1.0 (1.00-cm. cell), the cytochrome is eluted with a saturated solution of ammonium acetate. The pigment is collected in a small volume and dialyzed against cold 0.027% ammonium hydroxide. The preparation, about 0.5 g., is then lyophilized and stored at 5° . A table summarizing the isolation procedure has not been included here. The method is extremely simple and, furthermore, the assay technique is not exactly quantitative. Weisel and Allen 5 found the dry cells of Ustilago sphaerogena to contain up to 1% cytochrome c. Since the above fermentation yields about 2 kg. of dry cells, the 0.5 g. of cytochrome represents only a few per cent of the total enzyme in the cells. Purity of Enzyme. No evidence of heterogeneity has been obtained when this preparation is examined in the Tiselius apparatus in the region of the isoelectric point. In these experiments a red filter (Corning No. 2424) is placed between the light source and the Tiselius cell, and the camera is loaded with red-sensitive spectroscopic film. A single, symmetrical boundary is observed when the material is analyzed in the oil turbine ultra centrifuge of Svedberg.
Properties Molecular Weight. The oil turbine ultracentrifuge gives a sedimentation constant, $20, of 1.4 X 10-13 second. The diffusion constant, D20, is calculated by the height-area method 8 to be 7.7 × 10-7 cm. 2 sec. -1. Assuming that the partial specific volume, ~, is 0.74 cm2 g.-1, these data give M = 18,000. Iron analyses by the method of Lorber 7 give the value of 0.28 % corresponding to a minimum molecular weight of 20,000. These figures must be considered preliminary, since the determination of molecular weight by the above methods can be open to large errors for low molecular weight proteins. Absorption Spectrum. The absorption spectrum of Ustilago cytochrome c is remarkably similar to that of the beef heart product. It shows the same tendency to become autoxidizable at extreme pH values. This means that the prosthetic group is derived from iron protoporphy5 p. Weisel and P. Allen, Abstracts, Meeting of the American Institute of Biological Sciences, Minneapolis, September, 1951. e L. G. Longsworth, Ann. N. Y. Acad. Sci. 41, 267 (1941). TL. Lorber, Biochem. Z. 181, 391 (1927).
758
RESPIRATORY ENZYMES
[135]
rin I X and further that the heme is probably attached to the protein part in exactly the same manner as in beef cytochrome c. Electrophoretic Mobility. Electrophoresis of Ustilago cytochrome c in the buffers used by Theorell s show the protein to be isoelectrie near pH 7. Catalytic Activity. Mole for mole, the Ustilago cytochrome c appears to be about as active as beef heart cytochrome c in the succinic dehydrogenase system of Pot t er 2 s H. Theorell, Biochem. Z. 28fi, 207 (1936). 9 V. R. Potter, in "Manometric Techniques" (Umbreit, Burris, and Stauffer, eds.), rev. ed., p. 213, Burgess Publishing Co., Minneapolis, 1949.
[135] Cytochrome c and Cytochrome c Peroxidase from
Pseudomonas fluorescens By HOWARD M. LENHOFF and NATHAN O. •APLAN I. Cytochrome c
Assay Method Principle. The cytochrome c obtained from Pseudomonas fluorescens ~ is identical in spectrum to animal cytochrome c, but it differs from animal cytochrome c in some of its biochemical and adsorptive properties. The Pseudomonas pigment has never been separated from the cytochrome peroxidase present in the extracts of Ps. fiuorescens, although it appears that these two components are not activities of the same protein. ~ This inseparability is disadvantageous in that it is difficult to study the reactivity of the cytochrome with electron donors and acceptors from other organisms owing to the interfering cytochrome peroxidase activity. On the other hand, it is advantageous in that the peroxidase allows one to assay for the presence of small amounts of the cytochrome c. Procedure. SPECTROPHOTOMETRICASSAY. The reduced pigment has an band at 550 m~, a smaller ~ band at 520 m~, and a large "r band at 415 m~ in the Soret region. The oxidized pigment absorbs slightly at 530 m~ and has its Soret band at 408 m~. These spectra are nearly identical to the spectra of the respective states of animal cytochrome c. The reduced animal cytochrome c has an extinction coefficient at 550 m~ of approximately 27.0 × 103 cm.2/mM., whereas that of the oxidized form is 9.0; the difference is 18.0 X 108 cm./mM. By determining H. M. Lenhoff and N. O. Kaplan, Nature 172, 730 (1953). 2 H. M. Lenhoff, Doctoral Thesis, Johns Hopkins University, Baltimore, 1955.
CYTOCHROME c (USTILAGO)
755
25 °, m M K C N ) the cyanide cytochrome complex will contribute but little to the effect of cyanide on the respiration. With ferricytochrome c azide ion rapidly forms a compound with absorption bands at 540 m~ and 570 m~. 26 Because of the large dissociation constant (0.15 M at 25 °) also the azide effect on cytochrome c exerts little influence on the respiration. ~ B. L. Horecker and J. N. Stannard, J. Biol. Chem. 172, 589 (1948).
[134] C y t o c h r o m e
c (Ustilago)
By J. B. NEILANDS
Assay Method Principle. For isolation purposes a suitable approximation of the concentration of the hemoprotein m a y be obtained from the relative strength of the absorption bands of the reduced compound as seen with the hand spectroscope. Procedure. The material to be examined is suspended or dissolved in 0.1 M phosphate buffer and a "knife-point" of solid sodium hydrosulfite is added. The relative concentration m a y be estimated b y comparing the density of the 550-mr, band with t h a t of a similarly treated standard solution of beef heart cytochrome c. The concentration should be at t h a t level where the/~ band, in the region of 520 m~, is just barely visible. A Zeiss band spectroscope is suitable for the above observations.
Purification Procedure The procedure is essentially the same as t h a t described in the original publication. 1 Step 1. Cultivation of Ustilago sphaerogena. This aerobic organism is cultured under conditions which will provide maximum cell yields. The medium contains 1.0% glucose and 1.8% Difco yeast extract. ~ The culture is started in 100-ml. amounts of this medium in ten 500-ml. Erlenmeyer flasks. T h e inoculum is grown on a gentle rocker in a 25 ° room. Meanwhile a fermentation unit 3 is filled with 200 1. of the same medium, and the t a n k and contents are subjected to 15 lb. of steam pressure for 1j. B. Neilands, J. Biol. Chem. 197, 701 (1952). 2 Although Ustilago sphaerogena may be propagated on almost any type of natural material, maximum cytochrome formation has been obtained only on Difco yeast extract; see also the article by P. W. Grimm and P. Allen, Plant Physiol. 29, 369 (1954). s j. j. Stefaniak, F. B. Galley, C. S. Brown, and M. J. Johnson, Ind. Eng. Chem. 38, 666 (1946).
756
RESPIRATORY ENZYMES
[134]
1 hour. After heavy growth appears in the Erlenmeyer flasks" (about 2 days), the contents are pooled and aseptically transferred to the cooled fermentation unit. Aeration is maintained at the maximum rate that will not cause loss of medium as foam going through the exhaust. Small amounts of Dow Antifoam A may be added if foaming becomes too serious. The temperature of the fermentation is maintained at 25 + 1°. After 24 hours the pH of the medium is taken at 2-hour intervals, and when it has risen to 7.5 to 8.0 there is no further growth. At this point, about 36 hours after inoculation, the fermentation may be considered complete. Step 2. Extraction. The tank is opened and 7 kg. of Filter-Cel is thoroughly mixed with the broth. The suspension is pumped through a filter press, and the cake is washed with liberal quantities of tap water. Enough water is added to the filter cake to give a fluid suspension, and N NaOH is added to bring the pH to approximately 10. The mixture is stirred mechanically, NaOH being added from time to time to maintain the pH at about 10. After 4 hours of continuous stirring, the mixture is again pumped through the filter press, and this time the clear, brownish extract is saved. Step 3. Fractionation with Ammonium Sulfate. The pH of the extract is reduced to 7 with syrupy phosphoric acid, and 487 g. of ammonium sulfate is added per liter of solution (about 75 % saturation). The material is left at 5° for 3 to 4 hours, after which a white precipitate can be removed by filtration through fluted papers. The filtrate is completely saturated with ammonium sulfate and allowed to stand at 5 ° overnight. The next morning, the cytochrome, which has precipitated, is filtered off and dialyzed against cold 0.027 % ammonium hydroxide until free from sulfate ion. This preparation is lyophilized and stored at 5 °. The yield is about 0.6 g. Step 4. Chromatography on Amberlite IRC-50. Commercial Amberlite IRC-50 is pulverized with a mortar and pestle until the particle size is reduced to 200 to 400 mesh. 4 The resin is washed on the centrifuge in succession with 5% sulfuric acid, 5 % sodium hydroxide, distilled water, and 0.1 M phosphate buffer, pH 7.0. A chromatography tube, 2.0 X 20 cm., is closed with a one-hole rubber stopper; a layer of glass wool and a layer of asbestos are placed over the stopper in order to prevent the particles of Amberlite from escaping from the tube. The resin, suspended in phosphate buffer, is poured into the tube to form a column about 10 cm.-high. The column is then washed with phosphate buffer until the effluent has a negligible optical density at 280 m~. 4 This resin may now be obtained in a fine particle size (Amberlite XE-97) from R o h m and Haas Co., Philadelphia.
[134]
CYTOCHROME C (USTILAGO)
757
The sample, about 0.6 g., is dissolved in the least amount of buffer and placed on the column. On washing with the same buffer, 0.1 M phosphate at pH 7.0, the cytochrome remains rather strongly adsorbed while a brownish, noncytochrome fraction migrates rapidly down the column and into the effluent. After the optical density at 280 m~ in the effluent has fallen to a value of less than 1.0 (1.00-cm. cell), the cytochrome is eluted with a saturated solution of ammonium acetate. The pigment is collected in a small volume and dialyzed against cold 0.027% ammonium hydroxide. The preparation, about 0.5 g., is then lyophilized and stored at 5° . A table summarizing the isolation procedure has not been included here. The method is extremely simple and, furthermore, the assay technique is not exactly quantitative. Weisel and Allen 5 found the dry cells of Ustilago sphaerogena to contain up to 1% cytochrome c. Since the above fermentation yields about 2 kg. of dry cells, the 0.5 g. of cytochrome represents only a few per cent of the total enzyme in the cells. Purity of Enzyme. No evidence of heterogeneity has been obtained when this preparation is examined in the Tiselius apparatus in the region of the isoelectric point. In these experiments a red filter (Corning No. 2424) is placed between the light source and the Tiselius cell, and the camera is loaded with red-sensitive spectroscopic film. A single, symmetrical boundary is observed when the material is analyzed in the oil turbine ultra centrifuge of Svedberg.
Properties Molecular Weight. The oil turbine ultracentrifuge gives a sedimentation constant, $20, of 1.4 X 10-13 second. The diffusion constant, D20, is calculated by the height-area method 8 to be 7.7 × 10-7 cm. 2 sec. -1. Assuming that the partial specific volume, ~, is 0.74 cm2 g.-1, these data give M = 18,000. Iron analyses by the method of Lorber 7 give the value of 0.28 % corresponding to a minimum molecular weight of 20,000. These figures must be considered preliminary, since the determination of molecular weight by the above methods can be open to large errors for low molecular weight proteins. Absorption Spectrum. The absorption spectrum of Ustilago cytochrome c is remarkably similar to that of the beef heart product. It shows the same tendency to become autoxidizable at extreme pH values. This means that the prosthetic group is derived from iron protoporphy5 p. Weisel and P. Allen, Abstracts, Meeting of the American Institute of Biological Sciences, Minneapolis, September, 1951. e L. G. Longsworth, Ann. N. Y. Acad. Sci. 41, 267 (1941). TL. Lorber, Biochem. Z. 181, 391 (1927).
758
RESPIRATORY ENZYMES
[135]
rin I X and further that the heme is probably attached to the protein part in exactly the same manner as in beef cytochrome c. Electrophoretic Mobility. Electrophoresis of Ustilago cytochrome c in the buffers used by Theorell s show the protein to be isoelectrie near pH 7. Catalytic Activity. Mole for mole, the Ustilago cytochrome c appears to be about as active as beef heart cytochrome c in the succinic dehydrogenase system of Pot t er 2 s H. Theorell, Biochem. Z. 28fi, 207 (1936). 9 V. R. Potter, in "Manometric Techniques" (Umbreit, Burris, and Stauffer, eds.), rev. ed., p. 213, Burgess Publishing Co., Minneapolis, 1949.
[135] Cytochrome c and Cytochrome c Peroxidase from
Pseudomonas fluorescens By HOWARD M. LENHOFF and NATHAN O. •APLAN I. Cytochrome c
Assay Method Principle. The cytochrome c obtained from Pseudomonas fluorescens ~ is identical in spectrum to animal cytochrome c, but it differs from animal cytochrome c in some of its biochemical and adsorptive properties. The Pseudomonas pigment has never been separated from the cytochrome peroxidase present in the extracts of Ps. fiuorescens, although it appears that these two components are not activities of the same protein. ~ This inseparability is disadvantageous in that it is difficult to study the reactivity of the cytochrome with electron donors and acceptors from other organisms owing to the interfering cytochrome peroxidase activity. On the other hand, it is advantageous in that the peroxidase allows one to assay for the presence of small amounts of the cytochrome c. Procedure. SPECTROPHOTOMETRICASSAY. The reduced pigment has an band at 550 m~, a smaller ~ band at 520 m~, and a large "r band at 415 m~ in the Soret region. The oxidized pigment absorbs slightly at 530 m~ and has its Soret band at 408 m~. These spectra are nearly identical to the spectra of the respective states of animal cytochrome c. The reduced animal cytochrome c has an extinction coefficient at 550 m~ of approximately 27.0 × 103 cm.2/mM., whereas that of the oxidized form is 9.0; the difference is 18.0 X 108 cm./mM. By determining H. M. Lenhoff and N. O. Kaplan, Nature 172, 730 (1953). 2 H. M. Lenhoff, Doctoral Thesis, Johns Hopkins University, Baltimore, 1955.