[7]
NADPH-CYTOCHROME P-450 REDUCTASE
89
p-nitroanisole O-demethylase activity, N A D P H oxidase activity, and cytochrome P-450 content 29 when prepared by this method, as compared with ultracentrifugation techniques, and adrenal, ovaries, and testes microsomal steroid metabolizing activity is likewise unaltered, 3° the same is not true with all preparations. For example, microsomes prepared from the abdomen of insecticide-resistant and susceptible house flies, and from the midgut of the southern armyworm, showed significant differences when the two isolation procedures were compared; O-demethylation activity, NADPH oxidase activity, and cytochrome P-450 content were greatly diminished in the Ca2+-aggregated preparation. 29 Rat and rabbit lung microsomal preparations are apparently also susceptible to harm by Ca2+-aggregation, 31 since the treatment diminished microsomal NADPH-cytochrome c reductase activity, pchloro-N-methylaniline demethylase, and biphenyl-4-hydroxylase; these same activities were unimpaired in rat and rabbit kidney2 ~ These observations indicate that, before adapting the calcium aggregation procedure to preparation of microsomes of another tissue or species, it is necessary to first determine whether the method is deleterious. ~9 R. C. Baker, L. B. Coons, and E. Hodgson, Chem.-Biol. Interactions 6, 307 (1973). 3o A. Warchol and R. Rembiesa, Steroids Lipids Res. 5, 113 (1974). 31 C. L. Litterst, E. G. Mimnaugh, R. L. Reagan, and T. E. Gram, Life Sci. 17, 813 (1975).
[7] P u r i f i c a t i o n a n d P r o p e r t i e s o f N A D P H - C y t o c h r o m e P-450 Reductase 1 B y H E N R Y W . STROBEL a n d JOHN D A V I D D I G N A M
NADPH--cytochrome P-450 reductase, a flavoprotein component of the endoplasmic reticulum of liver and other organs, catalyzes the transfer of electrons from N A D P H to cytochrome P-450. Cytochrome P450 is the terminal oxidase of the drug metabolism system which hydroxylates a variety of compounds, such as alkanes, fatty acids, drugs, and steroids. ~ Several forms of this hemoprotein have been purified to homogeneity, differing in minimum molecular weight and substrate specificity. 3,a 1 Supported by Grant CA 19621 from the National Cancer Institute and DRG 1258 from the Damon Runyon Memorial Fund. 2 B. B. Brodie, J. R. Gillette, and B. N. LaDu, Annu. Rev. Biochem. 27, 427 (1958). 3 D. Ryan, A. Y. H. Lu, S. West, and W. Levin, J. Biol. Chem. 250, 2157 (1975). 4 D. A. Haugen, T. A. van der Hoeven, and M. J. Coon, J. Biol. Chem. 250, 3567 (1975).
90
M I C R O S O M AELECTRON L TRANSPORT AND CYT P-450
[7]
Earlier attempts to purify the reductase employed protease solubilization to release the enzyme from microsomal suspensions. These procedures 5-~ yielded a flavoprotein preparation capable of reducing various artificial electron acceptors, but unable, as was shown later, s to support cytochrome P-450-dependent substrate hydroxylation. This flavoprotein has been extensively studied and characterized. Williams and Kamin 5 obtained a molecular weight of 68,000 for the protein, and Iyanagi and Mason showed that this flavoprotein contained equimolar amounts of FMN and FAD. 9 Several laboratories (van der Hoeven and Coon, 1° Satake e t a l . , 11 Vermilion and Coon, 12 and Dignam and Strobe113) have isolated cytochrome P-450 reductase in varying degrees of purity by techniques that employed detergent solubilization to release the reductase from microsomes followed by various column and batch procedures. Unlike earlier procedures 5-r that utilized proteases to release the enzyme from microsomes, these detergent-solubilized preparations supported the cytochrome P-450-dependent hydroxylation of substrates. In addition, the apparent subunit molecular weight of the protein isolated by such procedureslZ,13 was about 10,000 greater than that of the protein isolated by protease-requiring, procedures. The procedure reported here describes the purification to apparent homogeneity of NADPH cytochrome P-450 reductase by solubilization with Renex 690 and affinity chromatography. ~4 The affinity column used is an NADP ligand attached to Sepharose 4B through adipic acid dihydrazide.
Assay Methods Cytochrome P-450 reductase transfers electrons to cytochrome P450, its native acceptor, which catalyzes the hydroxylation of drugs and other xenobiotics, and to a number of artificial electron acceptors, including cytochrome c, ferricyanide, and dichlorophenolindophenol. 5 C. H. Williams and H. Kamin, J. Biol. Chem. 237, 587 (1962). 6 A. H. Phillips and R. G. Langdon, J. Biol. Chem. 237, 2652 (1962). ZT. C. Pederson, J. A. Buege, and S. D. Aust, J. Biol. Chem. 248, 7134 (1973). g B. S. S. Masters, R. A, Prough, and H. Kamin, Biochemistry 14, 607 (1975). 9 T. Iyanagi and H. S. Mason, Biochemistry 12, 2297 (1973). ~0T. A. van der Hoeven and M. J. Coon, J. Biol. Chem. 249, 6302 (1974). 11 H. Satake, Y. lmai, and R. Sato, Abstr. Annu. Meeting Jpn. Biochem. Soc. (1972). ,z j. L. Vermilion and M. J. Coon, Biochem. Biophys. Res. Commun. 60, 595 (1974). 13 j. D. Dignam and H. W. Strobel, Biochem. Biophys. Res. Commun. 63, 845 (1975). a4 j. D. Dignam and H. W. Strobel, Biochemistry 16, 116 (1977).
[7]
NADPH-CYTOCHROME P-450 REDUCTASE
91
Reduction of Cytochrome P-450. The transfer of electrons by the reductase to cytochrome P-450 is determined by NADPH oxidation in a reconstituted system with benzphetamine as the substrate. The reaction mixture contains 100 t~mol of potassium phosphate buffer (pH 7.7), 1.0 ftmol of benzphetamine, cytochrome P-450, cytochrome P-450 reductase, dilauroylphosphatidylcholine, and 0.15 txmol of NADPH as a final addition. The optimal amounts of cytochrome P-450, reductase and phosphatidylcholine are empirically determined for each preparation, but the usual range per assay is 0.05 to 0.3 nmol of cytochrome P-450, 0.5 to 2.0 /xg of reductase, and 30 p,g of phosphatidylcholine. The reaction is followed spectrophotometrically at 340 nm, and the rate of NADPH oxidation is calculated using an extinction coefficient of 6.2 cm -1 mM-1. is The activity of the reconstituted system is dependent upon the presence of each of the protein and lipid components as well as substrate, NADPH, and oxygen. Alternatively, the reaction can be followed by determination of the amount of formaldehyde formed from benzphetamine through a colorimetric determination.16 The reduction of cytochrome P-450 can be determined directly under anaerobic conditions in the presence of carbon monoxide by following the formation of the peak at 450 nm in the reduced carbon monoxide difference spectrum. ~7,i~ Reduction of Cytochrome c. The reductase-catalyzed transfer of electrons to cytochrome c is determined spectrophotometrically by measuring the increase in absorbance at 550 nm due to the appearance of reduced cytochrome c. Phillips and Langdon demonstrated that the rate of cytochrome c reduction increases with increasing ionic strength, s Hence, the standard assay for cytochrome c reduction is conducted at 30 ° in semimicro cells containing 300 tzmol of potassium phosphate buffer (pH 7.7), 40.0 nmol of cytochrome c, and 0.1 izmol of NADPH in a final volume of 1.0 ml. The reaction is initiated by the addition of the NADPH. The rate of cytochrome c reduction is calculated using an extinction coefficient of 21 cm -1 mM -1 at 550 nm. ~ The reduction of other artificial electron acceptors can be measured spectrophotometrically under identical assay conditions (with the exception that 200 tzg of bovine serum albumin per milliliter are present) using extinction coeffi~z A. Y. H. Lu, H. W. Strobel, and M. J. Coon, Biochem. Biophys. Res, Commun. 36, 545 (1%9). le j. Cochin and J. Axelrod, J. Pharmacol. Exp. Ther. 125, 105 (1959). 17 H. W. Strobel, A. Y. H. Lu, J. Heidema, and M. J. Coon, J. Biol. Chem. 245, 4851 (1970). 18 M. J. Coon, A. P. Autor, and H. W. Strobel, Chem.-Biol. Interactions 3, 248 (1971).
92
MICROSOMAL ELECTRONTRANSPORTAND CYT P-450
[7]
cients of 21 cm -1 mM -1 at 600 nm for dichlorophenolindophenolTM and 1.02 cm -~ mM -~ at 420 for ferricyanide. ~° Purification Procedure The affinity column used in this procedure contains NADP linked to Sepharose 4B through an adipic acid dihydrazide spacer arm. Sepharose 4B is activated with cyanogen bromide according to Cuatrecasas and co-workers. 21"22 NADP and adipic acid dibydrazide are coupled to Sepharose 4B by a modification of the procedure of Lamed et al. zs Two hundred milliliters of cyanogen bromide-activated Sepharose 4B are added at 4 ° to 200 ml of 0.1 N NaHCO3 (pH 9.5) buffer saturated with adipic acid dihydrazide (18 g). This mixture is allowed to react overnight with stirring at 4 ° and is subsequently washed with 2 liters of 2.0 M NaCI. The washed side-arm resin can be used immediately or stored at 4 ° until needed. NADP (10 raM) is oxidized for 3 hr at 5 ° in a 20-ml reaction volume containing 0.1 M potassium phosphate buffer (pH 7.0) and 40 mM sodium periodate. Glycerol (5 ml) is added to eliminate excess periodate, and the mixture is stirred overnight at 5 °. The affinity resin is prepared by stirring 20 ml of oxidized nucleotide, 40 ml of packed side-arm resin, 25 ml of 0.4 M sodium acetate buffer (pH 5.0), and 15 ml of water for 4 hr at 4 °. The affinity resin is washed with 2 liters of 2.0 N NaCl and 500 ml of deionized water. A typical preparation of the affinity resin contains approximately 4/zmol of NADP per milliliter of gel. 28 Preparation
of Affinity Resin.
P r e p a r a t i o n o f M i c r o s o m e s . Male, 75-80 g Sprague-Dawley rats are induced by injection of phenobarbital sodium (75 mg/kg body weight) in 1.0 ml of 0.9% NaCl every 8 hr for 2 days prior to sacrifice. Livers are homogenized in a Waring Blendor in 1.14% (w/v) KCI containing l0 mM EDTA. Phenylmethylsulfonyl fluoride (PMSF) in absolute ethanol is added to a final concentration of 0.25 mM immediately prior to homogenization. Microsomes are prepared from the liver homogenate by differential centrifugation. The postmitochondrial (9000 g) supernatant fraction is made 0.25 mM in PMSF and centrifuged at 100,000 g for 90 rain. The microsomal pellet is resuspended in 1.14% KC1 containing 10
Steyn-Parveand H. Beinert,J. Biol. Chem. 233, 843 (1958). 20K. H. Schellenbergand L. Hellerman,J. Biol. Chem. 231, 547 (1958). 21p. Cuatrecasas, M. Wilchek,and C. B. Anfinsen,Proc. Natl. Acad. Sci. U.S.A. 61,636 (1968). 2z p. Cuatrecasas,J. Biol. Chem. 245, 3059 (1970), 2aR. Lamed, Y. Levin, and M. Wilchek,Biochim. Biophys. Acta 304, 231 (1973). ~9 E. P.
[7]
NADPH-CYTOCHROME P-450 REDUCTASE
93
mM EDTA made 0.25 mM in PMSF and recentrifuged at 100,000 g for 60 min. The washed microsomal pellets are resuspended in 0.25 M sucrose and stored at - 7 0 °. The use of EDTA and PMSF prevents proteolytic degradation of the microsomes. Microsomes prepared by this procedure can be stored for weeks at - 7 0 ° without loss of activity.
Step 1. Solubilization. Solubilization and all other operations in the purification procedure are performed at 4 °. Microsomal protein, 4-6 g, prepared not more than 3 weeks in advance, is suspended in 0.2 M Trischloride buffer (pH 7.7 at 4 °) containing 30% glycerol, 1 mM EDTA, and 0.1 mM dithiothreitol to a final concentration of 10 mg/ml. To solubilize the microsomal proteins, Renex 690 [polyoxyethylene (10)nonyl phenol ether] is added slowly with stirring as a 10% (v/v) solution to a final concentration of 1.5% (v/v). (The optimal concentration of Renex 690 required for solubilization may vary owing to variations in different lots of this detergent.) Immediately prior to the addition of detergent to the turbid microsomal suspension, PMSF is added to inhibit proteases that might be released during the solubilization process. Renex 690 solubilization causes little or no loss in reductase activity. Step 2. Protamine Sulfate Precipitation. To the clear solubilized microsomes protamine sulfate (Nutritional Biochemicals Corporation, 67-70% arginine) is added slowly as a 1.5% (w/v) solution to a final concentration of 0.03% (w/v). The cloudy suspension is centrifuged at 100,000 g for 60 rain. The sticky, grayish-white pellet is discarded. The protamine sulfate fractionation does not give a significant improvement in specific activity of the reductase, but use of this step makes possible the elution of the reductase as a sharp peak from DEAE-Sephadex A-25. Step 3. Chromatography on DEAE-Sephadex A-25 Column. The clear, red supernatant fraction resulting from high speed centrifugation of the protamine sulfate-treated, solubilized microsomes is loaded at 150 ml/hr onto a 1000-ml bed volume DEAE-Sephadex A-25 column (5 × 55 cm) previously equilibrated with 0.1 M Tris-chloride buffer (pH 7.7) containing 20% glycerol, 1 mM EDTA, 0.1 mM dithiothreitol, and 0.15% (v/v) Renex 690. The column is washed with about 800 ml of equilibration buffer at 200 ml/hr to elute cytochrome P-450, cytochrome bs, and NADH-dependent ferricyanide reductase activity. NADPH-cytochrome P-450 reductase is eluted with a linear 3 liter 0 to 0.3 M KC1 gradient in equilibration buffer. The reductase elutes as a sharp peak at about 0.1 M KC1. The reductase fraction after DEAE-Sephadex chromatography is free of cytochromes P-450 and b~ although heme is present. This column accomplishes a 20- to 40-fold purification of the reductase over the starting microsomes with a total yield of 70-100% of the initial activity.
94
MICROSOMAL ELECTRONTRANSPORTAND CYT P-450
[7]
Step 4. Affinity Chromatography on N A D P - - S e p h a r o s e 4B. Fractions with NADPH-cytochrome c reductase activity are pooled, and the pH of the pooled fractions is lowered to 7.0 (at 5 ~) by the dropwise addition of 1 M KH2PO4. Lowering the pH increases the binding of the reductase for the NADP ligand on the affinity column. Care must be taken not to expose the reductase to intense light, since the enzyme becomes lightsensitive at lower pHs. 24 The pooled fractions are pumped at 200 ml/hr onto a 20-ml bed volume NADP-Sepharose column previously equilibrated in 0.1 M Tris-chloride buffer (pH 7.0 at 4 ~) containing 20% glycerol, 1 mM EDTA, 0.1 mM dithiothreitol, and 0.15% (v/v) Renex 690. Approximately 20% of the enzyme does not bind to the column and is present in the flow-through. The affinity column is washed with 200 ml of 20 mM potassium phosphate buffer (pH 7.0) containing 20% glycerol, 1 mM EDTA, 0.1 mM dithiothreitol, and 0,1% sodium deoxycholate. The reductase is eluted with 0.5 mM NADP ÷ in 0.1 M potassium phosphate buffer (pH 7.7) containing 20% glycerol, 1.0 mM EDTA, 0.1 mM dithiothreitol, and 0.1% (w/v) sodium deoxycholate. The pooled fractions can be concentrated further by ultrafiltration using an Amicon XM-50 membrane. NADP is removed by dialysis or gel filtration on Sephadex G-25. The affinity column step removes the heme present in the reductase after chromatography on DEAE-Sephadex and gives an 8- to 12-fold purification of the enzyme. The usual yield for this step is 60-80% of the reductase activity present in the DEAE-Sephadex eluate. This purification procedure (summarized in Table I) is composed of two column steps and takes about 2 days to complete from microsomes to purified enzyme. The average preparation has an overall yield of 5060% of the reductase activity present in the starting microsomes. The overall purification of the reductase from microsomes is 250- to 300-fold. Yasukochi and Masters 2s have independently reported on the purification of this enzyme using 2',5'-ADP Sepharose. Recently, Coon and coworkers 2e have reported two forms of reductase from rabbit and rat liver microsomes differing in apparent minimum molecular weight, all of which catalyze electron transfer to cytochrome P-450.
Properties Flavin Content and Molecular Weight. NADPH-cytochrome P-450 rcductase contains both FMN and FAD in a ratio of 1 mol of each flavin
z4j. p. Baggotand R. G. Langdon,J. Biol. Chem. 245, 5888 (1970). 2~y. Yasukochiand B. S. S. Masters,J. Biol. Chem. 251, 5337 (1976). 28M. J. Coon,J. L. Vermilion,K. P. Vastis, J. S. French, W. L. Dean, and D. A. Haugen, in "Drug Metabolism Concepts" (D. M. Jerina, ed.), p. 46. Am. Chem. Soc., Washington, D.C.
[7]
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95
TABLE I PURIFICATION OF NADPH-CvToCaROME P-450 REDUCTASEFROM RAT LIVER MICROSOMES
Step Microsomes Solubilized microsomes treated with protamine sulfate DEAE-Sephadex A-25 pooled fractions NADP-adipic acid dihydrazide Sepharose 4B
Total protein (mg) 4200 3200
120 (100-300) 8.25 (7.0-24.0)
Total activity
Specific activity"
Yield (%)
1070 1065
0.255 0.323 (0.24-0.6)
100 99.4
1063 575
8.86 (5.0-12.0) 69 (62-70)
99.3 (70-100) 55 (40-66)
a Micromoles of cytochrome c reduced per minute per milligram of protein.
per mol of reductase in accord with the observations of Iyanagi and Mason 9 for protease-solubilized cytochrome c reductase. This complement of fiavin and the observation that reductase prepared by our method is not stimulated by preincubation with exogenous FMN, FAD, or both flavins is consistent with the suggestion that the reductase does not lose appreciable amounts of its flavin during the course of purification. The minimum molecular weight based on flavin content and L o w r y 27 protein determination is 74,100. When the reductase and proteins of known molecular weight are subjected to gel electrophoresis in the presence of sodium dodecyl sulfate (SDS), an apparent minimum molecular weight of 79,500 is obtained. 2s A minimum molecular weight of 76,500 is obtained from sedimentation equilibrium studies of the reductase in 6 M guanidine hydrochloride. 28 The minimum molecular weights determined by three different techniques are therefore in good agreement.
Stability. The reductase is stable indefinitely at - 7 0 ° in the presence of glycerol. It is also stable on ice in the presence of glycerol, but is relatively unstable on ice in the absence of glycerol. Activity. The reductase transfers electrons to its native acceptor, cytochrome P-450, as well as to a number of artificial electron acceptors. z70. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem. 193, 265 (1951). zs j. A. Knapp, J. D. Dignam, and H. W. Strobel, J. Biol. Chem. 252, 437 (1977).
96
[7]
MICROSOMAL ELECTRON TRANSPORT AND CYT P-450
Phosphatidylcholine is required for the transfer of electrons to cytochrome P-450 by the reductase, but not for electron transfer to artificial electron acceptors. The ability of the reductase to transfer electrons to both acceptors is summarized in Table II.
Degree of Purity. The reductase appears to be homogeneous by disc and slab gel electrophoresis in the presence of SDS. Studies on the free mobility of the reductase in SDS indicate that the reductase interacts with the detergent in a similar way as known protein standards, such as bovine serum albumin and ovalbumin. Inhibitors. Cytochrome P-450 reductase is inhibited by sulfhydryl reagents such as 5',5'-dithiobis-2-nitrobenzoic acid and 2-nitro-5-thiocyanatobenzoic acid. Other inhibitory compounds, such as sodium benzoate, 8-hydroxyquinoline, and the cytochrome P-450 inhibitor SKF525A (2-diethylaminoethyl-2,2-diphenylvalerate hydrochloride), have no effect on the rate of cytochrome c reduction catalyzed by the enzyme. TABLE II ELECTRON TRANSFER ACTIVITY OF NADPH-CYTOCHROME P-450 REDUCTASE Expt. No.
System Complete reconstituted a Minus cytochrome P-450 Minus phosphatidylcholine Minus reductase Reductase b + cytochrome c + dichlorophenolindophenol + potassium ferricyanide
Activity (/zmol/min/mg) 2.6 0 0 0 69 55 80
a Reaction mixture contains 150/~mol of potassium phosphate buffer (pH 7.7), 1.5/xmol of benzphetamine, cytochrome P-450 (0.35 nmol, 0.04 mg), reductase (0.63 p~g), phosphatidylcholine (30/zg), and 0.5 /~mol of NADPH in a final volume of 1.5 ml. Formaldehyde formation from benzphetamine was determined colorimetrically after a 10-min incubation. A substrate blank rate was subtracted from the substratedependent rate. b Reaction mixtures are described under assay methods.
NADPH-CYTOCHROME P-450 REDUCTASE
89
p-nitroanisole O-demethylase activity, N A D P H oxidase activity, and cytochrome P-450 content 29 when prepared by this method, as compared with ultracentrifugation techniques, and adrenal, ovaries, and testes microsomal steroid metabolizing activity is likewise unaltered, 3° the same is not true with all preparations. For example, microsomes prepared from the abdomen of insecticide-resistant and susceptible house flies, and from the midgut of the southern armyworm, showed significant differences when the two isolation procedures were compared; O-demethylation activity, NADPH oxidase activity, and cytochrome P-450 content were greatly diminished in the Ca2+-aggregated preparation. 29 Rat and rabbit lung microsomal preparations are apparently also susceptible to harm by Ca2+-aggregation, 31 since the treatment diminished microsomal NADPH-cytochrome c reductase activity, pchloro-N-methylaniline demethylase, and biphenyl-4-hydroxylase; these same activities were unimpaired in rat and rabbit kidney2 ~ These observations indicate that, before adapting the calcium aggregation procedure to preparation of microsomes of another tissue or species, it is necessary to first determine whether the method is deleterious. ~9 R. C. Baker, L. B. Coons, and E. Hodgson, Chem.-Biol. Interactions 6, 307 (1973). 3o A. Warchol and R. Rembiesa, Steroids Lipids Res. 5, 113 (1974). 31 C. L. Litterst, E. G. Mimnaugh, R. L. Reagan, and T. E. Gram, Life Sci. 17, 813 (1975).
[7] P u r i f i c a t i o n a n d P r o p e r t i e s o f N A D P H - C y t o c h r o m e P-450 Reductase 1 B y H E N R Y W . STROBEL a n d JOHN D A V I D D I G N A M
NADPH--cytochrome P-450 reductase, a flavoprotein component of the endoplasmic reticulum of liver and other organs, catalyzes the transfer of electrons from N A D P H to cytochrome P-450. Cytochrome P450 is the terminal oxidase of the drug metabolism system which hydroxylates a variety of compounds, such as alkanes, fatty acids, drugs, and steroids. ~ Several forms of this hemoprotein have been purified to homogeneity, differing in minimum molecular weight and substrate specificity. 3,a 1 Supported by Grant CA 19621 from the National Cancer Institute and DRG 1258 from the Damon Runyon Memorial Fund. 2 B. B. Brodie, J. R. Gillette, and B. N. LaDu, Annu. Rev. Biochem. 27, 427 (1958). 3 D. Ryan, A. Y. H. Lu, S. West, and W. Levin, J. Biol. Chem. 250, 2157 (1975). 4 D. A. Haugen, T. A. van der Hoeven, and M. J. Coon, J. Biol. Chem. 250, 3567 (1975).
90
M I C R O S O M AELECTRON L TRANSPORT AND CYT P-450
[7]
Earlier attempts to purify the reductase employed protease solubilization to release the enzyme from microsomal suspensions. These procedures 5-~ yielded a flavoprotein preparation capable of reducing various artificial electron acceptors, but unable, as was shown later, s to support cytochrome P-450-dependent substrate hydroxylation. This flavoprotein has been extensively studied and characterized. Williams and Kamin 5 obtained a molecular weight of 68,000 for the protein, and Iyanagi and Mason showed that this flavoprotein contained equimolar amounts of FMN and FAD. 9 Several laboratories (van der Hoeven and Coon, 1° Satake e t a l . , 11 Vermilion and Coon, 12 and Dignam and Strobe113) have isolated cytochrome P-450 reductase in varying degrees of purity by techniques that employed detergent solubilization to release the reductase from microsomes followed by various column and batch procedures. Unlike earlier procedures 5-r that utilized proteases to release the enzyme from microsomes, these detergent-solubilized preparations supported the cytochrome P-450-dependent hydroxylation of substrates. In addition, the apparent subunit molecular weight of the protein isolated by such procedureslZ,13 was about 10,000 greater than that of the protein isolated by protease-requiring, procedures. The procedure reported here describes the purification to apparent homogeneity of NADPH cytochrome P-450 reductase by solubilization with Renex 690 and affinity chromatography. ~4 The affinity column used is an NADP ligand attached to Sepharose 4B through adipic acid dihydrazide.
Assay Methods Cytochrome P-450 reductase transfers electrons to cytochrome P450, its native acceptor, which catalyzes the hydroxylation of drugs and other xenobiotics, and to a number of artificial electron acceptors, including cytochrome c, ferricyanide, and dichlorophenolindophenol. 5 C. H. Williams and H. Kamin, J. Biol. Chem. 237, 587 (1962). 6 A. H. Phillips and R. G. Langdon, J. Biol. Chem. 237, 2652 (1962). ZT. C. Pederson, J. A. Buege, and S. D. Aust, J. Biol. Chem. 248, 7134 (1973). g B. S. S. Masters, R. A, Prough, and H. Kamin, Biochemistry 14, 607 (1975). 9 T. Iyanagi and H. S. Mason, Biochemistry 12, 2297 (1973). ~0T. A. van der Hoeven and M. J. Coon, J. Biol. Chem. 249, 6302 (1974). 11 H. Satake, Y. lmai, and R. Sato, Abstr. Annu. Meeting Jpn. Biochem. Soc. (1972). ,z j. L. Vermilion and M. J. Coon, Biochem. Biophys. Res. Commun. 60, 595 (1974). 13 j. D. Dignam and H. W. Strobel, Biochem. Biophys. Res. Commun. 63, 845 (1975). a4 j. D. Dignam and H. W. Strobel, Biochemistry 16, 116 (1977).
[7]
NADPH-CYTOCHROME P-450 REDUCTASE
91
Reduction of Cytochrome P-450. The transfer of electrons by the reductase to cytochrome P-450 is determined by NADPH oxidation in a reconstituted system with benzphetamine as the substrate. The reaction mixture contains 100 t~mol of potassium phosphate buffer (pH 7.7), 1.0 ftmol of benzphetamine, cytochrome P-450, cytochrome P-450 reductase, dilauroylphosphatidylcholine, and 0.15 txmol of NADPH as a final addition. The optimal amounts of cytochrome P-450, reductase and phosphatidylcholine are empirically determined for each preparation, but the usual range per assay is 0.05 to 0.3 nmol of cytochrome P-450, 0.5 to 2.0 /xg of reductase, and 30 p,g of phosphatidylcholine. The reaction is followed spectrophotometrically at 340 nm, and the rate of NADPH oxidation is calculated using an extinction coefficient of 6.2 cm -1 mM-1. is The activity of the reconstituted system is dependent upon the presence of each of the protein and lipid components as well as substrate, NADPH, and oxygen. Alternatively, the reaction can be followed by determination of the amount of formaldehyde formed from benzphetamine through a colorimetric determination.16 The reduction of cytochrome P-450 can be determined directly under anaerobic conditions in the presence of carbon monoxide by following the formation of the peak at 450 nm in the reduced carbon monoxide difference spectrum. ~7,i~ Reduction of Cytochrome c. The reductase-catalyzed transfer of electrons to cytochrome c is determined spectrophotometrically by measuring the increase in absorbance at 550 nm due to the appearance of reduced cytochrome c. Phillips and Langdon demonstrated that the rate of cytochrome c reduction increases with increasing ionic strength, s Hence, the standard assay for cytochrome c reduction is conducted at 30 ° in semimicro cells containing 300 tzmol of potassium phosphate buffer (pH 7.7), 40.0 nmol of cytochrome c, and 0.1 izmol of NADPH in a final volume of 1.0 ml. The reaction is initiated by the addition of the NADPH. The rate of cytochrome c reduction is calculated using an extinction coefficient of 21 cm -1 mM -1 at 550 nm. ~ The reduction of other artificial electron acceptors can be measured spectrophotometrically under identical assay conditions (with the exception that 200 tzg of bovine serum albumin per milliliter are present) using extinction coeffi~z A. Y. H. Lu, H. W. Strobel, and M. J. Coon, Biochem. Biophys. Res, Commun. 36, 545 (1%9). le j. Cochin and J. Axelrod, J. Pharmacol. Exp. Ther. 125, 105 (1959). 17 H. W. Strobel, A. Y. H. Lu, J. Heidema, and M. J. Coon, J. Biol. Chem. 245, 4851 (1970). 18 M. J. Coon, A. P. Autor, and H. W. Strobel, Chem.-Biol. Interactions 3, 248 (1971).
92
MICROSOMAL ELECTRONTRANSPORTAND CYT P-450
[7]
cients of 21 cm -1 mM -1 at 600 nm for dichlorophenolindophenolTM and 1.02 cm -~ mM -~ at 420 for ferricyanide. ~° Purification Procedure The affinity column used in this procedure contains NADP linked to Sepharose 4B through an adipic acid dihydrazide spacer arm. Sepharose 4B is activated with cyanogen bromide according to Cuatrecasas and co-workers. 21"22 NADP and adipic acid dibydrazide are coupled to Sepharose 4B by a modification of the procedure of Lamed et al. zs Two hundred milliliters of cyanogen bromide-activated Sepharose 4B are added at 4 ° to 200 ml of 0.1 N NaHCO3 (pH 9.5) buffer saturated with adipic acid dihydrazide (18 g). This mixture is allowed to react overnight with stirring at 4 ° and is subsequently washed with 2 liters of 2.0 M NaCI. The washed side-arm resin can be used immediately or stored at 4 ° until needed. NADP (10 raM) is oxidized for 3 hr at 5 ° in a 20-ml reaction volume containing 0.1 M potassium phosphate buffer (pH 7.0) and 40 mM sodium periodate. Glycerol (5 ml) is added to eliminate excess periodate, and the mixture is stirred overnight at 5 °. The affinity resin is prepared by stirring 20 ml of oxidized nucleotide, 40 ml of packed side-arm resin, 25 ml of 0.4 M sodium acetate buffer (pH 5.0), and 15 ml of water for 4 hr at 4 °. The affinity resin is washed with 2 liters of 2.0 N NaCl and 500 ml of deionized water. A typical preparation of the affinity resin contains approximately 4/zmol of NADP per milliliter of gel. 28 Preparation
of Affinity Resin.
P r e p a r a t i o n o f M i c r o s o m e s . Male, 75-80 g Sprague-Dawley rats are induced by injection of phenobarbital sodium (75 mg/kg body weight) in 1.0 ml of 0.9% NaCl every 8 hr for 2 days prior to sacrifice. Livers are homogenized in a Waring Blendor in 1.14% (w/v) KCI containing l0 mM EDTA. Phenylmethylsulfonyl fluoride (PMSF) in absolute ethanol is added to a final concentration of 0.25 mM immediately prior to homogenization. Microsomes are prepared from the liver homogenate by differential centrifugation. The postmitochondrial (9000 g) supernatant fraction is made 0.25 mM in PMSF and centrifuged at 100,000 g for 90 rain. The microsomal pellet is resuspended in 1.14% KC1 containing 10
Steyn-Parveand H. Beinert,J. Biol. Chem. 233, 843 (1958). 20K. H. Schellenbergand L. Hellerman,J. Biol. Chem. 231, 547 (1958). 21p. Cuatrecasas, M. Wilchek,and C. B. Anfinsen,Proc. Natl. Acad. Sci. U.S.A. 61,636 (1968). 2z p. Cuatrecasas,J. Biol. Chem. 245, 3059 (1970), 2aR. Lamed, Y. Levin, and M. Wilchek,Biochim. Biophys. Acta 304, 231 (1973). ~9 E. P.
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NADPH-CYTOCHROME P-450 REDUCTASE
93
mM EDTA made 0.25 mM in PMSF and recentrifuged at 100,000 g for 60 min. The washed microsomal pellets are resuspended in 0.25 M sucrose and stored at - 7 0 °. The use of EDTA and PMSF prevents proteolytic degradation of the microsomes. Microsomes prepared by this procedure can be stored for weeks at - 7 0 ° without loss of activity.
Step 1. Solubilization. Solubilization and all other operations in the purification procedure are performed at 4 °. Microsomal protein, 4-6 g, prepared not more than 3 weeks in advance, is suspended in 0.2 M Trischloride buffer (pH 7.7 at 4 °) containing 30% glycerol, 1 mM EDTA, and 0.1 mM dithiothreitol to a final concentration of 10 mg/ml. To solubilize the microsomal proteins, Renex 690 [polyoxyethylene (10)nonyl phenol ether] is added slowly with stirring as a 10% (v/v) solution to a final concentration of 1.5% (v/v). (The optimal concentration of Renex 690 required for solubilization may vary owing to variations in different lots of this detergent.) Immediately prior to the addition of detergent to the turbid microsomal suspension, PMSF is added to inhibit proteases that might be released during the solubilization process. Renex 690 solubilization causes little or no loss in reductase activity. Step 2. Protamine Sulfate Precipitation. To the clear solubilized microsomes protamine sulfate (Nutritional Biochemicals Corporation, 67-70% arginine) is added slowly as a 1.5% (w/v) solution to a final concentration of 0.03% (w/v). The cloudy suspension is centrifuged at 100,000 g for 60 rain. The sticky, grayish-white pellet is discarded. The protamine sulfate fractionation does not give a significant improvement in specific activity of the reductase, but use of this step makes possible the elution of the reductase as a sharp peak from DEAE-Sephadex A-25. Step 3. Chromatography on DEAE-Sephadex A-25 Column. The clear, red supernatant fraction resulting from high speed centrifugation of the protamine sulfate-treated, solubilized microsomes is loaded at 150 ml/hr onto a 1000-ml bed volume DEAE-Sephadex A-25 column (5 × 55 cm) previously equilibrated with 0.1 M Tris-chloride buffer (pH 7.7) containing 20% glycerol, 1 mM EDTA, 0.1 mM dithiothreitol, and 0.15% (v/v) Renex 690. The column is washed with about 800 ml of equilibration buffer at 200 ml/hr to elute cytochrome P-450, cytochrome bs, and NADH-dependent ferricyanide reductase activity. NADPH-cytochrome P-450 reductase is eluted with a linear 3 liter 0 to 0.3 M KC1 gradient in equilibration buffer. The reductase elutes as a sharp peak at about 0.1 M KC1. The reductase fraction after DEAE-Sephadex chromatography is free of cytochromes P-450 and b~ although heme is present. This column accomplishes a 20- to 40-fold purification of the reductase over the starting microsomes with a total yield of 70-100% of the initial activity.
94
MICROSOMAL ELECTRONTRANSPORTAND CYT P-450
[7]
Step 4. Affinity Chromatography on N A D P - - S e p h a r o s e 4B. Fractions with NADPH-cytochrome c reductase activity are pooled, and the pH of the pooled fractions is lowered to 7.0 (at 5 ~) by the dropwise addition of 1 M KH2PO4. Lowering the pH increases the binding of the reductase for the NADP ligand on the affinity column. Care must be taken not to expose the reductase to intense light, since the enzyme becomes lightsensitive at lower pHs. 24 The pooled fractions are pumped at 200 ml/hr onto a 20-ml bed volume NADP-Sepharose column previously equilibrated in 0.1 M Tris-chloride buffer (pH 7.0 at 4 ~) containing 20% glycerol, 1 mM EDTA, 0.1 mM dithiothreitol, and 0.15% (v/v) Renex 690. Approximately 20% of the enzyme does not bind to the column and is present in the flow-through. The affinity column is washed with 200 ml of 20 mM potassium phosphate buffer (pH 7.0) containing 20% glycerol, 1 mM EDTA, 0.1 mM dithiothreitol, and 0,1% sodium deoxycholate. The reductase is eluted with 0.5 mM NADP ÷ in 0.1 M potassium phosphate buffer (pH 7.7) containing 20% glycerol, 1.0 mM EDTA, 0.1 mM dithiothreitol, and 0.1% (w/v) sodium deoxycholate. The pooled fractions can be concentrated further by ultrafiltration using an Amicon XM-50 membrane. NADP is removed by dialysis or gel filtration on Sephadex G-25. The affinity column step removes the heme present in the reductase after chromatography on DEAE-Sephadex and gives an 8- to 12-fold purification of the enzyme. The usual yield for this step is 60-80% of the reductase activity present in the DEAE-Sephadex eluate. This purification procedure (summarized in Table I) is composed of two column steps and takes about 2 days to complete from microsomes to purified enzyme. The average preparation has an overall yield of 5060% of the reductase activity present in the starting microsomes. The overall purification of the reductase from microsomes is 250- to 300-fold. Yasukochi and Masters 2s have independently reported on the purification of this enzyme using 2',5'-ADP Sepharose. Recently, Coon and coworkers 2e have reported two forms of reductase from rabbit and rat liver microsomes differing in apparent minimum molecular weight, all of which catalyze electron transfer to cytochrome P-450.
Properties Flavin Content and Molecular Weight. NADPH-cytochrome P-450 rcductase contains both FMN and FAD in a ratio of 1 mol of each flavin
z4j. p. Baggotand R. G. Langdon,J. Biol. Chem. 245, 5888 (1970). 2~y. Yasukochiand B. S. S. Masters,J. Biol. Chem. 251, 5337 (1976). 28M. J. Coon,J. L. Vermilion,K. P. Vastis, J. S. French, W. L. Dean, and D. A. Haugen, in "Drug Metabolism Concepts" (D. M. Jerina, ed.), p. 46. Am. Chem. Soc., Washington, D.C.
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95
TABLE I PURIFICATION OF NADPH-CvToCaROME P-450 REDUCTASEFROM RAT LIVER MICROSOMES
Step Microsomes Solubilized microsomes treated with protamine sulfate DEAE-Sephadex A-25 pooled fractions NADP-adipic acid dihydrazide Sepharose 4B
Total protein (mg) 4200 3200
120 (100-300) 8.25 (7.0-24.0)
Total activity
Specific activity"
Yield (%)
1070 1065
0.255 0.323 (0.24-0.6)
100 99.4
1063 575
8.86 (5.0-12.0) 69 (62-70)
99.3 (70-100) 55 (40-66)
a Micromoles of cytochrome c reduced per minute per milligram of protein.
per mol of reductase in accord with the observations of Iyanagi and Mason 9 for protease-solubilized cytochrome c reductase. This complement of fiavin and the observation that reductase prepared by our method is not stimulated by preincubation with exogenous FMN, FAD, or both flavins is consistent with the suggestion that the reductase does not lose appreciable amounts of its flavin during the course of purification. The minimum molecular weight based on flavin content and L o w r y 27 protein determination is 74,100. When the reductase and proteins of known molecular weight are subjected to gel electrophoresis in the presence of sodium dodecyl sulfate (SDS), an apparent minimum molecular weight of 79,500 is obtained. 2s A minimum molecular weight of 76,500 is obtained from sedimentation equilibrium studies of the reductase in 6 M guanidine hydrochloride. 28 The minimum molecular weights determined by three different techniques are therefore in good agreement.
Stability. The reductase is stable indefinitely at - 7 0 ° in the presence of glycerol. It is also stable on ice in the presence of glycerol, but is relatively unstable on ice in the absence of glycerol. Activity. The reductase transfers electrons to its native acceptor, cytochrome P-450, as well as to a number of artificial electron acceptors. z70. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem. 193, 265 (1951). zs j. A. Knapp, J. D. Dignam, and H. W. Strobel, J. Biol. Chem. 252, 437 (1977).
96
[7]
MICROSOMAL ELECTRON TRANSPORT AND CYT P-450
Phosphatidylcholine is required for the transfer of electrons to cytochrome P-450 by the reductase, but not for electron transfer to artificial electron acceptors. The ability of the reductase to transfer electrons to both acceptors is summarized in Table II.
Degree of Purity. The reductase appears to be homogeneous by disc and slab gel electrophoresis in the presence of SDS. Studies on the free mobility of the reductase in SDS indicate that the reductase interacts with the detergent in a similar way as known protein standards, such as bovine serum albumin and ovalbumin. Inhibitors. Cytochrome P-450 reductase is inhibited by sulfhydryl reagents such as 5',5'-dithiobis-2-nitrobenzoic acid and 2-nitro-5-thiocyanatobenzoic acid. Other inhibitory compounds, such as sodium benzoate, 8-hydroxyquinoline, and the cytochrome P-450 inhibitor SKF525A (2-diethylaminoethyl-2,2-diphenylvalerate hydrochloride), have no effect on the rate of cytochrome c reduction catalyzed by the enzyme. TABLE II ELECTRON TRANSFER ACTIVITY OF NADPH-CYTOCHROME P-450 REDUCTASE Expt. No.
System Complete reconstituted a Minus cytochrome P-450 Minus phosphatidylcholine Minus reductase Reductase b + cytochrome c + dichlorophenolindophenol + potassium ferricyanide
Activity (/zmol/min/mg) 2.6 0 0 0 69 55 80
a Reaction mixture contains 150/~mol of potassium phosphate buffer (pH 7.7), 1.5/xmol of benzphetamine, cytochrome P-450 (0.35 nmol, 0.04 mg), reductase (0.63 p~g), phosphatidylcholine (30/zg), and 0.5 /~mol of NADPH in a final volume of 1.5 ml. Formaldehyde formation from benzphetamine was determined colorimetrically after a 10-min incubation. A substrate blank rate was subtracted from the substratedependent rate. b Reaction mixtures are described under assay methods.
