Purification of cytochrome c oxidase by lysine-affinity chromatography.

A method for the purification of cytochrome c oxidase that is based on the affinity of this enzyme for polycations such as poly-L-lysine is described. When detergent extracts of bovine cardiac mitochondria were applied to either a poly-L-lysine-agarose or a lysine-Sepharose column at low ionic strength, cytochrome c oxidase was found to adhere tightly, whereas the bulk of the proteins were eluted by washing with the same buffer. The cytochrome c oxidase was eluted by application of a linear potassium chloride gradient to the columns. The resulting enzyme was identical to that obtained by more traditional purification methods in terms of its subunit composition, optical and resonance Raman spectra, and cytochrome c oxidizing activity. When detergent extracts of spheroplasts from Paracoccus denitrificans were applied to these columns, the cytochrome c oxidase from this organism was also found to adhere tightly. Thus this purification method appears applicable to both prokaryotic and eukaryotic forms of the enzyme. The advantages of this new purification method are that it is less labor intensive than the traditional procedure and less expensive than methods based on cytochrome c-affinity chromatography.     

Purification and partial characterization of NADPH-cytochrome P450 reductase from Gentiana triflora flowers

Reduced form of nicotineamide adenine dinucleotide phosphate (NADPH)-cytochrome P450 reductase was solubilized from a microsomal fraction of Gentiana triflora flowers by 3-[(3 Cholamidopropyl)-dimethylammonio]-1-propane sulfonate detergent and purified to electrophoretic homogeneity. The purification was achieved by adenosine 2', 5'-bisphosphate-Sepharose chromatography, followed by high-performance anion-exchange chromatography. A Mr value of 82,000 was obtained by SDS/polyacrylamide-gel electrophoresis. Western blot analysis showed that the purified protein cross-reacted with polyclonal antibody raised against rabbit anti-Gentiana triflora NADPH-cytochrome P450 reductase antibodies. The temperature and pH optimum for reduction of cytochrome c was 25 degrees C and 7.4 respectively. The Km values for the binding of NADPH and cytochrome c were 9.4 and 3.2 microM, respectively. In this paper, we present some results of the purification and partial characterization of microsomal NADPH-cytochrome P450 reductase from Gentiana triflora flowers.     

Respiratory proteins from the extremely thermophilic aerobic bacterium, Thermus thermophilus. Purification procedures for cytochromes c552, c555,549, and c1aa3 and chemical evidence for a single subunit cytochrome aa3

We have developed a chemically defined, minimal growth medium for Thermus thermophilus which is suitable for nutritional studies, isotopic enrichment, and genetic manipulation of the organism. Reliable procedures are described for the large scale purification of cytochrome c552 from the periplasm and for cytochrome c555,549 and cytochrome c1 aa3 from the plasma membrane. In contrast to a previous report (Fee, J. A., Choc, M. G., Findling, K. L., Lorence, R., and Yoshida, T. (1980) Proc. Natl. Acad. Sci. U. S. A. 77, 147-151) which suggested a molecular weight near 200,000, the cytochrome c1aa3 complex was shown by protein and amino acid analyses to have Mr approximately 93,000. Sodium dodecyl sulfate-urea-polyacrylamide gel electrophoresis and reversed phase high performance liquid chromatography, combined with amino acid analyses, revealed the presence of only two proteins in a 1:1 ratio: C-protein has Mr approximately 33,000, binds heme C, and is thought to correspond to cytochrome c1. A-protein has Mr approximately 55,000 and is thought to bind the four redox components (2 heme A and 2 Cu) of cytochrome aa3.     

Reconstitution of cytochrome P-450-dependent digitoxin 12 beta-hydroxylase from cell cultures of foxglove (Digitalis lanata EHRH.).

Cytochrome P-450-dependent digitoxin 12 beta-hydroxylase from cell cultures of foxglove (Digitalis lanata) was solubilized from microsomal membranes with CHAPS (3-[(3-cholamidopropyl)dimethylammonio]propane-1-sulphonic acid). Cytochrome P-450 was separated from NADPH: cytochrome c (P-450) reductase by ion-exchange chromatography on DEAE-Sephacel. NADPH:cytochrome c (P-450) reductase was further purified by affinity chromatography on 2',5'-ADP-Sepharose 4B. This procedure resulted in a 248-fold purification of the enzyme; on SDS/polyacrylamide-gel electrophoresis after silver staining, only one band, corresponding to a molecular mass of 80 kDa, was present. The digitoxin 12 beta-hydroxylase activity could be reconstituted by incubating partially purified cytochrome P-450 and NADPH:cytochrome c (P-450) reductase together with naturally occurring microsomal lipids and flavin nucleotides. This procedure yielded about 10% of the original amount of digitoxin 12 beta-hydroxylase.     

Cytochrome aa3 of Rhodobacter sphaeroides as a model for mitochondrial cytochrome c oxidase. Purification, kinetics, proton pumping, and spectral analysis.

Aerobically grown Rhodobacter sphaeroides synthesizes a respiratory chain similar to that of eukaryotes. We describe the purification of the aa3-type cytochrome c oxidase of Rb. sphaeroides as a highly active (Vmax > or = 1800 s-1), three-subunit enzyme from isolated, washed cytoplasmic membranes by hydroxylapatite chromatography and anion exchange fast protein liquid chromatography. The purified oxidase exhibits biphasic kinetics of oxidation of mammalian cytochrome c, similar to mitochondrial oxidases, and pumps protons efficiently (H+/e- = 0.7) following reconstitution into phospholipid vesicles. A membrane-bound cytochrome c is associated with the aa3-type oxidase in situ, but is removed during purification. The EPR spectra of the Rb. sphaeroides enzyme suggest the presence of a strong hydrogen bond to one or both of the histidine ligands of heme a. In other respects, optical, EPR, and resonance Raman analyses of the metal centers and their protein environments demonstrate a close correspondence between the bacterial enzyme and the structurally more complex bovine cytochrome c oxidase. The results establish this bacterial oxidase as an excellent model system for the mammalian enzyme and provide the basis for site-directed mutational analysis of its energy transducing function.     

Preparation of homogenous NADPH cytochrome c (P-450) reductase from house flies using affinity chromatography techniques.

NADPH-cytochrome c (P-450) reductase (EC 1.6.2.4) was purified to apparent homogeneity from microsomes of house flies, Musca domestica L. The purification procedure involves column chromatography on three different resins. The key step in the purification scheme is the chromatography of the enzyme mixture on an affinity column of agarose-hexane-nicotinamide adenine dinucleotide phosphate. The enzyme has an estimated molecular weight of 83,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and contains 1 mol each of FAD and FMN per mol of enzyme. The enzyme exhibited a Bi Bi ping-pong kinetic mechanism with NADPH and cytochrome c. The Vmax and Km for cytochrome c were 42.3 mumol min-1 mg-1 and 12.7 muM, respectively. Turnover numbers based on micromoles of enzyme were 2,600 min-1. NADP+ and 2'-AMP both inhibited the reductases with apparent Ki values of 6.9 and 187 muM, respectively. These preparations of NADPH-cytochrome c reductase were found to reduce purified house fly cytochrome P-450 in the presence of NADPH.     

Purification of NADPH:cytochrome c (cytochrome P-450) reductase from hamster liver microsomes by detergent extraction and affinity chromatography

NADPH:cytochrome c (cytochrome P-450) reductase (Fp) from hamster liver microsomes has been purified to near homogeneity using a simple and rapid method. Microsomes were treated with the detergent Chaps (3-[(3-cholamidopropyl)dimethylammonio]propanesulfonic acid) in combination with 0.07% protamine sulfate and then centrifuged to pellet insoluble material. While over 60% of the total microsomal protein was solubilized, all Fp activity remained in the pellet. Fp was extracted from the Chaps-insoluble material using a combination of the detergents sodium cholate and Lubrol PX. This treatment resulted in a fivefold increase in Fp specific activity and allowed direct processing of the enriched Fp fraction by 2',5'-ADP agarose affinity chromatography. The purified Fp had a total flavin content of 23 nmol/mg protein (flavin adenine dinucleotide:flavin mononucleotide ratio = 1:1), a specific activity of 26,000 units/mg protein at 22 degrees C using cytochrome c as electron acceptor, and migrated as a single band on sodium-dodecyl sulfate-polyacrylamide gel electrophoresis with a relative molecular weight of 76,000. The purity, specific activity, and yield were nearly identical to results obtained when the flavoprotein was purified by conventional methods. This procedure eliminates the need for anion-exchange chromatography and allows for the rapid purification of large amounts of Fp suitable for use in studies concerning cytochrome P-450-mediated drug metabolism. Importantly, this method is equally effective when used to purify Fp from rat liver microsomes.     

Affinity chromatography purification of cytochrome c oxidase and b-c1 complex from beef heart mitochondria. Use of thiol-sepharose-bound Saccharomyces cerevisiae cytochrome c

A method for simultaneous purification of cytochrome c reductase and cytochrome c oxidase using a cytochrome c affinity column is presented. Cytochrome c from Saccharomyces cerevisiae was linked to an activated thiol-Sepharose gel via its Cys-102 residue located far from the lysine residues on the front side of the molecule, responsible for the interaction with the reductase and oxidase. In previously reported affinity chromatography techniques these lysine residues most probably reacted with the column. Cytochrome c oxidase and reductase from bovine heart mitochondria bind specifically to the affinity column and can be recovered separately at different ionic strength in the elution buffer. The enzymes are highly pure and active.     

Purification and partial characterization of cytochrome c552 from Halobacterium salinarium

Cytochrome c552 was purified to near homogenity and partially characterized from Halobacterium salinarium JWS mutant, devoid of carotenoid pigments. The purification involved the extraction of membranes with 1% Triton X-100, followed by butylagarose, DEAE-Sepharose CL6B and hydroxyapatite column chromatography. The fold of purification was 16. The purified cytochrome showed maximum absorption at 552 nm. The molecular mass determined by SDS-PAGE was found to be 14.1 kD.     

Expression, purification, and characterization of recombinant forms of membrane-bound cytochrome c-550nm from Bacillus subtilis

Bacillus subtilis expresses a cytochrome c-550nm that participates in respiratory electron transfer and is an integral membrane protein. Analysis of the B. subtilis cytochrome c-550nm amino acid sequence predicts a single N-terminal transmembrane helix attached to a water-soluble heme binding domain [C. von Wachenfeldt and L. Hederstedt (1990) J. Biol. Chem. 265, 13939-13948]. We have purified cytochrome c-550nm from wild-type B. subtilis and B. subtilis transformed with the shuttle vector pHP13 containing the gene for B. subtilis cytochrome c-550nm (cccA). In B. subtilis transformed with pHP13/cccA there is better than eightfold more membrane-bound cytochrome c-550nm than in wild-type B. subtilis. The overexpressed cytochrome c-550nm can be purified by chromatography on hydroxylapatite and Q-Sepharose media. A six-histidine tag has been added to the C-terminus of cytochrome c-550nm from B. subtilis as a further aid for purification. This strain produces cytochrome c-550nm to a level fourfold greater than wild type and allows for one-step purification using metal affinity chromatography. UV-Vis spectroscopy detects no change in the heme C spectrum due to the addition of six histidines. Neither form of B. subtilis cytochrome c-550nm is stable in its reduced state in aerated buffer, unless EDTA is added. The two forms, wild-type and his-tagged, of cytochromes c have similar midpoint redox potentials of 195 and 185 mV, respectively, and are equally good substrates for B. subtilis cytochrome c oxidase. We conclude that the addition of the histidine tag eases the purification of cytochrome c-550nm from B. subtilis plasma membranes and that the additional metal binding site does not compromise the stability or functional properties of the protein.     

Use of immobilized cytochrome c as a ligand for affinity chromatography of thiosulfate dehydrogenase from Acidithiobacillus ferrooxidans

Three matrices were used for immobilizing the cytochrome c: Sepharose CL-4B, Silasorb SPH amine and a laboratory-prepared new matrix based on crosslinked triazine (2,4,6-tris(aminoethylamine)-1,3,5-triazine) (TAT). Cytochrome c was immobilized on the matrices by several procedures and the amount of incorporated cytochrome c was determined. Cytochrome c immobilized on Sepharose CL-4B with periodate activation, cytochrome c immobilized on Silasorb-amine with carbodiimide activation and cytochrome c immobilized on crosslinked triazine were suitable for purification of thiosulfate dehydrogenase from Acidithiobacillus ferrooxidans. The yield with all matrices was about 90%. The purification factor of the above matrices was about 15. A new matrix based on TAT with cytochrome c represented a suitable way for thiosulfate dehydrogenase purification.     

3,4,5,3',4'-Pentachlorobiphenyl as a useful inducer for purification of rat liver microsomal cytochrome P448.

An easy purification of rat liver microsomal cytochrome P448 was performed by using 3,4,5,3′,4′-pentachlorobiphenyl as an inducer. The cytochrome P448, a high spin form, was purified to 18.1 nmoles/mg protein with a good yield by ω-aminooctyl Sepharose 4B column chromatography followed by a hydroxyapatite column chromatography. This hemoprotein cross-reacted with antibody to cytochrome P448 from β-naphthoflavone-treated rats, but not with antibody to cytochrome P450 from phenobarbital-treated rats at all. The results of amino acid analyses suggested that this cytochrome P448 is similar to cytochrome P448 of 3-methylcholanthrene-treated rats.     

Cholesterol-hydroxylating cytochrome P-450 from bovine adrenal cortex mitochondria and human placenta: immunochemical properties and structural characteristics

An immunochemical comparison of components of cholesterol side chain cleavage system from bovine adrenocortical and human placental mitochondria has been carried out. Antibodies against cytochrome P-450scc, adrenodoxin reductase and adrenodoxin from bovine adrenocortical mitochondria were shown to cross-react with corresponding antigens of human placental mitochondria. A highly sensitive immunochemical method for cytochrome P-450scc determination has been developed. Limited proteolysis of cytochrome P-450scc of human placental mitochondria was studied, and the products of trypsinolysis were identified using antibodies against cytochrome P-450scc and fragments of its polypeptide chain: F1, F2 and F3. Immunochemical relatedness of ferredoxins from bovine adrenocortical and human placental mitochondria allowed one to develop a fast and efficient method for cytochrome P-450scc purification from human placental mitochondria by affinity chromatography on adrenodoxin-Sepharose.     

Biochemical characteristics of purified beef liver NADPH-cytochrome P450 reductase

NADPH-cytochrome P450 reductase, an obligatory component of the cytochrome P450 dependent monooxygenase system, was purified to electrophoretic homogeneity from beef liver microsomes. The purification procedure involved the ion exchange chromatography of the detergent-solubilized microsomes on first and second DEAE-cellulose columns, followed by 2',5'-ADP Sepharose affinity chromatography. Further concentration of the enzyme and removal of Emulgen 913 and 2'-AMP were accomplished on the final hydroxylapatite column. The enzyme was purified 239-fold and the yield was 13.5%. Monomer molecular weight of the enzyme was estimated to be 76000 +/- 3000 (N = 5) by SDS-PAGE. The absolute absorption spectrum of beef reductase showed two peaks at 455 and 378 nm, with a shoulder at 478 nm, characteristics of flavoproteins. The effects of cytochrome c concentration, pH, and ionic strength on enzyme activity were studied. Reduction of cytochrome c with the enzyme followed Michaelis-Menten kinetics, and the apparent K(m) of the purified enzyme was found to be 47.7 microM for cytochrome c when the enzyme activity was measured in 0.3 M potassium phosphate buffer (pH 7.7). Stability of cytochrome c reductase activity was examined at 25 and 37 degrees C in the presence and absence of 20% glycerol. The presence of glycerol enhanced the stability of cytochrome c reductase activity at both temperatures. Sheep lung microsomal cytochrome P4502B and NADPH-cytochrome P450 reductase were also purified by the already existing methods developed in our laboratory. Both beef liver and sheep lung reductases were found to be effective in supporting benzphetamine and cocaine N-demethylation reactions in the reconstituted systems containing purified sheep lung cytochrome P4502B and synthetic lipid, phosphatidylcholine dilauroyl.     

Synthesis and characterization of singly modified (carboxyferrocenyl)cytochrome c derivatives.

Carbodiimide-activated coupling chemistry has been used to covalently attach 1,1'-dicarboxyferrocene (dcFc) to the epsilon-amine of surface lysine residues of horse heart cytochrome c. Conditions have been found that optimize the production of singly modified (dcFc)cytochrome c derivatives and the presence of one free carboxylate per modification site allows separation and purification of about 10 of these derivatives by cation-exchange chromatography. Reversed-phase HPLC tryptic peptide mapping techniques have been used to identify the attachment sites of eight pure (dcFc)cytochrome c derivatives (at lysines 7, 8, 13, 25, 60, 72, 73, and 100). Through-space distances from these lysines to the nearest heme edge span the 6-16 A range and these derivatives should prove useful in exploring the distance dependence of long-range intramolecular electron transfer in cytochrome c.     

Partial purification of the cyanide-resistant alternative oxidase of skunk cabbage (Symplocarpus foetidus) mitochondria.

A partial purification of the cyanide-resistant, alternative oxidase from skunk cabbage (Symplocarpus foetidus L.) spadix mitochondria is described. Skunk cabbage mitochondria were solubilized in N,N-bis-(3-D-glucon-amido-propyl)deoxycholamide and the alternative oxidase was purified using a batch DEAE-cellulose treatment, followed by precipitation with Extracti-Gel and chromatography on Sephadex G-200. Following pooling and concentrating of the most active fractions from the gel filtration column, a 20- to 30-fold purification of the alternative oxidase was obtained, with no evidence of contamination by cytochrome c oxidase (complex IV) or cytochrome c reductase (complex III). Polyacrylamide gel electrophoresis of the partially purified oxidase showed major polypeptides at 36 and 29 kD, both of which react with monoclonal antibodies raised against the Sauromatum guttatum alternative oxidase. The purified oxidase fraction showed no absorbance in the visible spectral region, and addition of sodium borohydride induced no absorbance changes in the ultraviolet region. The purified alternative oxidase catalyzed the four-electron reduction of oxygen to water in the absence of citrate, but catalyzed an apparent two-electron reduction of oxygen to hydrogen peroxide in the presence of 0.7 M citrate.     

Purification of a cytochrome aa3 terminal oxidase from protoplast membrane vesicles of Micrococcus luteus

Abstract A cytochrome aa₃ terminal oxidase was isolated from protoplast membrane vesicles of Micrococcus luteus grown under aerobic conditions. The purified complex showed similarities to cytochrome c oxidase (EC 1.9.3.1) of the electron transport chain of mitochondria and many prokaryotes. The enzyme was solubilized by subsequent treatment with the detergents CHAPS and n-dodecyl-β-d-maltoside and purified by ion-exchange chromatography using poly-L-lysine agarose and TMAE-fractogel-650 (S) columns, followed by hydroxyapatite chromatography. The purified complex is composed of two major subunits with apparent molecular masses of 54 and 32 kDa. After purification the isolated enzyme contains 12.1 nmol of heme A (mg protein)⁻¹ and exhibits absorption maxima at 424 nm and 598 nm in the oxidized state and at 442 nm and 599 nm in the reduced state. The CO-difference spectrum shows peaks at 428 and 590 nm which is indicative of heme a ₃, furthermore oxygen consumption was found to be sensitive to cyanide.     

Purification and characterization of the NADPH-cytochrome P-450 (cytochrome c) reductase from higher-plant microsomal fraction.

NADPH-cytochrome P-450 (cytochrome c) reductase (EC 1.6.2.4) was solubilized by detergent from microsomal fraction of wounded Jerusalem-artichoke (Helianthus tuberosus L.) tubers and purified to electrophoretic homogeneity. The purification was achieved by two anion-exchange columns and by affinity chromatography on 2',5'-bisphosphoadenosine-Sepharose 4B. An Mr value of 82,000 was obtained by SDS/polyacrylamide-gel electrophoresis. The purified enzyme exhibited typical flavoprotein redox spectra and contained equimolar quantities of FAD and FMN. The purified enzyme followed Michaelis-Menten kinetics with Km values of 20 microM for NADPH and 6.3 microM for cytochrome c. In contrast, with NADH as substrate this enzyme exhibited biphasic kinetics with Km values ranging from 46 microM to 54 mM. Substrate saturation curves as a function of NADPH at fixed concentration of cytochrome c are compatible with a sequential type of substrate-addition mechanism. The enzyme was able to reconstitute cinnamate 4-hydroxylase activity when associated with partially purified tuber cytochrome P-450 and dilauroyl phosphatidylcholine in the presence of NADPH. Rabbit antibodies directed against plant NADPH-cytochrome c reductase affected only weakly NADH-sustained reduction of cytochrome c, but inhibited strongly NADPH-cytochrome c reductase and NADPH- or NADH-dependent cinnamate hydroxylase activities from Jerusalem-artichoke microsomal fraction.     

Fractionation and purification of hepatic microsomal cytochrome P-450 isoenzymes from phenobarbital-pretreated rats by h.p.l.c. A convenient tool for screening of isoenzymes inactivated by allylisopropylacetamide.

A procedure incorporating the salient features of ion-exchange column chromatography with ion-exchange h.p.l.c. is described for the fractionation and purification to homogeneity of several membrane-bound rat hepatic phenobarbital (PB)-inducible cytochrome P-450 isoenzymes, including the major PB-inducible species. The resolving power of this technique makes it a highly promising tool for the isolation and purification of closely related cytochrome P-450 isoenzymes. In addition, it may also be used for screening of individual isoenzymes either selectively induced or repressed by a variety of endobiotics or xenobiotics. Accordingly, we have exploited this particular feature to identify not only the PB-inducible cytochrome P-450 isoenzymes destroyed in vivo by allylisopropylacetamide, a suicide inactivator of cytochrome P-450, but also to distinguish those that are reparable by exogenous haemin from those that are irreparably damaged.     

A purification procedure for the soluble cytochrome oxidase and some other respiratory proteins from Pseudomonas aeruginosa.

The production of the soluble cytochrome oxidase/nitrite reductase in the bacterium Pseudomonas aeruginosa is favoured by anaerobic conditions and the presence of KNO3(20g/l) in the culture medium. Of three methods commonly used for the disruption of bacterial suspensions (ultrasonication, liquid-shear homogenization and glass-bead grinding), sonication proved the most efficient in releasing the Pseudomonas cytochrome oxidase. A polarographic assay of Pseudomonas cytochrome oxidase activity with sodium ascorbate as substrate and NNN'N'-tetramethyl-p-phenylenediamine dihydrochloride as electron mediator is described. A purification procedure was developed which can be used on the small scale (40-litre cultures) or the large scale (400-litre cultures) and provides high yields of three respiratory-chain proteins, Pseudomonas cytochrome oxidase, cytochrome c551 and azurin, in a pure state. A typical preparation of 250g of Ps.aeruginosa cell paste yielded 180mg of Pseudomonas cytochrome oxidase, 81 mg of Pseudomonas cytochrome c551 and 275mg of Pseudomonas azurin.