Cytochrome purification

No region of the cytochrome penetrates the membrane nevertheless, the cytochrome subunit is an integral part of this reaction center complex, held through protein-protein interactions similar to those in soluble globular multisubunit proteins. The protein-protein interactions that bind cytochrome in the reaction center of Rhodopseudomonas viridis are strong enough to survive the purification procedure. However, when the reaction center of Rhodohacter sphaeroides is isolated, the cytochrome is lost, even though the structures of the L, M, and H subunits are very similar in the two species. 

Squalene epoxidase, like most enzymes responsible for the later steps of sterol biosynthesis [43, 51], is membrane-bound which makes its purification in native form challenging. The purification is additionally complicated by the presence of a large number of cytochrome P450 and other enzymes that have similar hydro-phobicity and size as squalene epoxidase and are hence difficult to remove [52]. Most studies have been carried out with rat liver microsome squalene epoxidase either partially purified or as a homogenate of the cell membrane fraction. In vitro reconstitution of squalene epoxidase activity is absolutely dependent on molecular oxygen, NADPH, FAD, and NADPH-cytochrome c reductase [52, 53]. In this respect, squalene epoxidase resembles the cytochrome P450 enzymes described... 

Trower MK, FS Sariaslani, DP O Keefe (1989) Purification and characterization of a soybean flour-induced cytochrome P-450 from Streptomyces griseus. J Bacteriol 171 1781-1787. 

Sasaki M, A Akahira, L-i Oshiman, T Tsuchid, Y Matsumura (2005) Purification of cytochrome P450 and ferredoxin involved in bisphenol. A degradation from Sphingomonas sp. stain AOl. Appl Environ Microbiol 71 8024-8030. 

Tokimatsu T, Y Nagai, T Hattori, M Shimada (1998) Purification and characteristics of a novel cytochrome c dependent glyoxylate dehydrogenase from a wood-rotting fungus Tyromyces palustris. EEBS Lett 437 117-121. 

Louie TM, S Ni, L Xun, WW Mohn (1997) Purification, characterization and gene sequence analysis of a novel cytochrome c coinduced with reductive dechlorination activity in Desulfomonile tiedjei DCB-1. Arch Microbiol 168 520-527. 

STADLER, R., ZENK, M.H., The purification and characterization of a unique cytochrome P-450 enzyme from Berberis stolonifera plant cell cultures, J. Biol. Chem., 1993,268, 823-831. 

HALKIER, B.A., NIELSEN, H.L., KOCH, B., M0LLER, B.L., Purification and characterization of recombinant cytochrome P450TYR expressed at high levels in Escherichia coli, Arch. Biochem. Biophys., 1995,322, 369-377. 

GILLAM, E.M.J., BABA, T, KIM, B-R., OHMORI, S., GUENGERICH, F.P., Expression of modified human cytochrome P450 3A4 in Escherichia coli and purification and reconstitution of the enzyme, Arch. Biochem. Biophys., 1993, 305, 123-131. 

These findings lead to (he conclusion that the reduction of MHb by its reductase requires a natural cofactor, which is abolished during the purification procedure and can be replaced by methylene blue (G5, H22, H23, K8, K14). Since methylene blue and the other effective dyes are redox intermediates, it is obvious that the postulated cofactor interacts in the electron transport sequence of the MHbR reaction (H23). This is confirmed by the finding that oxygen and cytochrome c serve as well as terminal electron acceptor as does MHb (H22, H23, K14). Nevertheless, it had been possible to separate a cytochrome c reductase from MHbR in yeast extracts (A6). 

Patten CJ, Ning SM, Lu AYH, et al. 1986. Acetone-inducible cytochrome P-450 Purification, catalytic activity, and interaction with cytochrome b5. Arch Biochem Biophys 251 629-638. 

Haining RL, Hunter AP, Veronese ME, et al. Allelic variants of human cytochrome P450 2C9 baculovirus-mediated expression, purification, structural characterization, substrate stereoselectivity, and prochiral selectivity of the wild-type and I359L mutant forms. Arch Biochem Biophys 1996 333(2) 447 t58. 

Although the first purification of bNOS was a monomer, it is now clear that the enzyme in all cases is effective as a dimer. A purified macrophage iNOS was used by Baek and coworkers98 to separate the holoenzyme from the monomers. The subunits do not have NOS activity but do have the ability to oxidize reduced triphosphopyridine nucleotide with either ferricyanide, cytochrome c or dichlorophenolindophenol. When all of the missing factors are present, but not when any is missing, the authors find recombination, as shown in Figure 13. 

Coon, M.J., Ballou, D.P., Haugen, D.A., Krezoski, S.O., Nordblom, G.D. and White, R.E. Purification of membrane-bound oxygenases isolation of two electrophoretically homogeneous forms of liver microsomal cytochrome P-1+50. 

In this report we compare several properties of hepatic microsomal AHH activity in control and DBA-treated little skates (including metabolic profiles obtained from c-benzo(a)pyrene as elucidated by high pressure liquid chromatography [HPLC]), we describe the partial purification of two different forms of cytochrome P-450 (cytochrome P-448 and cytochrome P-451) from hepatic microsomes of DBA-pretreated little skates and we report polycyclic hydrocarbon-like induction in large numbers of winter flounder assayed in Maine during June, July, and August, which was quite different than data obtained with sheepshead examined in Florida during the same period. 

Solubilization and Partial Purification of Cytochrome P-450 from Hepatic Microsomes of Male, DBA-Pretreated Little Skates. Washed hepatic microsomes (3) from the livers of 10 skates were suspended in 0.25 M sucrose and frozen under nitrogen (-5 to -10°) at the Maine laboratory. They were then packed in dry ice and transported to NIEHS, Research Triangle Park, NC, within 14 days of preparation and were stored at -62°C until use. Microsomes... 

The hepatic microsomes were aliquots from pools of 10 control or pretreated fish that were also used for the partial purification of various forms of Cytochrome P-450 9,10-D, BP-9,10-dihydrodiol 4,5-D, BP-4,5-dihydrodiol 7,8-D, BP-7,8-dihydrodiol Q + E, BP-1,6-, -3,6-, and -6,12-quinones and BP-4,5-oxide 9-OH, 9-hydroxy-BP 3-OH, 3-hydroxy -BP. The DBA-dosing schedule and incubation conditions are described in Materials and... 

When all of the material absorbing at 418 nm (associated with the cytochrome P-448 fractions) was eluted from the DEAE-cellulose column (which in some experiments required more than 1 liter of Buffer II), elution was continued with a linear KC1 gradient (0-0.5 M) in Buffer II, as shown in Fig. 5. A different form(s) of cytochrome P-450 (fractions 130-155), having maximal absorption near 451 nm in the carbon monoxide ligated and reduced form (Fig. 6), was obtained although only 2- to 3-fold purification, relative to microsomes, was achieved. This form of cytochrome P-450 was extensively contaminated with epoxide hydratase activity. However, by combining fractions 130-150 (Fig. 5), it was possible to obtain cytochrome P-451 essentially free of cytochrome b5. The relative content of cytochrome P-448 and cytochrome P-451 Tn the DEAE-column eluates ranged from 1 1.1 to 1 1.6 in several different experiments. 

Very similar results to those described in Fig. 3-6 were obtained when sodium cholate solubilized hepatic microsomes from DBA-treated female little skates were subjected to chromatography on DEAE-cellulose as described above (data not shown). Also not shown are the results obtained with hepatic microsomes from untreated male and female little skates. With untreated animals, 80-90% of the cytochrome P-450 eluted from the DEAE-cellulose column only at higher ionic strength (i.e., with the KC1 gradient). However, in all preparations studied, an appreciable amount of cytochrome P-450 (10-20%), having its absorption maximum in the carbon monoxide-ligated and reduced state at 450 nm, was eluted from the column with buffer II, as was observed with cytochrome P-448 of hepatic microsomes from DBA-treated skates. The further purification of the various forms of cytochrome P-450 from control and DBA-pretreated little skate livers is currently in progress in our laboratory. 

Levin, W. Purification of liver microsomal cytochrome P-450 Hopes and promises. In "Microsomes and Drug Oxidations". Ullrich, V., Roots, I., Hildebrandt, A., Estabrook, R.W. and Conney, A.H. (Eds.), pp. 735-747. Pergamon Press. Oxford. 

Although mammalian CYPs are attractive candidates for use as commercial biocatalysts, many functional characteristics limit the opportunities to exploit such a system. Association of the enzymes with membranes prevents easy extraction and purification and limits the opportunities to produce useful recombinant enzymes by cloning the relevant genes for expression in microbial systems. All P450s have a porphyrin-haem active site that requires a second protein to reduce the iron component, often cytochrome P450 reductase or... 

After the production of the solution of proteins, the purification of the protein requires six different steps. Silica gel powder is added to the solution and filtered off, to adsorb large quantities of cytochrome C3. Several steps of column chromatography follow. 

Bergh AF, Strobel HW. 1992. Reconstitution ofthe brain mixed function oxidase system purification of NADPH-cytochrome P450 reductase and partial purification of cytochrome P450 from whole rat brain. J Neurochem 59 575-581. 

Bhamre S, Anandatheerathavarada HK, Shankar SK, Boyd MR, Ravindranath V. 1993. Purification of multiple forms of cytochrome P450 from a human brain and reconstitution of catalytic activities. Arch Biochem Biophys 301 251-255. 

Pikuleva lA, Bjorkhem 1, Waterman MR. 1997. Expression, purification, and enzymatic properties of recombinant human cytochrome P450c27 CYP27). Arch Biochem Bio-phys 343 123-130. 

Afkar E, Fukumori Y. 1999. Purification and characterization of triheme cytochrome c from the metal-redncing bacterinm, Geobacter metallireducens. FEMS Microbiol Lett 175 205-10. 

Dalet-Beluche 1, Boulenc X, Eabre G, Maurel P, Bonfils C (1992) Purification of two cytochrome P450 isozymes related to CYP2A and CYP3A gene families from monkey (baboon, Papio papio) liver microsomes. Cross reactivity with human forms. Eur J Biochem 204 641-648 Damaj Ml, Siu EC, Sellers EM, Tyndale RE, Martin BR (2007) Inhibition of nicotine metabolism by methoxysalen Pharmacokinetic and pharmacological studies in mice. J Pharmacol Exp Ther 320 250-257... 

Kaipainen P, Nebert DW, Lang MA (1984) Purification and characterization of a microsomal cytochrome P-450 with high activity of coumarin 7-hydroxylase from mouse liver, Eur J Biochem 144 425 31... 

Ohmori S, Horie T, Guengerich FP, Kiuchi M, Kitada M (1993b) Purification and characterization of two forms of hepatic microsomal cytochrome P450 from untreated cynomolgus monkeys. Arch Biochem Biophys 305 405 13... 

Gabriac B, Werck-Reichhart D, Teutsch H, Durst F (1991) Purification and immunochar-acterization of a plant cytochrome P450 the cinnamic acid 4-hydroxylase. Arch Biochem Biophys 288(l) 302-309... 

Mizutani M, Ohta D, Sato R (1993) Purification and characterization of a cytochrome P450 (rran -cinnamic acid 4-hydroxylase) from etiolated mung bean seedlings. Plant Cell Physiol 34 481-488... 

Recombinant Cytochrome C553 E. coli periplasm AOT/isoocatne Extraction and purification [104]... 

Tween 85 is used extensively for RME [84]. Russell and coworkers [234] used Tween 85/isopropanol microemulsions in hexane to solubilize proteins and not only showed >80% solubilization of cytochrome C at optimum conditions, but also proved that Tween 85 does not have a detrimental effect on the structure, function, and stability of subtilisin and cytochrome C. There are other reports available on the extraction and purification of proteins using Tween 85-RMs and also on the stability of protein activity in these systems [234]. It has also been shown that Tween 85-RMs can solubilize larger amounts of protein and water than AOT. Tween 85 has an HLB of 11, which indicates that it is soluble in organic solvents. In addition, it is biodegradable and can be successfully used as an additive in fertihzers [235,236]. Pfammatter et al. [35] have demonstrated that RMs made of Tween 85 and Span 80 can be successfully used for the solubilization and growth of whole cells. Recently, Hossain et al. [84] showed an enhanced enzymatic activity of Chromobacterium viscosum Hpase in AOT/Tween 85 mixed reverse micellar systems when compared to that in classical AOT-RMs. This is due to the modification of the interface in AOT-RMs caused by the co-adsorption of Tween 85, and increased availability of the oHve oil molecules (substrate) to the enzyme. 

Extraction and purification of proteins by employing RMs have been extensively studied using model systems (proteins from commercial suppliers) such as cytochrome C, ribonuclease, a-chymotrypsin, etc. However, very few reports are available on the extraction studies of proteins from the real systems of fermentation broths using RMs [ 15,43]. As we know that fermentation broths containing proteins are very much more complicated media compared to model proteins and mixture of model proteins, there is a need to investigate extraction behavior of proteins present in these real systems. 

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