Two-step purification of cytochrome P-450 from rat liver microsomes using high-performance liquid chromatography

Cytochrome P-450 from rat liver microsomes treated with phenobarbital (PB) was separated into six fractions, as was cytochrome P-450 treated with 3-methylcholanthrene (MC), by high-performance liquid chromatography (HPLC) with an anion-exchange column. PB and MC induced three forms and one form of cytochrome P-450, respectively. The major forms induced by PB and by MC were further purified to apparent homogeneity based on sodium dodecyl(lauryl)sulphate--polyacrylamide gel electrophoresis by HPLC using a hydroxyapatite column. These new HPLC techniques are simple, rapid and useful for the purification of major forms of cytochrome P-450 from solubilized microsomes.     

Detection of the enzymatic activity of cytochrome P-450 enzymes by high-performance liquid chromatography.

The reactions catalysed by the various cytochrome P-450 enzymes are reviewed with respect to the analysis of products by high-performance liquid chromatography (HPLC). Especially biotransformation reactions of purified cytochrome P-450 enzymes in a reconstituted system and in microsomes mainly of rat liver origin are considered. Emphasis is put on the specificity of product formation due to the individual isozymes of cytochrome P-450. It is shown that the presence of eight cytochrome P-450 isozymes can be monitored and determined by specific product formation after HPLC analysis, which is an important parameter in toxicological studies.     

Analytical fractionation of microsomal cytochrome P-450 isoenzymes from rat liver by high-performance ion-exchange chromatography

Ion-exchange Fast Protein Liquid Chromatography (FPLC) on Mono Q and Mono S was optimized for the analytical separation of microsomal cytochrome P-450 species from rat liver. The effects of detergent, pH, gradient profile and column load on resolution are demonstrated. Successive application of anion- and cation-exchange chromatography leads to eleven separated P-450 fractions. The altered microsomal P450 pattern after treatment of rats with various inducers is reflected by distinct elution profiles. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis and enzymatic analysis imply that several FPLC fractions contain more than one P-450 species. Preliminary results are presented showing the suitability of immobilized metal affinity chromatography (MAC) for general P-450 fractionation and thus for the further resolution of Mono Q and Mono S fractions. Scale-up for preparative P-450 fractionation is easily done by adapting the optimized analytical FPLC procedures to Q- and S-Sepharose Fast Flow.     

Determination of metabolites of cytochrome P-450 model systems using high-performance liquid chromatography

High-performance liquid chromatographic techniques were developed for the simultaneous detection of metabolites in a cytochrome P-450 model system composed of NADH, haemoglobin and methylene blue. Monohydroxylated metabolites were determined following aniline, acetanilide and phenol hydroxylations. 4-Aminoantipyrine, 7-hydroxycoumarin and p-nitrophenol were determined after dealkylation of 4-N,N-dimethylamino-antipyrine, 7-ethoxycoumarin and p-nitroanisole. These substrates are commonly used for measuring cytochrome P-450 activities. Treatment of the samples was minimal, consisting of a simple deproteinization, and did not involve any organic extraction. Separations were carried out on reversed-phase columns and the products were detected by UV adsorption. Separations were completed in less than 15 min and the detection limits were between 0.5 and 4 microM.     

Purification of cytochromes P-450 derived from liver microsomes of untreated and 2,3,7,8-tetrachlorodibenzo-p-dioxin-treated marmoset monkeys

The purification of multiple forms of cytochrome P-450 (P450) from 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-treated marmosets using fast protein liquid chromatography (FPLC) is described. The main aim was to achieve a better separation of certain closely related P450 sub-forms from each other than that previously obtained using conventional chromatography. An 8-aminooctyl-Sepharose fraction of cholate-solubilized microsomes was obtained first and, after fast desalting on Sephadex G-25, loaded on to a preparative Mono Q column. Five of the six gradient peaks contained P450 and were each rechromatographed on an analytical Mono Q column. The pass-through peak was fractionated further using a Mono S column. Other HPLC-quality anion- and cation-exchange gels were compared. For removal of excess of non-ionic detergent, five types of hydroxyapatite gels were compared. Seven purified forms of P450 and cytochrome b5 and P420 were isolated and characterized according to PHAST sodium dodecylsulphate-polyacrylamide gel electrophoretic apparent molecular masses, catalytic, spectral and magnetic properties and also TCDD-binding capacity (molar ratio of [14C]TCDD to P450). There are at least two sub-forms which appear to be TCDD inducible, one showing a substantial ethoxyresorufin-O-deethylase activity and the other having a high TCDD-binding molar ratio. Two other forms appear to be constitutive, as deduced from comparisons with forms purified from untreated animals.     

Determination of the cytochrome P-450 IV marker, omega-hydroxylauric acid, by high-performance liquid chromatography and fluorimetric detection

The formation of omega-hydroxylauric acid from lauric acid is an indicator of the activity of cytochrome P-450 IV family proteins. The two main metabolites of lauric acid, (omega-1)-and omega-hydroxylauric acid, have been completely separated by reversed-phase high-performance liquid chromatography. Measurement of lauric acid hydroxylase activity in microsomal liver samples, based on derivatization of the substrate and metabolites with the fluorescent agent 4-bromomethyl-6,7-dimethoxycoumarin, is a precise method (coefficient of variation = 7.6 and 10% for omega and (omega-1) metabolites, respectively) with good sensitivity (signal-to-noise ratio in microsomal samples of untreated rats greater than 20). In microsomal fractions from livers of rats treated with di-(2-ethylhexyl)phthalate the extent of omega-hydroxylation of lauric acid increased dose-dependently (ca. ten-fold). The (omega-1)-hydroxylase activity was not altered. A strong correlation between immunochemically determined cytochrome P-450 IVA1 and lauric acid omega-hydroxylase activity was found (r = 0.94, n = 30).     

Measurement of enzyme activities of cytochrome P-450 isoenzymes by high-performance liquid chromatographic analysis of products.

A method is described for the qualitative and quantitative determination of the isoenzymes of cytochrome P-450 from rat liver microsomes. Microsomes are incubated with the endogenous steroid 17 beta-testosterone, which results in the formation of a number of stereo-specific hydroxylation products of testosterone. The hydroxylated products were identified using standards or by comparison with data from the literature. The products can be analysed by reversed-phase gradient high-performance liquid chromatography. The assay has been optimised for pH, linearity and time of incubation. An evaluation of the assay was performed for different kinds of microsomes, microsomal dilution and specificity for particular cytochrome P-450 isoenzymes.     

Metabolism of 24,25-dihydrolanosterol analogs by partially purified cytochrome P-450(14DM) from rat liver microsomes

27-Nor-24,25-dihydrolanosterol (27-nor-DHL), 26,27-dinor-24,25- dihydrolanosterol (26,27-dinor-DHL), and 25,26,27-trinor-24,25-dihydrolanosterol (25,26,27-trinor-DHL), analogs of 24,25-dihydrolanosterol (DHL) which have no C-27 carbon, C-26,27 carbons and C-25,26,27 carbons, were converted to the corresponding 14-demethylated products using a reconstituted monooxygenase system from rat liver microsomes which contained cytochrome P-450(14DM) catalyzing lanosterol 14-demethylation and reduced nicotinamide adenine dinucleotide phosphate (NADPH)-cytochrome P-450 reductase in the presence of NADPH and molecular oxygen. Each metabolite showed a relative retention time (RtR) of 0.72 with respect to each substrate in high-performance liquid chromatography (HPLC) on a reversed-phase column. Comparison of each gas chromatography-mass spectrum and RtR value with those of the metabolite of DHL, 4,4-dimethyl-5 alpha-cholesta-8,14-dien-3 beta-ol, indicated that the metabolites could be inferred to be 27-nor-4,4-dimethyl-5 alpha-cholesta-8,14-dien-3 beta-ol, 26,27-dinor-4,4- dimethyl-5 alpha-cholesta-8,14-dien-3 beta-ol, and 25,26,27-trinor-4,4-dimethyl-5 alpha-cholesta-8,14-dien-3 beta-ol. However, 24,25,26,27-tetranor- and 23,24,25,26,27-pentanor analogs of DHL and 20-iso-24,25-dihydrolanosterol were not metabolized by the reconstituted enzyme system.     

Metabolism of 32-oxo-24,25-dihydrolanosterols by partially purified cytochrome P-450(14DM) from rat liver microsomes

Metabolism of 32-oxo-24,25-dihydrolanosterols (3 beta-hydroxylanost-8-en-32- al (4,delta 8-CHO) and 3 beta-hydroxylanost-7-en-32-al (5,delta 7-CHO)) was studied in a reconstituted system consisting of rat liver partially purified cytochrome P-450, which catalyzes lanosterol 14-demethylation (P-450(14DM)), and reduced nicotineamide adenine dinucleotide phosphate (NADPH)-cytochrome P-450 reductase. The reconstituted system converted delta 8-CHO to 4,4-dimethyl-5 alpha-cholesta-8,14-dien-3 beta-ol (2, 8, 14-Diene), which corresponds to the 14-deformylated product. delta 7-CHO, the isomer of delta 8-CHO, was not converted to the corresponding 14-deformylated product. The apparent Km value of cytochrome P-450(14DM) for delta 8-CHO was about 1/20 of that for 24,25-dihydrolanosterol (1, DHL). The metabolism of delta 8-CHO was inhibited by 7-oxo-24,25-dihydrolanosterol (6, 7-oxo-DHL), which is a potent inhibitor of cholesterol biosynthesis from lanosterol or DHL. However, the metabolism of delta 8-CHO was less inhibited by 7-oxo-DHL than that of DHL.     

Isolation of teratogenic alkaloids by reversed-phase high-performance liquid chromatography

Reversed-phase high-performance liquid chromatography was used for both analytical and preparative separations of several steroidal alkaloids which occur in extracts of Veratrum californicum. The inclusion of 0.1% trifluoroacetic acid in the mobile phase improved the efficiency of the chromatography and the solubility of the compounds in aqueous acetonitrile. Nuclear magnetic resonance was used to assist the identification of the isolated steroidal alkaloids. The effect of the interaction of trifluoroacetic acid with the alkaloids could be clearly seen by changes in the chemical shifts in the nuclear magnetic resonance spectra.     

Analysis of tea components by high-performance liquid chromatography and high-performance capillary electrophoresis

Tea is one of the most popular beverages in the world. The number of reports on the analysis of tea components, especially for catechins, has recently been increasing. We review the recent reports on the analysis of tea components using the analytical methods of high-performance liquid chromatography and high-performance capillary electrophoresis.     

Isolation of experimental anti-AIDS glycerophospholipids by micro-preparative reversed-phase high-performance liquid chromatography.

The experimental anti-AIDS glycerophosphatidic acid: nucleoside (sn-1/sn-2 diacylglycerol:dideoxynucleotide) drugs 3'-azido-3'-deoxythymidine monophosphate diglyceride (AZT-MP-DG) and 2',3'-dideoxycytidine monophosphate diglyceride (ddC-MP-DG) were isolated and purified by reversed-phase high-performance liquid chromatography (HPLC). The chromatographic separation was based on the glycerophospholipid moiety of the drugs and detection of the nucleoside component. The separations were optimized on method development columns packed with the stationary phase to be used in the micro-preparative column and monitored by a UV detector. Fractions were collected and analyzed for purity by analytical-scale HPLC and by thin-layer chromatography (TLC). The purity of the recovered drugs based on UV and light-scattering detection and on TLC was greater than 99%. The purified compounds were isolated for studies on structure confirmation, physical, biophysical and formulation properties and anti-HIV efficacy in culture.     

Kinetics of amine-inhibited naphthalene hydroxylation by rat liver microsomal cytochrome P-450

Inhibition constants for amine-inhibited naphthalene hydroxylation by rat liver microsomal cytochrome P-450 have been determined. Irrespective of the nature of the amine added, inhibition has a competitive character. The inhibition mechanism of cytochrome P-450 dependent hydroxylation is discussed.

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