Isolation of cytochrome P-450 components from marmoset liver microsomes by high-performance liquid chromatography.

A fast protein liquid chromatographic (FPLC) system with pre-packed and laboratory-packed columns was used for the analytical and preparative isolation of marmoset monkey cytochrome P-450 (P450) and NADPH-P450-reductase. Chromatographic separations also allowed the recovery of cytochrome b5, NADH-b5-reductase and epoxide hydratase. Cholate-solubilized liver microsomes from phenobarbital-induced marmosets were crudely purified on 8-aminooctyl-Sepharose or 6-aminohexyl-Sepharose and then fractionated into several isoenzyme groups using hydroxyapatite. Further purification on Mono S or CM-Sepharose and finally on phenyl-Superose, phenyl-Sepharose or octyl-Sepharose yielded a P450 fraction which was apparently homogeneous as judged by sodium dodecyl sulphate-polyacrylamide gel electrophoresis in the automated Phast system using silver staining. Removal of excess of non-ionic detergent was effected by hydroxyapatite columns, and this was compared with other methods. For the isolation of P450 isoenzymes from untreated marmosets, Mono Q columns were employed and yielded at least two highly purified forms. NADPH-P450-reductase was recovered from the 8-aminooctyl-Sepharose column or crudely fractionated on DEAE-Sepharose Fast Flow. Subsequent purification via 2',5'-ADP-Sepharose and Superose 12 chromatography resulted in a homogeneous preparation.     

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.     

Purification of the glycoprotein glucose oxidase from Penicillium amagasakiense by high-performance liquid chromatography

Fast protein liquid chromatography (FPLC) in combination with ion-exchange chromatography on a Mono Q column was used to purify glucose oxidase from Penicillium amagasakiense to homogeneity. Purification was performed with a mixed pH and salt gradient, with 20 mM phosphate buffer (pH 8.5) as starting buffer (A) and 50 mM acetate buffer (pH 3.6) with 0.1 M NaCl as elution buffer (B). Elution conditions were optimized to permit the simultaneous purification and separation of the glucose oxidase isoforms. Three peaks, each consisting of 1-2 isoforms and exhibiting a homogeneous titration curve profile, were resolved with a very flat linear gradient of 5.0-5.1% B in 40 ml. Three more peaks, each consisting of several isoforms, were eluted at 10%, 30% and 100% B. Optimization of the elution conditions and separation of the glucose oxidase isoforms was only possible because of the rapidity of each purification step and the high resolution provided by FPLC and Mono Q.     

Isolation and partial characterization of angiotensinase A and aminopeptidase M from urine and human kidney by lectin affinity chromatography and high-performance liquid chromatography

Angiotensinase A (ATA) and aminopeptidase M (APM) were partially purified from human urine specimens and human kidney particles using wheat germ lectin affinity chromatography, anion-exchange Fast Protein Liquid Chromatography (FPLC) (Mono Q), chromatofocusing (Mono P, FPLC) and Superose 12 gel filtration. APM, a globular 5-nm glycoprotein, is localized in the brush border membrane of the proximal tubule; angiotensin II-degrading ATA is present on glomerular endothelia and podocytes and, to lesser extent, in the brush border. For the first time, both peaks of ATA and APM activity from urine samples were separated by the above-mentioned techniques with only slight overlap; ATP (146,000 dalton: pI4.8) was enriched more than 20-fold and APM (153,000 dalton, pI4.7) more than 50-fold compared with the activity of the starting material. Using similar separation steps, ATA and APM solubilized from kidney particles could not be resolved into two distinct peak fractions, however, except after hydrophobic interaction chromatography. Thus urine is a major source for the preparation of individual ATA and APM fractions, necessary to generate specific anti-enzyme antibodies for diagnostic purposes.     

Immobilized metal affinity chromatography as a means of fractionating microsomal cytochrome P-450 isozymes.

Fractionation of microsomal cytochrome P-450s is usually done by chromatography on ion-exchange resins and hydroxyapatite. The resolution of the great number of similar P-450 isozymes, however, requires additional methods based on different separation parameters. For this purpose immobilized-metal affinity chromatography (IMAC) was applied to the separation of P-450 isozymes. The method in its application to rat liver microsomes is described in detail. For method optimization and for the reproducibility of analytical fractionations a completely automatic fast protein liquid chromatographic system especially designed for IMAC is presented. Optimization is done with respect to the choice of the immobilized metal ion and the elution conditions. The chromatographic resolution is markedly enhanced by using segmented vs. linear gradients. The efficiency of P-450 resolution is demonstrated by sodium dodecyl sulphate polyacrylamide gel electrophoresis and immunoblotting, verifying the different retention behaviours of the isozymes. However, for all the isozymes analysed so far, reactivity with one particular polyclonal antibody is observed with more than two IMAC fractions of a single run. This may be explained in part by the occurrence of isozymic forms distinguishable by the pattern of chymotryptic peptides. Hence IMAC appears to be suitable for the separation of closely related isozyme forms.     

Potential for the speciation of Zn using fast protein liquid chromatography (FPLC) and convective interaction media (CIM) fast monolithic chromatography with FAAS and electrospray (ES)-MS-MS detection

Analytical procedures were developed for the speciation of Zn using fast protein liquid chromatography (FPLC), flame atomic absorption spectrometry (FAAS) and convective interaction media (CIM) fast monolithic chromatography with FAAS and electrospray (ES)-MS-MS detection. The investigation was performed on synthetic solutions (2 microg cm-3 Zn) of hydrated Zn2+ species and Zn complexes with citrate, oxalate and EDTA (ligand-to-Zn molar ratio 100:1) over a pH range from 5.4 to 7.4. It was found that Zn interacts with various buffers and the careful adjustment of the pH with diluted solutions of KOH is, therefore, required. FPLC separations were carried out on a Mono Q HR 5/5 strong anion-exchange column, applying an aqueous 1 mol dm(-3) NH4NO3 linear gradient elution over 15 min, at a flow rate of 1.0 cm3 min(-1). The separated Zn species were determined in 1.0 cm3 eluate fractions "off line" by FAAS. Speciation of Zn was also performed on a weak anion-exchange CIM DEAE fast monolithic disc by applying an aqueous 0.4 mol dm(-3) NH4NO3 linear gradient elution over 7.5 min, at a flow rate of 2.0 cm3 min(-1) and determination of the separated Zn species in 1.0 cm3 eluate fractions "off line" by FAAS. Zn-binding ligands in separated fractions were also characterized by electrospray (ES)-MS-MS analysis. The CIM DEAE disc was found to be more efficient in the separation of negatively charged Zn complexes than the Mono Q FPLC column. On the CIM DEAE disc Zn-citrate was separated from both Zn-oxalate and from Zn-EDTA. All these species were also separated from hydrated Zn2+, which was eluted with the solvent front. This method has an advantage over commonly used analytical techniques for the speciation of Zn which are only able to distinguish between labile and strong Zn complexes. Good repeatability of the measurements (RSD 2-4%), tested for six parallel determinations (2 microg cm(-3) Zn) of Zn-EDTA, Zn-citrate and Zn-oxalate was found at a pH of 6.4 on a CIM DAEA disc. The limit of detection (3s) for the separated Zn species was 10 ng cm(-3). The proposed analytical procedure was applied to the speciation of Zn in aqueous soil extracts and industrial waste water from a lead and zinc mining area.     

Preparation of trout liver microsomes for iron speciation in P-450 enzymes by AE–FPLC with ICP–(ORS)MS detection

Cytochromes P-450 are members of a superfamily of hemoproteins involved in the oxidative metabolism of various physiological and xenobiotic compounds in eukaryotes and prokaryotes. The multiplicity of this group of enzymes has been widely studied by chromatographic techniques, mainly high-performance liquid chromatography (HPLC). Because these enzymes are membrane-bound proteins, sample preparation for chromatographic separation of P-450 enzymes requires a solubilization step. The sample-preparation procedures are critical, because detergents affect not only the efficiency of protein solubilization but also their further chromatographic resolution. Trout liver microsomes have been taken here as a model sample to investigate iron speciation in cytochrome P-450. Trouts were treated intraperitoneally with -naphthoflavone, a potent inducer of some P-450 enzymes, and a microsomal suspension containing 7.4±0.1 nmol mL^–1 P-450 enzymes was obtained by ultracentrifugation. Lubrol PX was selected as detergent for solubilization, resulting in about 90% solubilization recovery. The solubilized cytochromes P-450 were further separated by AE–FPLC, with UV detection, or coupled to ICP–MS with an octapole reaction system, ICP–(ORS)MS (monitoring Fe signals at masses 54, 56, and 57). A sampling procedure and chromatographic conditions are developed and were successfully applied to iron speciation in trout liver P-450 enzymes. ICP–(ORS)MS detection of P-450 enzymes is Fe-specific and so will give accurate information on the prosthetic group of the protein, which can constitute an advantageous alternative to classical methods for detection of these hemoproteins.     

Rapid purification of cytochrome c oxidase from Paracoccus denitrificans

Two methods are described for the purification of cytochrome c oxidase from Triton X-100 extracts of the periplasma membrane of Paracoccus denitrificans. The first is a large-scale procedure for the preparation of 100-250 nmol of cytochrome c oxidase (10-20 mg) in 1 week. The second is a rapid procedure for isolating up to 25 nmol in 2-3 days. Owing to the high yields given by fast protein liquid chromatography (FPLC) on Mono Q columns, the overall yield is about 20%, whereas the yield in many other previously published procedures does not exceed 10%. The use of FPLC on Mono Q also offers a considerable saving of time.     

Possibilities for speciation of Al-citrate and other negatively charged Al complexes by anion-exchange FPLC-ICP-AES

An anion-exchange fast protein liquid chromatographic-inductively coupled plasma atomic emission spectrometric procedure (FPLC-ICP-AES) was developed for speciation of Al-citrate and other negatively charged Al complexes. FPLC separations were carried out on a Mono Q HR 5/5 strong anion-exchange FPLC column over a pH range from 3.5 to 11.0. An aqueous-NaNO(3) (4 mol dm(-3)) linear gradient elution was applied over 10 min for separation of a particular Al species. The separated Al species were determined in 0.5 cm(3) eluate fractions ;off line' by ICP-AES. Under optimal analytical procedures Al-citrate was separated from Al-oxalate and Al-EDTA in a neutral pH range. Good reproducibility of the FPLC-ICP-AES procedure was obtained for determination of a particular Al species at optimal measurement conditions (RSD +/-2%). Al(3+) and neutral Al-citrate species were strongly adsorbed on the column resin and did not interfere with the separation of negatively charged Al complexes. Al(OH)(4)(-) species were separated from Al-citrate in an alkaline pH region, but quantitatively determined only at a pH of 11.0. The distribution of Al species over a pH range from 3.5 to 11.0 agreed with the reported calculated data. The limit of detection (3sigma basis) for separated Al species was 0.1 mug cm(-3).     

Application of high-performance liquid chromatography to the purification of the putative intestinal peptide transporter

A membrane protein of relative molecular mass (Mr) 127,000 was identified by photoaffinity labelling as (a component of) the uptake system for small peptides and beta-lactam antibiotics in rabbit small intestine. This binding protein is a microheterogeneous glycosylated integral membrane protein which could be solubilized with non-ionic detergents and enriched by lectin affinity chromatography on wheat germ lectin agarose. For the final purification of this protein and separation from aminopeptidase N of Mr 127,000, fast protein liquid chromatography (FPLC) was used. Gel permeation, hydroxyapatite and hydrophobic interaction chromatography were not successful for the purification of the 127,000-dalton binding protein. By anion-exchange chromatography on a Mono Q column with either Triton X-100 or n-octylglucoside as detergent, a partial separation of the 127,000-dalton binding protein from aminopeptidase N was achieved. By cation-exchange chromatography on a Mono S HR 5/5 column at pH 4.5 using Triton X-100 as detergent also only a partial separation from aminopeptidase N could be achieved. If, however, Triton X-100 was replaced with n-octylglucoside, the binding protein for beta-lactam antibiotics and small peptides of Mr 127,000 could be completely separated from aminopeptidase N. These results indicate that Triton X-100 should be avoided for the purification of integral membrane proteins because mixed protein-detergent micelles of high molecular weight prevent a separation into the individual membrane proteins. The putative peptide transport protein was finally purified by rechromatography on Mono S and was obtained more than 95% pure as determined densitometrically after sodium dodecyl sulphate gel electrophoresis. By application of FPLC even microheterogeneous membrane glycoproteins from the intestinal mucosa can be purified to such an extent that a sequence analysis and immunohistochemical localization with antibodies prepared from the purified protein is possible.     

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).     

Rapid analysis of bovine milk proteins by fast protein liquid chromatography

The separation of bovine milk proteins by fast protein liquid chromatography has been investigated by ion-exchange chromatography on Mono Q and Mono S columns and by gel filtration on a column of Superose 12. The four major casein components (alpha s1, alpha s2, beta and kappa) as well as the minor gamma-caseins were generally well separated on the Mono S column with urea-containing buffers at pH 3.8 in as short a time as 7 min, although there was considerable overlap between alpha s1- and alpha s2-casein peaks. Peak area measurements indicated that the four caseins alpha s1, alpha s2, beta and kappa were present in total casein in the approximate proportions of 3.0:0.5:3.4:0.9, in good agreement with other literature values. Whey proteins were not separated on the Mono S column, but were all well resolved by rapid analysis on the Mono Q column at pH values between 6 and 8 in buffers free of urea or 2-mercaptoethanol. Both urea and 2-mercaptoethanol were required for casein analyses on the Mono Q column, but all the casein components were then separable over a broad pH range (5.0-11.0). While urea levels of 4.5-8.0 M and pH values of 7.0 to 8.0 were most generally useful, the resolution of some components was affected by urea concentration or pH, so conditions may have to be modified for specific analysis problems. The caseins were too similar in size to be separated on the Superose 12 column but high-speed gel filtration in as little as 15 min separated all the whey proteins well, molecular weight values obtained being in good agreement with literature values.     

Analytical- and preparative-scale separation of molecular variants of alpha-fetoprotein by anion-exchange chromatography on Monobead resins.

A rapid and reliable purification procedure is described that is useful for both analytical detection and quantitative recovery of milligram amounts of individual molecular variants of mouse alpha-fetoprotein (AFP). The appropriate separation conditions were developed with an analytical-size Mono Q anion-exchange column linked to an automated Fast Protein Liquid Chromatography system. Effective separations of fetal-derived AFP variants was accomplished within 20 min under mild conditions with an L-histidine buffer. Employing the optimal separation conditions established on the Mono Q HR 5/5 column we upscaled the procedure by using a preparative Mono Q HR 16/10 column in order to obtain milligram quantities of each molecular variant of AFP. Seven distinct isomeric forms of AFP could be recovered on the preparative anion exchanger in a highly reproducible manner. Each of the seven protein peaks eluted from the Mono Q column were confirmed to be distinct isoforms of AFP by isoelectric focusing and Western blotting developed with monospecific anti-AFP antisera. This method in its scaled up version offers the benefit of providing milligram quantities of immunochemically pure AFP isomers for structure and function studies.     

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.     

Determination of nitrated-polyaromatic hydrocarbons (nitro-PAHs) in environmental samples by high resolution chromatographic techniques

An analytical procedure is described for the fractionation of organic compounds present in environmental samples and the determination of nitro polyaromatic hydrocarbons (nitro-PAHs). Both low and high resolution liquid chromatography are employed for the prefractionation of the soluble organic fraction (SOF) extracted from particulate matter or gaseous pollutants collected on adsorption traps. High resolution gas chromatography is used to analyze four fractions containing alkanes, PAHs, nitro-PAHs, and other polar PAHs. Nitrogen-containing species are separated by GC and detected specifically using an alkali flame (NPD) detector. Flame ionization (FID) detection, GC-MS of positive ions, and negative ion chemical ionization MS of the whole fraction is used for the identification and quantitation of the various components. The composition of SOF extracted from particulate matter emitted from diesel exhausts is elucidated and a large number of nitro-PAHs identified by the combination of the various techniques.     

Fractionation by High Performance Liquid Chromatography of Microsomal Cytochrome P-450 Induced by Hexachlorobiphenyl Isomers

Abstract

High performance liquid chromatography has been employed to fractionate rat liver microsomes under nondenaturing conditions. Selective detection at 405 nm allowed resolution of microsomal heme proteins into three peaks (A, B, and C). Cytochromes in the peaks retain their native property of binding CO after HPLC. Peak-A, first eluting, contains P-450 and is rich in cytochrome P-420. Peak-B is largely hemoglobin and peak-C is a major cytochrome P-450. The ratio of peak-C to A is increased by treatment of rats with phenobarbitone, β-naphthoflavone, 2,3,5,2′,3′,5′-hexachlorobiphenyl and 3,4,5,3′,4′,5′-hexachlorobiphenyl as compared to controls. The highest increment in the ratio is observed on feeding 3,4,5,3′,4′,5′-hexachlorobiphenyl. NADPH cytochrome c reductase elutes earlier than peak-C but cytochrome b₅ is not separated from the major cytochrome P-450 peak. The separations obtained are highly reproducible and considerably faster than conventional gel permeation chromatography. The data presented here are very promising in establishing the role of HPLC in the studies of insoluble proteins and enzymes in general and cytochrome P-450 in particular.     

Angiotensinase A (aminopeptidase A): properties of chromatographically purified isoforms from human kidney

Angiotensin-II-cleaving angiotensinase A (aminopeptidase A, E.C. 3.4.11.7, ATA) plays an important role in glomerular haemodynamics. the pathophysiology of essential arterial hypertension and the induction of vascular disorders. In order to study biochemical and immunological properties of ATA, two isoforms (I and II) of the glycoprotein were isolated for the first time from human kidney cortex. Kidney cortex homogenate, digested with bromelain, was fractionated by ammonium sulphate precipitation and subsequent hydrophobic interaction chromatography, using a fast protein liquid chromatographic (FPLC) system. By anion-exchange FPLC (Mono Q column), the isoforms of ATA were eluted in two distinct peaks and were further purified by size-exclusion FPLC and preparative polyacrylamide gel electrophoresis. Biochemical, immunological and immunohistological characterization disclosed differences in the intrarenal localization, glycosylation Michaelis constant and apparent molecular mass (native and sodium dodecyl sulphate gel electrophoresis) but similar properties in the double-immunodiffusion technique. Polyclonal rabbit antibodies, raised against ATA isoforms I and II, precipitated an analogous antigen in urine from patients with renal tubular damage.     

Separation of a glycosylated and non-glycosylated fraction of caseinomacropeptide using different anion-exchange stationary phases

Caseinomacropeptide (CMP), a heterogeneous group of peptides regarding the degree of glycosylation and phosphorylation, has so far not been effectively fractionated into its two major fractions: the glycosylated gCMP and the non-glycosylated aCMP. Therefore, an anion-exchange chromatography (AEC) process for the fractionation of CMP in those two fractions was developed and optimized. Furthermore, the method was applied to compare membrane adsorption chromatography (MAC) devices with classical bead-based column chromatography. It was shown that MAC devices can separate gCMP from aCMP, however, at a lower level of binding capacity and chromatographic resolution compared to classical chromatography. On the other hand, a fractionation is achieved at a four times faster separation speed.     

Quantitative determination of trace amounts of Al-citrate by anion-exchange FPLC-ETAAS

An anion-exchange fast protein liquid chromatographic-electrothermal atomic absorption spectrometric procedure (FPLC-ETAAS) was developed for determination of trace amounts of negatively charged Al-citrate in the pH range 3.5-8.0. Aqueous-4 mol dm(-3) NH(4)NO(3) linear gradient elution at a flow rate of 1 cm(3) min(-1) was applied for 10 min to separate Al-citrate on a FPLC Mono Q HR 5/5 column. The separated aluminium species were determined 'off line' by ETAAS in 0.5 cm(3) fractions. After separation the column was regenerated for 5 min with 4 mol dm(-3) NH(4)NO(3) and equilibrated with water. All reagents used in the separation procedure were cleaned with a silica based LiChrosorb RP-18 HPLC column to remove traces of aluminium. The main advantage of NH(4)NO(3) as eluent lies in its ability to decompose quantitatively in the graphite tube during the ashing step, which enables reproducible analysis of aluminium in the separated fractions. Using the procedure developed reproducible (RSD+/-2.0%) and quantitative determination of negatively charged Al-citrate at a retention time of 4.5 min was obtained. The LOD was found to be 2.0 ng cm(-3) of Al-citrate. The technique was successfully applied for the determination of Al-citrate in human serum. Spiked samples (50-150 ng Al(3+) cm(-3)) were microultrafiltered through a membrane filter (cut-off 30 000 Da) to separate aluminium bound to transferrin from low molecular weight aluminium complexes. It was found that 15-19% of aluminium in spiked samples from healthy volunteers passed through the membrane. By applying FPLC separation it was proved that all the aluminium in the filtrate corresponded to Al-citrate. The analytical technique developed enabled quantitative and reproducible determination (RSD+/-3.0%) of Al-citrate in spiked human serum at levels which could be found in patients undergoing long term haemodialysis.