Dimer-monomer conformational equilibrium of gramicidin A in 1-alkanols as studied by h.p.l.c. and fluorescence spectroscopy

High performance size-exclusion chromatograph (HPSEC) has been successfully used to characterized the dimer-monomer conformational equilibrium of gramicidin A (GA) in a series of 1-alkanols, from methanol to 1-pentanol. The chromatographic methodology proposed has allowed a rapid and accurate determination of kinetic and thermodynamic constants in the different alcohols assayed. The validity of this chromatographic approach has been tested by comparing the values of the kinetic and thermodynamic constants obtained with those reported in the literature deduced from spectroscopic techniques. Taking advantage of the chromatographic results, the differential fluorescent features of the monomer relative to the dimer have been investigated in terms of the quantum yields ratio of the individual species, as a function of solvent polarity. Finally, the possibility of applying this HPSEC approach to the study of other auto-associating polypeptides and of GA incorporated in liposomes is also considered.     

Reverse-phase h.p.l.c. separation, quantification and preparation of bilirubin and its conjugates from native bile. Quantitative analysis of the intact tetrapyrroles based on h.p.l.c. of their ethyl anthranilate azo derivatives.

We describe a facile and sensitive reverse-phase h.p.l.c. method for analytical separation of biliary bile pigments and direct quantification of unconjugated bilirubin (UCB) and its monoglucuronide (BMG) and diglucuronide (BDG) conjugates in bile. The method can be 'scaled up' for preparative isolation of pure BDG and BMG from pigment-enriched biles. We employed an Altex ultrasphere ODS column in the preparative steps and a Waters mu-Bondapak C18 column in the separatory and analytical procedures. Bile pigments were eluted with ammonium acetate buffer, pH 4.5, and a 20 min linear gradient of 60-100% (v/v) methanol at a flow rate of 2.0 ml/min for the preparative separations and 1.0 ml/min for the analytical separations. Bile pigments were eluted in order of decreasing polarity (glucuronide greater than glucose greater than xylose conjugates greater than UCB) and were chemically identified by t.l.c. of their respective ethyl anthranilate azo derivatives. Quantification of UCB was carried out by using a standard curve relating a range of h.p.l.c. integrated peak areas to concentrations of pure crystalline UCB. A pure crystalline ethyl anthranilate azo derivative of UCB (AZO . UCB) was employed as a single h.p.l.c. reference standard for quantification of BMG and BDG. We demonstrate that: separation and quantification of biliary bile pigments are rapid (approximately 25 min); bile pigment concentrations ranging from 1-500 microM can be determined 'on line' by using 5 microliters of bile without sample pretreatment; bilirubin conjugates can be obtained preparatively in milligram quantities without degradation or contamination by other components of bile. H.p.l.c. analyses of a series of mammalian biles show that biliary UCB concentrations generally range from 1 to 17 microM. These values are considerably lower than those estimated previously by t.l.c. BMG is the predominant, if not exclusive, bilirubin conjugate in the biles of a number of rodents (guinea pig, hamster, mouse, prairie dog) that are experimental models of both pigment and cholesterol gallstone formation. Conjugated bilirubins in the biles of other animals (human, monkey, pony, cat, rat and dog) are chemically more diverse and include mono-, di- and mixed disconjugates of glucuronic acid, xylose and glucose in proportions that give distinct patterns for each species.     

Single-step purification and h.p.l.c. analysis of glutathione transferase 8-8 in rat tissues.

GSSG selectively elutes two GSH transferases from a mixture of rat GSH transferases bound to a GSH-agarose affinity matrix. One is a form of GSH transferase 1-1 and the other is shown to be GSH transferase 8-8. By using tissues that lack this form of GSH transferase 1-1 (e.g. lung), GSH transferase 8-8 may thus be purified from cytosol in a single step. Quantitative analysis of the tissue distribution of GSH transferase 8-8 was obtained by h.p.l.c.     

High genetic diversity and fine-scale spatial structure in the marine flagellate Oxyrrhis marina (Dinophyceae) uncovered by microsatellite loci

Free-living marine protists are often assumed to be broadly distributed and genetically homogeneous on large spatial scales. However, an increasing application of highly polymorphic genetic markers (e.g., microsatellites) has provided evidence for high genetic diversity and population structuring on small spatial scales in many free-living protists. Here we characterise a panel of new microsatellite markers for the common marine flagellate Oxyrrhis marina. Nine microsatellite loci were used to assess genotypic diversity at two spatial scales by genotyping 200 isolates of O. marina from 6 broad geographic regions around Great Britain and Ireland; in one region, a single 2 km shore line was sampled intensively to assess fine-scale genetic diversity. Microsatellite loci resolved between 1-6 and 7-23 distinct alleles per region in the least and most variable loci respectively, with corresponding variation in expected heterozygosities (H(e)) of 0.00-0.30 and 0.81-0.93. Across the dataset, genotypic diversity was high with 183 genotypes detected from 200 isolates. Bayesian analysis of population structure supported two model populations. One population was distributed across all sampled regions; the other was confined to the intensively sampled shore, and thus two distinct populations co-occurred at this site. Whilst model-based analysis inferred a single UK-wide population, pairwise regional F(ST) values indicated weak to moderate population sub-division (0.01-0.12), but no clear correlation between spatial and genetic distance was evident. Data presented in this study highlight extensive genetic diversity for O. marina; however, it remains a substantial challenge to uncover the mechanisms that drive genetic diversity in free-living microorganisms.     

Characterization of methylation of rat liver cytosolic glutathione S-transferases by using reverse-phase h.p.l.c. and chromatofocusing.

Glutathione S-transferase (GST) subunits in rat liver cytosol were separated by reverse-phase h.p.l.c.; five major proteins were isolated and identified as subunits 1, 2, 3, 4 and 8. F.p.l.c. chromatofocusing resolved the affinity-purified GST pool into nine different isoenzymes. The five basic (Alpha class) dimeric peaks of GST activity were 1-1, 1-2a, 1-2b, 2-2a and 2-2b. Reverse-phase h.p.l.c. analysis revealed that subunit 8 was also present in the protein peaks designated 1-1, 1-2a and 1-2b. The four neutral (Mu class) isoenzymes were 3-3, 3-4, 3-6 and 4-4. The GST pool was methylated in vitro before reverse-phase h.p.l.c. or f.p.l.c. chromatofocusing. Chromatofocusing indicated that the Mu class isoforms (3-3, 3-4 and 4-4) were the primary GSTs methylated, and h.p.l.c. analysis confirmed that subunits 3 and 4 were the major methyl-accepting GST subunits. The addition of calmodulin stimulated the methylation in vitro of GST isoenzymes 3-3, 3-4 and 4-4 by 3.0-, 7.5- and 9.9-fold respectively. Reverse-phase h.p.l.c. also indicated that only the methylation of GST subunits 3 and 4 was stimulated by calmodulin. Basic GST isoenzymes were minimally methylated and the methylation was not enhanced by calmodulin. Investigation of the time course of methylation of GST subunits 3 and 4 indicated that at incubation times less than 4 h the methylation of both Mu class subunits was stimulated by calmodulin, and that under such conditions subunit 4 was the preferred substrate. In contrast, there was essentially no calmodulin-stimulated methylation at incubation times of 4 or 6 h, and the methylation of subunit 3 was predominant. Kinetic parameters at 2 h of incubation were determined in the presence and in the absence of calmodulin. The addition of calmodulin doubled the Vmax. for methylation of both subunits 3 and 4 and decreased the Km of subunit 4 for S-adenosyl-L-methionine 3.6-fold. Finally, methylation was substoichiometric and after 6 h of incubation ranged from 2.8 to 7.6% on a mole-to-mole basis for subunits 4 and 3 respectively.     

A new h.p.l.c. isolation procedure for chicken and goose erythrocyte histones.

Total chicken erythrocyte histones were separated by reversed-phase h.p.l.c. using a multi-step acetonitrile gradient in a very short time (35 min). The proteins were eluted in the following order: H1, H5, H2B, H2A.2, H4, H2A.1 and H3.2. Applying a special gradient system adapted for the separation of very-lysine-rich histones, chicken erythrocyte H5 was resolved into two subfractions. Their electrophoretic mobilities were identical in both SDS and acetic acid/urea/Triton polyacrylamide-gel electrophoresis, but different in free-flow electrophoresis. Amino-acid-sequence analyses revealed that the two components only differ with respect to position 15, one having glutamine in that position and the other arginine. A separation of histones prepared from goose erythrocytes disclosed no H5 subfractionation. Furthermore, histones obtained from anaemic-chicken blood were analysed by the above-mentioned h.p.l.c. conditions. An alteration in the relation of H1 to H5 was detected, but no further differences in the number and quantity of the histones and histone variants were observed as compared with the corresponding proteins processed from normal-chicken blood.     

Separation of rabbit mammary-gland prolactin receptors by ion-exchange chromatography, h.p.l.c.-gel filtration and ultracentrifugation.

Rabbit mammary-gland prolactin (Prl) receptors in the microsomal fraction were solubilized in 7.5 mM-Chaps) or 1% Triton X-100 and analysed by ion-exchange chromatography using DEAE-Bio-Gel A. Prl receptors in the presence of 7.5 mM-Chaps were separated into two different fractions (Fr. A and B), both of which showed identical specificity of binding to peptide hormones as those in the Chaps or Triton extract. oPrl and human growth hormone (hGH) bound to the same site, but other non-lactogenic hormones (follicle-stimulating hormone, oGH, luteinizing hormone and insulin) failed to bind to the Prl receptors. The dissociation constant (Kd) for Prl binding to the receptors in Fr. A was about 50% of those in Fr. B, suggesting that the rabbit mammary gland contains two types of Prl receptors, one with a high, and one with a low, Kd for Prl binding. A decrease in the concentration of Chaps in the column buffer to 4 mM caused aggregation of the receptors in Fr. A. H.p.l.c.-gel filtration, using Shim pack 150 and 300 columns connected in series, separated the receptor as a protein with an Mr of 74,000 +/- 4,900 (mean +/- S.D.) in the presence of 5 mM-Chaps, or of 36,800 +/- 2,100 in the presence of 7.5 mM-Chaps. Sucrose-gradient-centrifugation analysis showed that the Prl-receptor complexes in the presence of 5 mM-Chaps were sedimented between gamma-globulin and bovine serum albumin (5.56 +/- 0.22 S). As the Chaps concentration was increased to 7.5 mM, a further peak of the Prl-receptor complexes (4.01 +/- 0.23 S) appeared below ovalbumin. The present data suggest that the binding subunit causes the monomeric subunit to aggregate with itself or with another specific associated protein of similar Mr.     

Detection of radical species in haematin-catalysed retinoic acid 5,6-epoxidation by using h.p.l.c.-e.p.r. spectrometry.

E.p.r. signals were detected in an all-trans-retinoic acid/haematin incubation mixture by using an e.p.r. spin-trapping technique. The spin adducts are presumably attributable to some intermediates in haematin-catalysed retinoic acid 5,6-epoxidation, since addition of nitrosobenzene to the reaction mixture dose-dependently inhibited the epoxidation. Analysing the reaction mixture by h.p.l.c.-e.p.r. spectrometry resulted in the detection of three peaks (III-1, III-2, IV) ascribable to the radical species. Two (peaks III-1 and -2) of the three peaks, which appeared 10 min after the reaction had started, seem to be attributable to the radical species directly participating in the epoxidation. The radicals trapped by nitrosobenzene do not appear to be derived from active oxygen, since none of these peaks were detected in a similar h.p.l.c. analysis of O2- and OH.-generating systems. They are presumably derived from retinoic acid. This view is also supported by the following results: none of these peaks were detected in the h.p.l.c. elution profile of the reaction mixture when retinoic acid was absent; peaks III-1 and 2 were detected even under anaerobic conditions, and their peak heights were unchanged under aerobic conditions.     

Isolation of four forms of acetone-induced cytochrome P-450 in chicken liver by h.p.l.c. and their enzymic characterization.

The purpose of this study was to purify and characterize the forms of cytochrome P-450 induced in chicken liver by acetone or ethanol. Using high performance liquid ion-exchange chromatography, we were able to isolate at least four different forms of cytochrome P-450 which were induced by acetone in chicken liver. All four forms of cytochrome P-450 proved to be distinct proteins, as indicated by their N-terminal amino acid sequences and their reconstituted catalytic activities. Two of these forms, also induced by glutethimide in chicken embryo liver, appeared to be cytochromes P450IIH1 and P450IIH2. Both of these cytochromes P-450 have identical catalytic activities towards benzphetamine demethylation. However, they differ in their abilities to hydroxylate p-nitrophenol and to convert acetaminophen into a metabolite that forms a covalent adduct with glutathione at the 3-position. Another form of cytochrome P-450 induced by acetone is highly active in the hydroxylation of p-nitrophenol and in the conversion of acetaminophen to a reactive metabolite, similar to reactions catalysed by mammalian cytochrome P450IIE. Yet the N-terminal amino acid sequence of this form has only 30-33% similarity with cytochrome P450IIE purified from rat, rabbit and human livers. A fourth form of cytochrome P-450 was identified whose N-terminal amino acid sequence and enzymic activities do not correspond to any mammalian cytochromes P-450 reported to be induced by acetone or ethanol.     

A novel metal-dye detection system permits picomolar-range h.p.l.c. analysis of inositol polyphosphates from non-radioactively labelled cell or tissue specimens.

A novel complexometric dye- and transition-metal-based post-column detection system for polyanions, called 'metal-dye detection' has been developed. This technique, combined with a new h.p.l.c. separation protocol, permits a direct highly-isomer-selective determination of bis- to poly-phosphorylated non-radioactively labelled compounds in the picomolar range, a sensitivity hitherto unknown for these substances. The application of the technique in the quantitative microanalysis of inositol polyphosphates from milligram amounts of cells or tissue specimens is described. The technique promises to answer hitherto unresolved questions about the role of inositol phosphates, especially those in intact tissues, which are not readily amenable to analysis by radioisotopic techniques.     

H.p.l.c. analysis of di- and tri-carboxylic porphyrins in porphyric patients.

New h.p.l.c. methods have been developed for the quantitative determination of di- and tri-carboxylic porphyrin methyl esters, and applied to the analysis of faecal extracts from patients with four different types of porphyria.     

Improved assay method for activity of thyroid peroxidase-catalysed coupling of iodotyrosine residues of thyroglobulin utilizing h.p.l.c. for analysis of iodothyronines.

The coupling of iodotyrosine residues of thyroglobulin (Tg) catalysed by thyroid peroxidase (TPO) has scarcely been studied with respect to the TPO of abnormal human thyroid glands. The present paper proposes a rapid and convenient assay method applicable for determining the coupling activity of a sample of less than 500 mg from each patient's thyroid. The main characteristics of the method are as follows: (i) mitochondrial/microsomal fractions of thyroid glands were treated with sodium cholate plus trypsin, and the supernatants obtained by ultracentrifugation were directly used for the assay of coupling and peroxidase activity of TPO; (ii) the formation of iodotyrosine residues catalysed by TPO was performed by using chemically iodinated Graves'-disease Tg containing 41 iodine atoms per molecule and with a high iodotyrosine and a low iodothyronine content; (iii) newly synthesized iodothyronine residues (thyroxine, 3,5,3'-tri-iodothyronine, and 3,3',5'-tri-iodothyronine) were analysed by h.p.l.c. after hydrolysis of Tg with proteinases and extraction of iodothyronines with ethyl acetate.     

Separation of rat tissue histone H1 subtypes by reverse-phase h.p.l.c. Identification and assignment to a standard H1 nomenclature.

H1 histones from rat liver and rat testis were separated by reverse-phase h.p.l.c. Within 40 min six subfractions (H1(0), H1b, H1a, H1d, H1e + H1c and H1c) and seven subfractions (H1(0), H1b, H1a, H1d, H1e + H1c, H1c and H1t) respectively were isolated by using a linear acetonitrile gradient. Each individual H1 subtype was identified either by comparing the H1 variants (contained in both tissues but in different quantities) or by SDS/PAGE and acetic acid/urea/PAGE. Moreover, all H1 variants were characterized by amino acid analyses. The amino acid compositions of rat histone subfractions H1(0), H1b and H1e were determined for the first time. It was possible to classify unambiguously the H1 subfractions obtained by h.p.l.c. by following the standardized H1 nomenclature for electrophoretic systems recommended by Lennox, Oshima & Cohen [(1982) J. Biol. Chem. 257, 5183-5189]. Incorrect assignments that have been made in various publications are discussed.     

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.     

Skeletal muscle mitochondrial beta-oxidation. A study of the products of oxidation of [U-14C]hexadecanoate by h.p.l.c. using continuous on-line radiochemical detection.

Well-coupled mitochondrial fractions were prepared from rat skeletal muscle without the use of proteolytic enzymes. The products of [U-14C]hexadecanoate oxidation by rat skeletal muscle mitochondrial fractions were analysed by h.p.l.c. with on-line radiochemical detection. In the presence of 1 mM-carnitine, 70% of the products is acetylcarnitine. In agreement with Veerkamp et al. [Veerkamp, van Moerkerk, Glatz, Zuurveld, Jacobs & Wagenmakers (1986) Biochem. Med. Metab. Biol. 35, 248-259] 14CO2 release is shown to be an unreliable estimate of flux through beta-oxidation in skeletal muscle mitochondrial fractions. The flux through beta-oxidation is recorded unambiguously polarographically in the presence of 1 mM-carnitine and the absence of citrate cycle intermediates.     

The chloroplast pigments of the algal classes Eustigmatophyceae and Xanthophyceae. II. Xanthophyceae

The chloroplast pigments of Pleurochloris meiringensis Vischer, Mischococcus sphaerocephalus Vischer and Tribonema aequale Pascher have been analysed by a variety of chromatographic methods. There are significant differences between these xanthophycean algae and the eustigmatophycean algae formerly classified with them. In particular the former contain diadinoxanthin as the major xanthophyll whereas diadinoxanthin is absent from the latter and violaxanthin occurs as the major xanthophyll pigment. The other pigments of the Xanthophyceae sensu stricto include chlorophyll a, β-carotene, vaucheriaxanthin-ester, heteroxanthin, diatoxanthin, cryptoxanthin epoxide and a neoxanthin-like pigment.     

The quantitative spectrum of inositol phosphate metabolites in avian erythrocytes, analysed by proton n.m.r. and h.p.l.c. with direct isomer detection.

The spectrum of inositol phosphate isomers present in avian erythrocytes was investigated in qualitative and quantitative terms. Inositol phosphates were isolated in micromolar quantities from turkey blood by anion-exchange chromatography on Q-Sepharose and subjected to proton n.m.r. and h.p.l.c. analysis. We employed a h.p.l.c. technique with a novel, recently described complexometric post-column detection system, called 'metal-dye detection' [Mayr (1988) Biochem. J. 254, 585-591], which enabled us to identify non-radioactively labelled inositol phosphate isomers and to determine their masses. The results indicate that avian erythrocytes contain the same inositol phosphate isomers as mammalian cells. Denoted by the 'lowest-locant rule' [NC-IUB Recommendations (1988) Biochem. J. 258, 1-2] irrespective of true enantiomerism, these are Ins(1,4)P2, Ins(1,6)P2, Ins(1,3,4)P3, Ins(1,4,5)P3, Ins(1,3,4,5)P4, Ins(1,3,4,6)P4, Ins(1,4,5,6)P4, Ins(1,3,4,5,6)P5, and InsP6. Furthermore, we identified two inositol trisphosphate isomers hitherto not described for mammalian cells, namely Ins(1,5,6)P3 and Ins(2,4,5)P3. The possible position of these two isomers in inositol phosphate metabolism and implications resulting from absolute abundances of inositol phosphates are discussed.