Evaluation of the efficiency of extraction of ultraviolet-absorbing pollen allergens and organic pollutants from airborne dust samples by capillary electromigration methods

Capillary electromigration methods, zone electrophoresis (CZE) and micellar electrokinetic chromatography (MEKC), have been used for evaluation of the efficiency of different extraction agents applied to the extraction of pollen allergens and organic pollutants from dust samples collected during different periods (before, during and after pollen seasons) and in different locations in air-filtration devices (car-traffic tunnel in Prague and a metro station in Paris). Water and acetic acid extracts were analyzed by CZE using acetic acid as background electrolyte (BGE). Water and alkaline water-SDS-buffer extracts were analyzed by MEKC in Tris-phosphate BGE with anionic detergent sodium dodecylsulfate (SDS) micellar pseudophase. More material was extracted and more components were found in the water-buffer extracts than in the water extracts, and better resolution of the components was achieved by MEKC than by CZE. Significant differences have been found in the analyses of dust extracts of different origin. More material and more components have been found in the extracts of the dust collected in the pollen-rich period (March, April) than in the pollen-free period (December, January).     

Determination of purity degree and counter-ion content in lecirelin by capillary zone electrophoresis and capillary isotachophoresis

Capillary zone electrophoresis (CZE) and capillary isotachophoresis (CITP) were applied for the determination of peptide purity degree and counter-ion content in lecirelin, the synthetic analogue of luteinizing hormone-releasing hormone (LHRH). CZE analyses were carried out in acidic background electrolyte (100 mM H3PO4, 50 mM Tris, pH 2.25) in bare fused silica capillary using UV-absorption detection at 206 nm. CITP analyses were performed in the electrophoretic analyzer with column coupling, equipped with contactless conductivity detectors both in preseparation capillary and in analytical capillary, and with UV-absorption detector (220 and 254 nm) in analytical capillary. Determinations of peptide purity were carried out in cationic mode with leading electrolyte (LE), 10 mM KOH/AcOH, pH 4.5, and terminating electrolyte (TE), 10 mM beta-alanine (BALA)/AcOH, pH 4.4. Degree of peptide purity determined by both CZE and CITP was in the range 60.1-80.9% for crude preparations of lecirelin and in the range 96.4-99.9% for HPLC purified batches. Concentrations of contaminating counter-ions, the anions of trifluoromethanesulfonic acid (TFMSA), trifluoroacetic acid (TFA) and acetic acid (AcOH), were determined by CITP analyses in anionic mode with LE 10 mM HCl/His, pH 6.0, and TE 10 mM 2-(N-morpholino)-ethanesulfonic acid (MES), pH 4.0, by the calibration curve method. Mass percentages of the counterion contents in the analyzed lecirelin batches varied from zero to ca. 9% (TFMSA), 3% (TFA) and 11% (AcOH), respectively.     

Mono-N-acyl-2,6-diaminopimelic acid derivatives: Analysis by electromigration and spectroscopic methods and examination of enzyme inhibitory activity

Thirteen mono-N-acyl derivatives of 2,6-diaminopimelic acid (DAP)—new potential inhibitors of the dapE-encoded N-succinyl-l,l-diaminopimelic acid desuccinylase (DapE; EC 3.5.1.18)—were analyzed and characterized by infrared (IR) and nuclear magnetic resonance (NMR) spectroscopies and two capillary electromigration methods: capillary zone electrophoresis (CZE) and micellar electrokinetic chromatography (MEKC). Structural features of DAP derivatives were characterized by IR and NMR spectroscopies, whereas CZE and MEKC were applied to evaluate their purity and to investigate their electromigration properties. Effective electrophoretic mobilities of these compounds were determined by CZE in acidic and alkaline background electrolytes (BGEs) and by MEKC in acidic and alkaline BGEs containing a pseudostationary phase of anionic detergent sodium dodecyl sulfate (SDS) or cationic detergent cetyltrimethylammonium bromide (CTAB). The best separation of DAP derivatives, including diastereomers of some of them, was achieved by MEKC in an acidic BGE (500 mM acetic acid [pH 2.54] and 60 mM SDS). All DAP derivatives were examined for their ability to inhibit catalytic activity of DapE from Haemophilus influenzae (HiDapE) and ArgE from Escherichia coli (EcArgE). None of these DAP derivatives worked as an effective inhibitor of HiDapE, but one derivative—N-fumaryl, Me-ester-DAP—was found to be a moderate inhibitor of EcArgE, thereby providing a promising lead structure for further studies on ArgE inhibitors.     

Analysis of synthetic derivatives of peptide hormones by capillary zone electrophoresis and micellar electrokinetic chromatography with ultraviolet-absorption and laser-induced fluorescence detection

Capillary zone electrophoresis (CZE) and micellar electrokinetic chromatography (MEKC) were used for the analysis of new synthetic derivatives of hypophysis neurohormones--vasopressin and oxytocin, and pancreatic hormone--human insulin (HI) and its octapeptide fragment, derivatized by fluorescent probe, 4-chloro-7-nitrobenzo[1,2,5]oxadiazol (NBD). The suitable composition of background electrolytes (BGEs) was selected on the basis of calculated pH dependence of effective charge of analyzed peptides. Basic ionogenic peptides were analyzed by CZE in the acidic BGE composed of 100 mM H3PO4, 50 mM Tris, pH 2.25. The ionogenic peptides with fluorescent label, NBD, were analyzed in 0.5 M acetic acid, pH 2.5. The best MEKC separation of non-ionogenic peptides was achieved in alkaline BGE, 20 mM Tris, 5 mM H3PO4, with micellar pseudophase formed by 50 mM sodium dodecylsulfate (SDS), pH 8.8. Selected characteristics (noise, detectability of substance, sensitivity of detector) of the UV-absorption detectors (single wavelength detector, multiple-wavelength photodiode array detector (PDA), both of them operating at constant wavelength 206 nm) and laser-induced fluorescence (LIF) detector (excitation/emission wavelength 488/520 nm) were determined. The detectability of peptides in the single wavelength detector was 1.3-6.0 micromol dm(-3) and in the PDA detector 1.6-3.1 micromol dm(-3). The LIF detection was more sensitive, the applied concentration of NBD derivative of insulin fragment in CZE analysis with LIF detection was three orders lower than in CZE with UV-absorption detector, and the detectability of this peptide was improved to 15.8 nmol dm(-3).     

Enrichment of the basic/cationic urinary proteome using ion exchange chromatography and batch adsorption

Anionic or acidic proteins are the main compositions of normal urinary proteome. Efforts to characterize human urinary proteome, thus, have focused mainly on the anionic compartment. The information of cationic or basic proteins present in the normal urine is virtually unknown. In the present study, we applied different methods to enrich cationic urinary proteome. Efficacies of these methods were compared using equal volume (1 L) of urine samples from the same pool obtained from 8 normal healthy individuals. Cation exchange chromatography using RESOURCE-S column provided the least amount of the recovered proteins, whereas batch adsorption using SP Sepharose 4 Fast Flow beads equilibrated with acetic acid (pH 4.8) provided the greatest yield of protein recovery. The recovered proteins were then resolved with 2-DE (pI 7-11) and identified by peptide mass fingerprinting using MALDI-TOF MS. There were several isoforms of immunoglobulin heavy and light chains enriched by these methods. In addition, three isoforms of interferon alpha-3 (IFNalpha3) and six isoforms of eosinophil-derived neurotoxin (EDN), were also enriched. The enrichment of IFNalpha3 and EDN was particularly effective by batch adsorption using SP Sepharose 4 Fast Flow beads equilibrated with acetic acid (pH 6.0). Initial depletion of anionic components using DEAE batch adsorption reduced the recovery yield of these two proteins and did not improve recovery of any other cationic urinary proteins. We conclude that batch adsorption using SP Sepharose Fast Flow beads equilibrated with acetic acid (pH 6.0) is the method of choice to examine the basic/cationic urinary proteome, as this protocol provided the satisfactory yield of protein recovery and provided the greatest amount as well as maximal number of IFNalpha3 and EDN isoforms. Our data will be useful for further highly focused study targeting on cationic/basic urinary proteins. Moreover, the techniques described herein may be applicable for enrichment of cationic proteomes in other body fluids, cells, and tissues.     

Inhibition of anion and glucose permeabilities by anesthetics in erythrocytes. The mechanisms of action of positively and negatively charged drugs

(1) The mode of action of anesthetics as inhibitors of Cl- and glucose transports in human red cells was studied. The term anesthetic is taken in its broad meaning as defined by Seeman (Seeman, P. (1972) Pharmacol. Rev. 24, 583-655) and covers anionic and cationic liposoluble compounds which reversibly block the rising phase of the action potential, without effect on the resting membrane potential. (2) Phenothiazine derivatives were chosen as prototypes of anesthetics because they represent a set of compounds having the same basic chemical structure, the phenothiazine ring, but with either a positive or a negative charge. (3) The Cl- self-exchange is inhibited by both cationic and anionic derivatives. However, to obtain the same level of inhibition, it is necessary to use a concentration 10-100 times higher with cationic than with anionic drugs. (4) At a concentration which inhibits Cl- permeability, cationic derivatives induce a very strong morphological change (cup-shaped cells: stomatocytes or spherostomatocytes) and protect erythrocytes against osmotic hemolysis, signifying that the membrane is fully expanded. Conversely, with anionic derivatives, inhibition occurs at a concentration which does not induce any apparent shape change or protect against osmotic hemolysis: there is no significant membrane expansion. (5) Glucose permeability, measured by glucose exit, is inhibited by cationic and anionic phenothiazine, but always at a concentration which fully expands the membrane as indicated by morphological changes and anti-hemolytic effects. It is interesting to point out that whilst glucose exit shows inhibition by cationic derivatives, glucose exchange flux is scarcely altered. (6) It is concluded that cationic and anionic anesthetics are general inhibitors of transmembrane solute movements involving a facilitated-diffusion process. However, the mechanism of inhibition is not identical for all: inhibition of glucose permeability by anionic and cationic anesthetics, as well as inhibition of Cl- permeability by cationic anesthetics may be of a non-specific nature and result from their interaction with the bilayer (this indirect effect is discussed); on the other hand, inhibition of Cl- permeability by anionic anesthetics may result from a specific perturbation of the transport mechanism according to recent evidence in some cases (Cousin, J.L. and Motais, R. (1979) J. Membrane Biol. 46, 125-153; Zaki, L., Ruffing, W. G?rtner, E.M., Fasold, H., Motais, R. and Passow, H. (1977) 11th FEBS Meeting, Copenhagen, A4 17-671.     

Chloride fluxes in lily pollen tubes: a critical reevaluation

Microelectrodes, made from a Cl(-)-selective liquid ion exchanger previously used to measure putative Cl- fluxes in Lilium longiflorum pollen tubes, were characterized. The electrodes were poorly selective, possessing only about 10-fold selectivity for Cl- over other anions tested. They had only 2.4-fold selectivity for Cl- over the anionic form of the H+ buffer, MES, indicating that the electrode can indirectly detect H+ gradients. Apparent anion influx was detected along the pollen tube shafts and at the grains while apparent anion efflux was detected near the tip of the tube. During oscillating growth, the peak of the oscillating apparent anion efflux at the tip occurred, on average, 7.9 sec after the peak of the growth oscillations. Consideration of the previously characterized H+ fluxes in lily pollen grains and tubes, as well as the poor anion selectivity of the Cl- electrodes, indicates that the putative Cl- fluxes are in fact changes in the anionic concentration of the buffer resulting from H+ gradients and not changes in Cl- concentration. The claim of a central role for Cl- in lily pollen tube growth is further undermined by the fact that these tubes grow at the same rate if the Cl- content of the growth medium is reduced to trace levels (< or =31 microM), and that the grains have only small reserves of Cl-. These results lead to the conclusion that Cl- fluxes are not a significant component of pollen tube growth and Cl- itself is not required for growth.     

Carrier ampholytes as potential buffers in electrophoresis: physico-chemical study

Joule heating is a limiting factor when separating proteins in capillary zone electrophoresis (CZE). Low conductivity buffers, are required for high-speed separations. We investigated the use of carrier ampholytes (CA) as background electrolytes (BGE) in CZE. We prepared 25 "narrow pH cuts" of wide pH range (3-10) CA mixture in order to know if these fractions were suitable to be used as BGE in CZE. Each fraction was characterised by CZE analysis, giving an idea of its heterogeneity (number and relative abundance of molecular ampholytes). Conductivities and buffering capacities of each fraction have been also measured. Our conclusion is that "narrow pH cuts" of CA might be well suited buffers for electrophoretic separations.     

Use of novel cationic bile salts in cholesterol crystallization and solubilization in vitro

Unnatural bile salts have been synthesized with a cationic group at the side chain of natural bile acids. These cationic bile salts aggregate in water and aqueous salt solutions in a manner similar to their natural counterparts. The critical micellar concentrations of the cationic bile salts were measured using a fluorescence method. Cationic bile salts aggregated at a concentration lower than natural deoxycholic acid. Since dihydroxy bile salt micelles are well known for cholesterol dissolution/removal, the dissolution in the cationic micelles has been evaluated. The cationic analogs dissolve approximately 70 mg/dL of cholesterol, which is comparable to taurochenodeoxycholate micelle under identical bile salt concentrations. Cholesterol dissolution in cationic bile salt micelle enhanced upon adding various amounts of PC. Cholesterol crystallization was studied in model bile at various cationic bile salt concentrations. The addition of 5, 15 and 30 mM of the cationic bile salts attenuated the crystallization process, without influencing the crystal observation time or decreasing the final amount of crystals formed. All these effects were comparable to those observed with cholic acid. These findings suggest that cationic bile salts have physico-chemical properties analogous to those of natural anionic bile salts, and thus may have therapeutic potential.     

Effect of homologous series of n-alkyl sulfates and n-alkyl trimethylammonium bromides on low molecular mass protein tyrosine phosphatase activity

The effect of anionic and cationic surfactants on acid phosphatase denaturation has been extensively studied. Low molecular mass (LMr) protein tyrosine phosphatase (PTP), a key regulatory enzyme involved in many different processes in the cell, was distinctly affected by anionic (homologous series of n-alkyl sulfates (C8-C14)) and cationic (n-alkyl trimethylammonium bromides (C12-C16)) surfactants. At concentrations 10-fold lower critical micellar concentration (cmc) values, the enzyme was completely inactivated in the presence of anionic surfactants, in a process independent of the pH, and dependent on the chain length of the surfactants. Under the same conditions, the effect of cationic surfactants on the enzyme activity was pH-dependent and only at pH 7.0 full inactivation was observed at concentrations 10-fold higher cmc values. In contrast to cationic surfactants the effect of anionic surfactants on the enzyme activity was irreversible and was not affected by the presence of NaCl. Inorganic phosphate, a known competitive inhibitor of PTP, protected the enzyme against inactivation by the surfactants. Our results suggest that the inactivation of the LMr PTP by anionic and cationic surfactants involved both electrostatic and hydrophobic interactions, and that the interactions enzyme-surfactants probably occurred at or near the active site.     

An accumulation of insulin-like growth factor I (IGF-I) in human myometrium and uterine leiomyomas in various stages of tumour growth

It is commonly thought that uterine leiomyomas result from hyperstimulation of myometrium by ovarian hormones. Some observations suggest that cytokines and growth factors are intermediate elements through which the ovarian hormones may exert their growth-stimulatory effects on leiomyomas. Human myometrium and uterine leiomyomas of various weights were homogenised and extracted with 1 M acetic acid or with 0.05 M Tris/HCl, pH 7.6. The extracts were assayed for IGF-I using the ELISA technique. It was found that 0.05 M Tris/HCl extracts contained several times more IGF-I than the 1 M acetic acid extracts. Nanogram amounts of IGF-I were found in both control myometrium and in leiomyomas. It was found that the amounts of IGF-I extracted from leiomyomas were distinctly higher in comparison to control myometrium and they increased as a function of tumour growth. Polyacrylamide gel electrophoresis, followed by Western immunoblotting, demonstrated that IGF-I in acidic and alkaline extracts exists as stable complexes, probably with extracellular matrix components. No free IGF-I was detected. Furthermore, it was found that some components of both the acidic and alkaline extracts were able to bind exogenous (125)I-labeled IGF-I. It is suggested that IGF-I plays an important role both in myometrium biology and in the growth of uterine leiomyomas.     

Retention of bile salts in micellar electrokinetic chromatography: relation of capacity factor to octanol–water partition coefficient and critical micellar concentration

The capacity factors of 16 anionic cholates (from six bile salts, including their glyco- and tauro-conjugates) were determined in a micellar electrokinetic chromatography (MEKC) system consisting of buffer, pH 7.5 (phosphate–boric acid; 20 mmol/l) with 50 mmol/l sodium dodecyl sulfate (SDS) as micelle former and 10% acetonitrile as organic modifier. The capacity factors of the fully dissociated, negatively charged analytes (ranging between 0.2 and 60) were calculated from their mobilities, with a reference background electrolyte (BGE) without SDS representing “free” solution. For comparison, the capacity factors were derived for a second reference BGE where the SDS concentration (5 mmol/l) is close to the critical micellar concentration (CMC). The capacity factors are compared with the logarithm of the octanol–water partition coefficient, log P_OW, as measure for lipophilicity. Clear disagreement between these two parameters is found especially for epimeric cholates with the hydroxy group in position 7. In contrast, fair relation between the capacity factor of the analytes and their CMC is observed both depending strongly on the orientation of the OH groups, and tauro-conjugation as well. In this respect the retention behaviour of the bile salts in MEKC seems to reflect their role as detergents in living systems, and might serve as model parameter beyond lipophilicity.     

Determination of microcystins in environmental samples using capillary electrophoresis

The applicability of capillary zone electrophoresis (CZE) and micellar electrokinetic chromatography (MEKC) methods for the simultaneous determination of two frequently occurring microcystins (MCs-LR and -YR) and a new variant (MC-YA) in crude extracts of Hungarian bloom samples and cyanobacterial cultures was studied. It was found that the comparison of the results obtained by both CZE and MEKC measurements (due to the differences in their separation mechanisms) for the same sample can guarantee the reliability of the quantitative results. In our work environmental samples like lake waters, water bloom samples, cyanobacterial isolates were analysed. The three microcystins could be directly determined in water bloom samples collected from Hungarian lakes and laboratory culture samples of cyanobacteria.     

Transport of Cl– across the Plasma Membrane during Pollen Grain Germination in Tobacco

The role of Cl- transport across the plasma membrane was studied in an early step of pollen grain germination in tobacco Nicotiana tabacum L. The Cl- channel blockers, 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) and niflumic acid, completely suppress the germination with IC(50) approximately 8 micro M. At this concentration NPPB reduces the rate of Cl- efflux out of pollen grain by 1.8-fold in the interval 5-12 min, and niflumic acid reduces the rate 1.2-fold. 4,4;-Diisothiocyanatostilbene-2,2;-disulfonic acid, a known inhibitor of Cl- channels and antiporters, completely suppresses germination as well (IC(50) = 240 micro M), but has no effect on the rate of Cl- efflux. Inhibitors of chloride co-transporters, such as furosemide, bumetanide, and bis(1,3-dibutylbarbituric acid)pentamethine oxonol, suppress the germination by less than 50%. This set of data suggests that NPPB-sensitive anion channels are involved in the activation of pollen grains in the early stage of germination.     

Mechanism of protein salting in and salting out by divalent cation salts: balance between hydration and salt binding

The preferential interactions of proteins with solvent components were studied in concentrated aqueous solutions of the sulfate, acetate, and chloride salts of Mg2+, Ba2+, Ca2+, Mn2+ and Ni2+ [except for CaSO4, BaSO4, Mn-(OAc)2, and Ni(OAc)2], and results were compared with those of the Na+ salts. It was found that, for all the salts, the preferential hydration increased in the order of Cl- less than CH3-COO- less than SO42- regardless of the cationic species used, in agreement with the anionic lyotropic series, and that the same parameter exhibited a tendency to increase in the order of Mn2+, Ni2+ less than Ca2+, Ba2+ less than Mg2+ less than Na+. The salting-out and stabilizing or salting-in and destabilizing effectiveness of the salts were interpreted in terms of the observed preferential interactions. The surface tension increment of salts, which is a major factor responsible for the preferential interactions of the Na+ salts, had no correlation with those of the divalent cation salts. It was shown that the binding of divalent cations to the proteins overcomes the salt exclusion due to the surface tension increase, leading to a decrease in the preferential hydration. In conformity with this mechanism, the preferential interaction of MgCl2 was strongly pH dependent, because of the protein charge-dependent affinity of Mg2+ for proteins, while NaCl showed no pH dependence of the preferential interaction. The proposed mechanism was supported by a strong correlation between the preferential interaction results and the interaction of these salts with the model peptide compound acetyltetraglycine ethyl ester, described by Robinson and Jencks.     

Influence of Surfactant Type and Concentration on Electrospinning of Chitosan–Poly(Ethylene Oxide) Blend Nanofibers

Electrospun blend nanofibers were fabricated from chitosan (1,000 kDa, 80% DDA) and poly(ethylene oxide) (PEO; 900 kDa) at a ratio of 3:1 dispersed in 50% and 90% acetic acid. The influence of surfactants on the production of electrospun nanofibers was investigated by adding nonionic polyoxyethylene glycol dodecyl ether (Brij 35), anionic sodium dodecyl sulfate, or cationic dodecyl trimethyl ammonium bromide below, at, and above their specific critical micellar concentration to the polymer blend solution. Viscosity, conductivity, and surface tension of polymer solutions, as well as morphology and composition, of nanofibers containing surfactants were determined. Pure chitosan did not form fibers and was instead deposited as beads. Addition of PEO and an increasing concentration of surfactants induced spinnability and yielded larger fibers with diameters ranging from 10 to 240 nm. Surfactants affected morphology yielding needle-like, smooth, or beaded fibers. Compositional analysis revealed that nanofibers consisted of both polymers and surfactants with concentration of the constituents in nanofibers differing from that in polymer solutions. Results suggest that surfactants may modulate polymer–polymer interactions thus influencing the morphology and composition of deposited nanostructures.     

The Occurrence of <Emphasis Type="Italic">p</Emphasis>-coumaric Acid and Ferulic Acid in Fossil Plant Materials and their Use as UV-proxy

The applicability of p-coumaric acid and ferulic acid concentrations or ratios in (sub)fossil plant remnant as UV-B proxies relies on various aspects, which are discussed in this paper and will be illustrated with some experimental data. A newly developed THM-micropyrolysis–gas chromatography–mass spectrometry method was tested on various spores, pollen and other plant remains, which were analysed for the presence of the UV-absorbing compounds p-coumaric acid and ferulic acid. This revealed that these supposed building-blocks of sporopollenin appear to be present in pollen of many plant species but also in moss spores. The development of this micropyrolysis method paved the way for the quantitative analysis of UV-absorbing compounds in case only a small amount of analyte is available, for example for fossil pollen and spores but also other small palynomorphs and plant fossils. The use of this technique will provide a better insight in the plant responses to UV-radiation, the chemistry of pollen and spores, their fossil counterparts and furthermore the means for a further development of a proxy for the reconstruction of past UV-B radiation.     

Synthesis of chitosan-based polymeric surfactants and their adsorption properties for heavy metals and fatty acids

Chitosan-based polymeric surfactants (CBPSs) were prepared by N-acylation of chitosans (chitosan 10 and 500) with several acid anhydrides such as hexanoic (C6), lauric (C12), and palmitic (C16) anhydrides. Among the CBPS samples, CBPSs having a good solubility at pH 4.0 were selected and observed for viscosity, surface tension, and adsorption of heavy metals (Cd2+, Co2+, Cr2O7(2-), and Pb2+) as well as the fatty acid (n-octanoic acid). The 1H NMR spectrum of chitosan 10 modified with C16 at the substitution ratio of 0.4 (CBPS10-C16,0.4) showed 85% of acylation in 1% DCl/D2O solutions. CBPS10 with the substitution ratio less than 0.4 showed a good solubility because of shorter repeating units and lesser amounts of hydrophobic substituents. The intrinsic viscosity of CBPS10 was slightly increased, while that of CBPS500 was decreased. As the substitution ratio and length of the carbon chain increased, the surface tension of CBPS10 tended to decrease. CBPS10-C16,0.2 had high adsorption ability for cationic metal ions such as Cd2+, Co2+, and Pb2+ comparable to chitosan. Interestingly, CBPS(10)-C(16,0.2) showed a unique pH optimum for the anionic metal ion such as Cr2O7(2-). In addition, CBPS10-C16,0.2 exhibited the highest adsorption ability for n-octanoic acid among the tested CBPS10 with different carbon chains.     

Determination of microcystins in environmental samples using capillary electrophoresis

The applicability of capillary zone electrophoreses (CZE) and micellar electrokinetic chromatography (MEKC) methods for the simultaneous determination of two frequently occurring microcystins (MCs-LR and -YR) and a new variant (MC-YA) in crude extracts of Hungarian bloom samples and cyanobacterial cultures was studied. It was found that the comparison of the results obtained by both CZE and MEKC measurements (due to the differences in their separation mechanisms) for the same sample can guarantee the reliability of the quantitative results. In our work environmental samples like lake waters, water bloom samples, cyanobacterial isolates were analysed. The three microcystins could be directly determined in water bloom samples collected from Hungarian lakes and laboratory culture samples of cyanobacteria.     

Oxidation of tyrosine by peroxidase isozymes derived from peanut suspension culture medium and by isolated cell walls

A comparative study on tyrosine oxidation was made with a pure cationic and anionic peroxidase from peanut cell culture medium. The results showed that both isozymes possessed almost identical capacity to oxidize tyrosine to dityrosine, isodityrosine and polytyrosine with the main difference being the pH optimum (pH 4 for the anionic and pH 7 for the cationic isozyme). Variation of reaction time after 1.5 h incubation had little effect on the quantity and quality of the oxidation products. On the other hand, increase of enzyme units correspondingly increased tyrosine-oxidation. The removal of heme and carbohydrate moieties from the holoenzyme arrested the reaction thereby suggesting the role played by these moieties in stabilizing the active site of peroxidase isoenzymes. Isolated cell wall extracts catalyzed the tyrosine-oxidation equally well as the purified peroxidase. Even though polyclonal antibodies against anionic peroxidase inhibited the in vitro tyrosine reaction they did not affect the tyrosine oxidation by the cell walls, while the cationic antibodies did.Abbreviations A.PRX anionic peanut peroxidase - C.PRX cationic peanut peroxidase - PcAb polyclonal antibodies - ELISA enzyme-linked-immuno-sorbent-assay - TFMS trifluoromethane sulfonic acid