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F Kilár

Publications and source records attributed to F Kilár.

At least 19 recordsLinked to original sources
PubMed1998

New set-up for capillary isoelectric focusing in uncoated capillaries.

Substituted aminomethylphenol dyes, low-molecular-mass isoelectric point (pI) markers and hemoglobin samples from normal individuals and diabetic patients were used to test a new set-up of capillary isoelectric focusing (cIEF) in uncoated capillaries. In previous cIEF methods, a mixture of sample components and carrier ampholytes was applied in the capillary and analyzed. In the new set-up a fractionated injection protocol is used to apply a 'sandwich' ampholyte-sample-ampholyte plug in the capillary for analysis. This new set-up allows the separation of amphoteric compounds having pI values outside the pH region of the ampholytes applied in the capillary with high precision. The high resolution power of this technique was proven with the analysis of hemoglobin variants.

Diabetes Mellitus↗
PubMed1998

Stereoselective interaction of drug enantiomers with human serum transferrin in capillary zone electrophoresis (II).

Further studies on chiral resolution of drugs with different chemical structures by capillary zone electrophoresis using iron-free human serum transferrin are described. The substances passed a highly concentrated pseudo-stationary protein zone applied in a coated capillary and the possible chiral separation of the optical isomers was followed. Eighteen drugs with different structures were screened, and the enantiomers of clofedanol, buphenine, acebutolol and chlorphenamine were resolved. Several, but not all drugs, showed longer migration times while passing the protein zone, indicating an interaction with transferrin, although chiral resolution was not observed in all cases. The observations provided further information about the properties of the surface interaction sites of transferrin.

Electrophoresis, Capillary↗
PubMed1998

Chiral separation of alpha-amino acids by ligand-exchange capillary electrophoresis using N-(2-hydroxy-octyl)-L-4-hydroxyproline as a selector.

The direct chiral resolution of underivatized alpha-amino acids by capillary zone electrophoresis (CZE) based on the principle of ligand exchange is described. An N-(2-hydroxyoctyl)-L-4-hydroxyproline/Cu(II) complex was used as a chiral selector. Besides amino acids containing aromatic residues, the basic amino acid histidine was resolved. Baseline separations were obtained for all amino acids investigated. The influence of selector concentration, electrolyte composition and pH on the resolution was investigated. It was found that there is a correlation between pI of the amino acids and the optimal pH.

Amino Acids↗
PubMed1998

Protein profile characterization of bacterial lysates by capillary electrophoresis.

A fast and reproducible method was developed to characterize cell lysates by their electrophoretic profiles using capillary electrophoresis (CE). Characteristic and reproducible patterns were recorded for each bacterial strains when "dynamic sieving" CE, using a polymer solution in the capillary, was applied to distinguish four strains of the Enterobacteriaceae family. The electropherograms showed distinct differences when comparing them to the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) protein profiles. This is certainly a result of the differences in the separation principles and in the detection methods of the two techniques.

Bacterial Proteins↗
PubMed1998

Changes in outer membrane protein profiles of bacteria after meropenem-induced postantibiotic effect studied by capillary electrophoresis.

Persistent inhibition of bacterial growth, called postantibiotic effect (PAE), after a short exposure to a new carbapenem, meropenem, was determined in different strains of the Enterobacteriaceae family. Capillary electrophoresis (CE), as well as sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) were used to study the outer membrane protein (OMP) profiles before and after meropenem treatment. CE proved to be suitable for the characterization of the OMP profiles of bacteria. Significant changes in the electrophoretic patterns were observed, showing the consequential effect of meropenem on bacteria.

Bacterial Outer Membrane Proteins↗
PubMed1996

Stereoselective interaction of drug enantiomers with human serum transferrin in capillary zone electrophoresis.

Stereoselective interaction of drugs with human serum transferrin in capillary zone electrophoresis is described. The substances passed a pseudo-stationary protein zone applied in a coated capillary and the possible chiral separation of the optical isomers was followed. Drugs with different structures were screened and the enantiomers of bupivacaine, propranolol and promethazine as well as the diastereomers of labetalol were resolved. Racemic mixtures of atenolol and pindolol enantiomers could not be resolved under these conditions.

Bupivacaine↗
PubMed1995

Separation of tryptophan-derivative enantiomers with iron-free human serum transferrin by capillary zone electrophoresis.

Enantiomers can be separated by using human serum transferrin as a chiral phase. With the help of the native protein we were able to separate enantiomers with high efficiency, using a low ionic strength 2-(N-morpholino)ethanesulfonic acid (MES) buffer, pH 6, in capillary zone electrophoresis. Tryptophan methyl, ethyl and butyl ester enantiomers-moving towards the cathode at pH 6-were resolved by passing through an iron-free transferrin zone in coated capillaries. Since the isoelectric point of the iron-free transferrin is a little higher than 6, the protein zone is either not moving in the experiment or is slowly moving towards the anode. Under the simplest experimental conditions the highest resolution was obtained for the butyl ester enantiomers and the lowest for the methyl ester ones. By changing the experimental conditions, however, this order could be reversed. The results indicate that the lengths of the alkyl chains in the enantiomers have a significant effect on the resolution, i.e., on the interaction between the protein and the separands.

Alkanesulfonic Acids↗
PubMed1994

Complementary Mössbauer and EPR studies of iron(III) in diferric human serum transferrin with oxalate or bicarbonate as synergistic anions.

The spectral and magnetic properties of iron(III) bound in the metal binding sites of human serum transferrin with oxalate or bicarbonate as synergistic anions has been studied with Mössbauer spectroscopy and electron paramagnetic resonance (EPR). The Mössbauer spectra of the iron(III) in diferric transferrin with oxalate have been described using a spin Hamiltonian with the values of the zero field splitting parameter, D = 0.55 +/- 0.05 cm-1, and the rhombicity of the crystal field, E/D = 0.045 +/- 0.005. The EPR spectrum can be described with D = 0.58 cm-1 and E/D = 0.057, using a g-strain model for the lineshape that is based on a Gaussian distribution of D and E/D with Gaussian widths sigma(D) = 0.35 cm-1 and sigma(E/D) = 0.013, respectively. The rhombicity of the iron surroundings for the transferrin-oxalate complex is almost one order of magnitude smaller than for the bicarbonate complex and the zero field splitting parameter is twice as large in the oxalate as in the bicarbonate complex. We conclude that the crystal field symmetry of the iron site is almost tetragonal in the oxalate complex but rhombohedral in the bicarbonate complex, reflecting the different geometries of the oxalate and bicarbonate coordination. The isomer shift delta = 0.56 +/- 0.01 mm s-1 and the quadrupole splitting delta EQ = 0.2 +/- 0.1 mm s-1, on the other hand, are very close to the values found for the bicarbonate complex. No differences between the Mössbauer spectra of the two iron(III) ions in diferric transferrin with oxalate were found. The homogeneity of the diferric transferrin samples was controlled by capillary zone electrophoresis in the presence of urea.

Bicarbonates↗
PubMed1993

Unfolding of human serum transferrin in urea studied by high-performance capillary electrophoresis.

High-performance capillary electrophoresis (HPCE) was used to monitor the progress of the unfolding of human serum transferrin in urea. Denaturation curves of the transferrin forms were constructed plotting the migration times corrected for the viscosity vs. the concentration of urea in the buffer. The practical advantage of capillary zone electrophoresis is the short analysis time, 5-15 min, as compared with slab-gel experiments, which require overnight runs for similar purposes. The resolution increased with the urea concentration, and hence high concentrations are beneficial for quantitative and qualitative analysis of mixtures of transferrin forms. Unfolding intermediates of the isoforms, which interconvert to the unfolded state slowly compared with the time scale of the electrophoretic separation, and also the completely unfolded isoforms were resolved and detected simultaneously when iron-free transferrin was subjected to denaturation by urea at concentrations between 3 and 6 M. However, no unfolding intermediates were observed with transferrin isoforms containing two iron atoms (i.e. diferric transferrin molecules), which accordingly are strongly resistant to urea denaturation. The unfolding of the transferrin isoforms depends on the iron content of the complexes, but not the carbohydrate content. HPCE in the presence of urea in this mode has the potential to become an analytical tool for diagnosis of diseases in which the transferrin patterns change.

Chromatography, High Pressure Liquid↗
PubMed1991

In situ hybridization histochemistry of mRNAs for hormones and chromogranins in normal pituitary tissue and pituitary adenoma.

In situ hybridization histochemistry was employed to detect mRNAs of pituitary hormones and chromogranins in normal pituitary gland and pituitary adenomas. Oligonucleotide probes specific to the mRNAs for prolactin, growth hormone, proopiomelanocortin, the alpha- and beta-subunits of the glycoprotein hormones and chromogranins A and B were used in the hybridization experiments. The oligonucleotides of 27 to 51 bases were labelled radioactively with dATP[alpha-35S] at the 3'-end using terminal deoxynucleotidyl transferase. Positive hybridization reactions were visualized by autoradiography in the normal pituitary gland with all of the probes. The clinically diagnosed pituitary adenomas (prolactinoma, acromegaly, Cushing's disease, FSH-secreting tumour) showed positive hybridization with the corresponding oligonucleotide probes. In some cases positive hybridization was also obtained with other probes, suggesting multihormone-producing character of the tumour cells. A microprolactinoma was found in a pituitary gland obtained from a patient without any known pituitary disorders. Examination of mRNAs for chromogranin A and B revealed that the normal pituitary gland contains a larger number of cells expressing chromogranin B and a lower number expressing chromogranin A and, moreover, the microprolactinoma lacked the expression of mRNA for chromogranin A but expressed that of chromogranin B.

Adenoma↗
PubMed1989

Different segmental flexibility of human serum transferrin and lactoferrin.

X-ray diffraction studies show that the diferric (holo) forms of human serum transferrin and lactoferrin have almost the same conformation in crystal. In solution, however, the two proteins exhibit different characteristics. The differences are even more pronounced in the apo forms. Small-angle X-ray and neutron scattering data show that lactoferrin is less compact, in apo and holo forms, than the corresponding forms of transferrin in solution. The comparison of primary structures of the two proteins suggests that one of the interdomain hinge regions is significantly longer in lactoferrin than its counterpart in transferrin. The difference in flexibility due to the long hinge region in lactoferrin may be responsible for many of the differences in the physicochemical characteristics of the two proteins.

Humans↗
PubMed1989

Zero-field Mössbauer studies of diferric human transferrin.

Diferric transferrin samples labelled with 57Fe at the N- or the C-terminal binding sites are compared by Mössbauer spectroscopy at 15 K and in zero magnetic field. The spectra of the samples are similar but the fitting of single Lorenzian lines to the data shows that some of the line positions differ in the two cases. According to this we can not exclude a difference between the chemical structures of the binding sites that can arise for example from the participation of different forms of the anion and/or water in the two lobes of transferrin. All other line parameters (line-width, intensity) are the same within the limits of errors.

Binding Sites↗
PubMed1989

Fast and high resolution analysis of human serum transferrin by high performance isoelectric focusing in capillaries.

Human serum transferrin is a mixture of isoforms (isoproteins) having different amounts of carbohydrates. Each isoform may exist in iron-free and iron-complexed molecular form. The genetic variations in different populations increase the number of combinations of the different forms of transferrin. To resolve the many components in transferrin preparations, the new high performance capillary technique was employed for isoelectric focusing. Iron-free transferrin and transferrin samples of known iron content were examined. The above method gives an exceptionally rapid analysis (within 15-25 min) of small amounts of samples (less than 1 microgram protein) and as good as or better resolution than other isoelectric focusing techniques previously used for transferrin analysis. By monitoring the focused protein zones at both 280 and 460 nm the molecular forms of transferrin (iron-free, monoferric and differic complexes) can easily be identified. Both steps of isoelectric focusing in capillaries (i.e., prefocusing and mobilization) can be used for analysis. We observed that chelating agents (e.g., carrier ampholytes, nitrilotriacetate) may release iron from microsyringes having metal pistons causing the formation of iron-transferrin complexes.

Capillary Action↗
PubMed1989

Surface hydrophobicity and electrophoretic mobilities of staphylococcal exotoxins with special reference to toxic shock syndrome toxin-1.

The surface hydrophobicities of eleven staphylococcal toxins were estimated and compared with those of standard proteins on an octyl agarose column by high-performance hydrophobic-interaction chromatography (HP-HIC). Staphylococcal enterotoxins (SE) D, C3, C2, C1 and B showed a low surface hydrophobicity whereas alpha-toxin and gamma-toxin had a moderate surface hydrophobicity. SEA, toxic shock syndrome toxin-1 (TSST-1) and staphylococcal epidermolytic toxin (SET) showed high surface hydrophobicity and delta-toxin was the most hydrophobic protein. The electrophoretic mobility of the toxins was determined by free zone electrophoresis (FZE). All toxins except SEC1 and one of the two SEA species showed negative charge at pH 8.6. Charge heterogeneity was observed in SEA, SEC1, SEC3 and TSST-1: SEA and SEC1 had two overlapping components, whereas SEC3 and TSST-1 were resolved into two distinct components. The mobilities of the two TSST-1 components were estimated at -2.12 x 10(-5) and -3.60 x 10(-5) cm2v-1s-1, respectively, at 10 degrees C, and both fractions were immunologically indistinguishable as tested by specific TSST-1 antibodies with ELISA. An asymmetric peak was obtained in hydrophobic-interaction chromatography of TSST-1 indicating heterogeneity.

Animals↗
PubMed1987

Carrier-free zone electrophoresis, displacement electrophoresis and isoelectric focusing in a high-performance electrophoresis apparatus.

A characteristic feature of high-performance electrophoresis (HPE), the electrophoretic counterpart of high-performance liquid chromatography (HPLC), is that the separation chamber is a thin-walled, narrow-bore (0.05-0.3 mm) glass or fused-silica capillary tube for rapid dissipation of the Joule heat in order to minimize thermal zone deformation even at high field strengths. This paper is centered around the usefulness of HPE for separation in a carrier-free medium (i.e., in buffer alone) and deals with both zone electrophoresis, isoelectric focusing and displacement electrophoresis. Examples are given of analytical and micropreparative separations of inorganic and organic ions, proteins, viruses and bacteria. The run times are 5-30 min. Discontinuous buffer systems have up to now been used exclusively for the separation of proteins by electrophoresis in polyacrylamide gels ("disc electrophoresis"). However, the Ornstein and Davis discontinuous buffer system has been modified to adapt it to carrier-free zone electrophoresis in order to achieve automatic sharpening of the starting zone. Very high resolution of serum proteins was obtained when they were subjected to free high-performance disc electrophoresis in such a modified buffer system. To show that the HPE apparatus permits electrophoresis also in a gel medium, a polyacrylamide electrophoresis in SDS is presented. This experiment illustrates the difference between electropherograms obtained in free solution and in a molecular-sieving medium. Detection can be performed both on- and off-tube. The latter technique permits the rapid identification of the solutes by photodiode array spectrophotometry and the collection of fractions for further studies. The former detection method is simpler but mainly useful for analytical purposes. Non-UV-absorbing ions can be monitored with the aid of an on-tube UV detector if the run is performed in a UV-absorbing buffer.

Blood Proteins↗
PubMed1987

Application of high-performance chromatographic and electrophoretic methods to the purification and characterization of glucose oxidase and catalase from Penicillium chrysogenum.

The high resolving power of the preparative and analytical high-performance chromatographic and electrophoretic methods recently developed in this laboratory for the separation of biopolymers has been demonstrated by the purification and characterization of glucose oxidase and catalase from Penicillium chrysogenum. Crude glucose oxidase was purified to homogeneity in one step by high-performance hydrophobic-interaction chromatography (HIC) on a pentylagarose column. Crude catalase was purified by a combination of HIC and high-performance anion-exchange chromatography on 3-diethylamino-2-hydroxypropylagarose. The homogeneity of the enzymes was monitored by high-performance electrophoresis and free zone electrophoresis. The pI values of these two enzymes determined by isoelectric focusing in the high-performance electrophoresis apparatus were 4.2 and 6.5, respectively. Their molecular weights were determined by high-performance molecular sieve chromatography on an agarose column. Glucose oxidase has a molecular weight of 175,000 and probably consists of two identical subunits, as sodium dodecyl sulphate polyacrylamide gel electrophoresis gave a molecular weight of around 72,000. The molecular weight of catalase, which is probably composed of non-identical subunits, as indicated by sodium dodecyl sulphate electrophoresis, is around 320,000. Some other characteristics of these two enzymes were also investigated, e.g., electrophoretic mobility, pH stability and optimum pH.

Amino Acids↗
PubMed1987

Quantitative analysis of the interaction between immune complex and C1q complement subcomponent. The role of interdomain interactions in rabbit IgG in binding of C1q to immune precipitates.

A novel method was developed for the analysis of the interaction of large multivalent ligands with surfaces (matrices) to analyse the binding of complement subcomponent C1q to immune precipitates. Our new evaluation method provides quantitative data characteristic of the C1q-immune-complex interaction and of the structure of the immune complex as well. To reveal the functional role of domain-domain interactions in the Fc part of IgG the binding of C1q to different anti-ovalbumin IgG-ovalbumin immune complexes was studied. Immune-complex precipitates composed of rabbit IgG in which the non-covalent or covalent bonds between the heavy chains had been eliminated were used. Non-covalent bonds were abolished by splitting off the CH3 domains, i.e. by using Facb fragments, and the covalent contact was broken by reduction and alkylation of the single inter-heavy-chain disulphide bond. The quantitative analysis of the binding curves provides a dissociation constant (K) of 200 nM for the interaction between C1q and immune precipitate formed from native IgG. Surprisingly, for immune precipitates composed of Facb fragments or IgG in which the inter-heavy-chain disulphide bond had been selectively reduced and alkylated, stronger binding (K = 30 nM) was observed. In this case, however, changes in the structure of the immune-complex matrix were also detected. These structural changes may account for the strengthening of the C1q-immune-complex interaction, which can be strongly influenced by the flexibility and the binding-site pattern of the immune-complex precipitates. These results suggest that domain-domain interactions in the Fc part of IgG affect the segmental mobility of IgG molecules and the spatial arrangement of the immune-complex matrix rather than the affinity of individual C1q-binding sites on IgG.

Animals↗