Biomedical subjects

M Filipiak

Publications and source records attributed to M Filipiak.

5 recordsLinked to original sources

PubMed2006

Electrochemical response of oligonucleotides on carbon paste electrode.

Electrochemical response of synthetic oligonucleotides with different DNA bases sequences was investigated to find relationships between a chain composition and a signal. All DNA mononucleotides present electroactivity at a carbon paste electrode yielding anodic peaks at potentials: 1.00 (GMP), 1.28 (AMP), 1.47 (TMP) and 1.53 V (CMP). Also 15-mer homooligonucleotides show respective anodic peaks. Electrochemical response of 15- and 19-mer oligonucleotides consisting of all four DNA bases in different amounts was determined by the composition of oligonucleotide chain. When the contribution of different bases in oligonucleotide was balanced two anodic peaks were obtained that can be attributed to guanine and adenine moieties. Thymine residue is shown as a separate peak in voltammogram when its content in oligonucleotide chain is close to 50% of the total number of bases. Cytosine also yields a peak at its significant contribution in oligonucleotide chain and both pyrimidinic moieties produce catalytic waves easier when one of them is dominating or when only one pyrimidine derivative is present in a chain. Guanine is the easiest oxidized base and it produces a peak even at its minimal contribution (one guanine residue in 19-mer oligonucleotide). Guanine peak potential is dependent on oligonucleotide concentration and oligonucleotide composition. The lowest oligonucleotide concentration detected by guanine peak was 12.5 nM whereas detected by thymine peak was 90 nM.

Base Sequence↗

PubMed2004

DNA sensor for o-dianisidine.

o-Dianisidine (3,3'-dimethoxybenzidine) is applied in the production of some dyes and also used in analytical tests. However, this compound is anticipated to be a human carcinogen. An analytical strategy utilizing square wave voltammetry for the determination of o-dianisidine is presented. An electrochemical system was consisted of three electrodes: carbon paste working electrode, platinum wire counter electrode and silver-silver chloride (Ag/AgCl) reference electrode. However, square wave voltammograms of direct measurements of o-dianisidine were found to be hardly reproducible, exhibiting few peaks due to some labile short-lived intermediates with the only exception of a quite stable peak at +0.7 V vs. Ag/AgCl. Quantitative determination of o-dianisidine gave satisfactory results only when the carbon paste working electrode was replaced by deoxyribonucleic acids (DNA) electrode obtained by immobilization of double-stranded (ds) DNA on carbon electrode. Square wave voltammogram of DNA showed two peaks attributed to adenine and guanine and the latter was used as analytical signal. After interaction with o-dianisidine, guanine oxidation peak was reduced to the extent related to the concentration of the analyte. Initial reduction of guanine peak took place already at the concentration of o-dianisidine equal to 0.4 microM; high concentrations (above 100 microM) of the analyte quenched completely a guanine response. The presented electrochemical system enables a specific detection of o-dianisidine by the presence of an oxidation peak at +0.7 V and its quantitative determination by measuring a reduction of guanine peak by means of a DNA sensor.

Adenine↗

PubMed1997

Direct electrochemistry of Megasphaera elsdenii iron hydrogenase. Definition of the enzyme's catalytic operating potential and quantitation of the catalytic behaviour over a continuous potential range.

The Fe-hydrogenase from Megasphaera elsdenii undergoes direct electron exchange with glassy carbon electrodes. Cyclic voltammetry defines the catalytic-performance of the enzyme over a continuous but precisely defined range of potentials. In the presence of H2 and protons the bias of the enzyme towards H2 production is readily visualised. Variation of the response with pH indicates that protein ionisations with pK of approximately 6.7 and 8.3 regulate the catalytic activity. Possible origins for these observations in the chemistry of the H2-activating site are discussed. The mid-wave potential of the catalytic response, Emid, is defined as the catalytic operating potential of the enzyme. Under an atmosphere of hydrogen Emid = -421 +/- 10 mV, pH 7 with a variation of -21 +/- 4 mV pH-1, 22 degrees C. Deviation of Emid from the thermodynamic potential of the hydrogen/proton couple reflects the enzyme's influence over the catalysed reaction. Emid is the reduction potential of the H2-activating centre (H-cluster) in the absence of kinetic bottle-necks at other steps in the reaction mechanism.

Air↗

PubMed1996

Enzymatic membranes for determination of some disaccharides by means of an oxygen electrode.

Membranes for determination of maltose, lactose and sucrose with the use of an oxygen electrode are described. They were obtained by immobilization of glucose oxidase with suitable disaccharide hydrolase in gelatin or albumin. For the sucrose determination mutarotase was also used. The membranes contained one, two or three enzymes working in sequence. The enzyme composition of the membranes influenced the linear range slightly and the value of electrode response considerably. The electrode response of the maltose membranes was also affected by the immobilization material. The measurements with the use of enzymatic membranes were very sensitive to temperature and, to some degree, the pH of the sample. Stability of the membranes was differentiated. The albumin membranes for sucrose determination generated almost the same signal after 1 month of frequent use, whereas the gelatin membranes for lactose determination lost about 85% of the initial activity after 1 week of operation. The electrode response of both the gelatin and albumin maltose membranes decreased in the same time by about 50%.

Albumins↗

PubMed1989

Hydrodynamic, structural and magnetic properties of Megasphaera elsdenii Fe hydrogenase reinvestigated.

Megasphaera elsdenii hydrogenase has been purified to homogeneity using an FPLC procedure as the final step. The protein gives a single band in SDS/PAGE with an apparent molecular mass of 57-59 kDa. There is no second hydrogenase activity in the soluble fraction of M. elsdenii. The hydrodynamics of the enzyme have been compared to those of the two-subunit Fe hydrogenase from Desulfovibrio vulgaris (Hildenborough) in the analytical ultracentrifuge using the absorption of the intrinsic iron-sulfur clusters as the monitor. Sedimentation-velocity experiments indicate the M. elsdenii enzyme (s20,w = 4.95 S) to be essentially globular, while the D. vulgaris enzyme (s20,w = 4.1 S) has a less symmetric shape. From the sedimentation equilibrium measurements under a variety of conditions an average molecular mass is calculated of 58 kDa (M. elsdenii) and 54 kDa (D. vulgaris), respectively. Pure, maximally active M. elsdenii hydrogenase has A405/A280 = 0.36 and has a specific H2-production activity of 400 mumol H2.min-1.(mg protein)-1 at 30 degrees C and pH 8.0. The enzyme contains some 13-18 iron and acid-labile sulfur ions/58-kDa monomer. Eight of these Fe-S are present as two electron-transferring ferredoxin-like cubanes with Em approximately greater than -0.3 V, as indicated by pH-dependent EPR spectroscopy on the H2-reduced enzyme. In the (re)oxidized state the remainder iron gives rise to a single S = 1/2 rhombic EPR signal. Hydrogen-production activity, content of remainder iron and rhombic EPR signal intensity are mutually correlated. Purified hydrogenase appears to exist as a mixture of fully active holoenzyme and inactive protein still carrying the two cubanes but deficient in active-site iron.

Bacteria, Anaerobic↗