Chanchal K. Mitra

Electrochemistry of reconstituted glucose oxidase on carbon paste electrodes

Abstract Flavine Adenine Dinucleotide (FAD) was covalently immobilised onto glassy carbon matrix using a 13-carbon atom long spacer arm. FAD modified electrodes offer a convenient handle for immobilising glucose oxidase (GOD) enzyme for direct electron transfer. The electrochemical characteristics of immobilised FAD were compared with free FAD in solution at blank paste electrode surface. The prominent peaks at −475 mV and −601 mV indicate the positive coupling of FAD onto the carbon matrix. In our case we observed that reduction peaks are well separated from the oxidation and the immobilised FAD shows more reversible behavior compared to the free FAD in solution. GOD apoenzyme has been prepared by acidification of GOD solution (5 mg/ml) in buffer (100 mM sodium acetate) using ammonium sulfate solution at pH 1.4. The apoenzyme was coupled to the FAD modified matrix by incubating the matrix with the solution of apoenzyme for 4–12 h. The paste electrode with reconstituted GOD was investigated for its electrochemical characteristics and for its response with substrate (glucose solution). Our major finding is that the reconstituted enzyme shows better electron transfer rates compared to normal enzyme. The reason for this can be attributed to the long spacer arm holding the electroactive FAD which facilitates better electron transfer between enzyme redox center and electrode surface. The above technique appears to be a promising approach to be used in biosensor application.

Direct Electrochemistry of Horseradish Peroxidase-Gold Nanoparticles Conjugate

We have studied the direct electrochemistry of horseradish peroxidase (HRP) coupled to gold nanoparticles (AuNP) using electrochemical techniques, which provide some insight in the application of biosensors as tools for diagnostics because HRP is widely used in clinical diagnostics kits. AuNP capped with (i) glutathione and (ii) lipoic acid was covalently linked to HRP. The immobilized HRP/AuNP conjugate showed characteristic redox peaks at a gold electrode. It displayed good electrocatalytic response to the reduction of H2O2, with good sensitivity and without any electron mediator. The covalent linking of HRP and AuNP did not affect the activity of the enzyme significantly. The response of the electrode towards the different concentrations of H2O2 showed the characteristics of Michaelis Menten enzyme kinetics with an optimum pH between 7.0 to 8.0. The preparation of the sensor involves single layer of enzyme, which can be carried out efficiently and is also highly reproducible when compared to other systems involving the layer-by-layer assembly, adsorption or encapsulation of the enzyme. The immobilized AuNP-HRP can be used for immunosensor applications.

Bioelectrochemical studies on catalase modified glassy carbon paste electrodes

Abstract The enzyme catalase (EC: 1.11.1.6) has been covalently coupled onto the surface of glassy carbon (GC) powder matrix using a 16 atom spacer arm. The enzyme coupled powder was made into a paste electrode that was used to study the electrochemical properties. Standard electrochemical techniques like cyclic voltammetry, differential pulse voltammetry and flow injection analysis studies were carried out using this paste electrode. The cyclic voltammogram of the modified paste exhibited a clear increase in the reduction peak at −180 mV in the presence of hydrogen peroxide. The potential at which maximum Faradaic activity was observed was determined using differential pulse voltammetry, which showed a clear peak at −100 mV. This potential was used to monitor the response of the electrode to varying substrate concentrations using a home made setup for flow injection analysis. A linear increase in the current values in the range 0.1–1 mM hydrogen peroxide concentration was observed in our system.

Conserved Short Sequences in Promoter Regions of Human Genome

Abstract Recognition of promoter elements by the transcription factors is one of the early initial and crucial steps in gene expression and regulation. In prokaryotes, there are clear signals to identify the promoter regions like TATAAT at around −10 and TTGACA at −35 positions from transcription start site (TSS). In eukaryotes the promoter regions are structurally more complex and there are no conserved or consensus sequences similar to the ones found in prokaryotic promoters. We have located a set of GC rich short sequences (<8 nt) that are relatively common in human promoter sequences around the TSS (±100 relative to TSS). These sequences were sorted based on their frequency of occurrence in the database and the most common 50 sequences were used for further studies. Sigmoidal behavior of the high end of the frequency distribution of these sequences suggests presence of some internal co-operativity. These short sequences are distributed on both sides of TSS, suggesting that probably the transcription factors recognize these sequences on both upstream and downstream of TSS. As eukaryotic promoters lack any conserved sequences, we expect that these short sequences may help in recognition of promoter regions by relevant transcription factors prior to the initiation of transcription process. We postulate that a cluster of genes with common short sequences in the promoter region can be recognized by a particular transcription factor. We also found that most of these short sequences are fairly common within miRNA (both mature and stem-loop sequences). Our studies indicate that eukaryotic transcription is more complex than currently believed.

Modelling the Krebs cycle and oxidative phosphorylation

The Krebs cycle and oxidative phosphorylation are the two most important sets of reactions in a eukaryotic cell that meet the major part of the total energy demands of a cell. In this paper, we present a computer simulation of the coupled reactions using open source tools for simulation. We also show that it is possible to model the Krebs cycle with a simple black box with a few inputs and outputs. However, the kinetics of the internal processes has been modelled using numerical tools. We also show that the Krebs cycle and oxidative phosphorylation together can be combined in a similar fashion – a black box with a few inputs and outputs. The Octave script is flexible and customisable for any chosen set-up for this model. In several cases, we had no explicit idea of the underlying reaction mechanism and the rate determining steps involved, and we have used the stoichiometric equations that can be easily changed as and when more detailed information is obtained. The script includes the feedback regulation of the various enzymes of the Krebs cycle. For the electron transport chain, the pH gradient across the membrane is an essential regulator of the kinetics and this has been modelled empirically but fully consistent with experimental results. The initial conditions can be very easily changed and the simulation is potentially very useful in a number of cases of clinical importance.

Covalent enzyme immobilization onto glassy carbon matrix-implications in biosensor design

The aim of the present work is to design an electrode for biosensors by covalent immobilization of the redox enzyme. In the covalently modified electrode, the biocatalyst is located close to the electrode surface and this is expected to enhance the electron transfer rate from the enzyme to the electrode. Several methods of covalent immobilization of enzymes onto a glassy carbon surface are described. We have chosen horse radish peroxidase enzyme in our study but any other suitable enzyme can be immobilized depending on the intended use. A three step procedure that includes (i) heat treatment of matrix at l00-l10°C to remove volatiles and absorbates, (ii) chemjcal pretreatment to introduce functional groups like -OH, -NO2, -Br etc. followed by (iii) glutaraldehyde coupling of the enzyme (for the nitrated matix after subsequent reduction) or modification of the matrix by carboxymethylation and enzyme coupling using carbodiimide (for hydroxylated matrix) was followed. The amount of enzyme immobilized onto the carbon surface was estimated by spectrophotometric enzymatic activity assay, commonly used for the soluble enzyme. We found that simple nitration did not introduce any significant amount of functional groups and the matrix with hydrogen peroxide pretreatment showed the highest enzyme loading of 0.05 U/mg of carbon matrix. The HRP enzyme electrode was tested in a rotating disk experiment for its response with the substrate.

Brief communication: Comparative analysis of core promoter region: Information content from mono and dinucleotide substitution matrices

We have studied the core promoter region in five sets of promoter sequences by calculating the average mutual information content H (relative entropy). We have used specially constructed substitution matrices to calculate mono and dinucleotide replacements in a given block of aligned sequences. These substitution matrices use log-odds form of scores, which are in bits of information. Here, we constructed and applied nucleotide substitution matrices for the core promoter region to calculate the information content to study the Transcription Start Site (TSS), TATA-box and downstream regions. As expected, the information content decreases with increasing block size. This clearly implies that the TSS region is likely to be 5-10 bases in size (length). We also notice that both in the case of mouse and humans, both TATA-boxes and TSS regions are likely to play important roles in proper transcriptional initiation.

Protein sequences as random fractals

The analysis of primary sequences from a protein sequence data base suggests that the sequences can be considered as examples of constrained random fractals. Fractal dimensions of the positional distributions of the 20 residues along the chain have been calculated. These fractal dimensions can be used as indices of intrinsic preferences of various residues.

Low Frequency Impedance Studies on Covalently Modified Glassy Carbon Paste

Cyclic voltammetry and impedance studies have been carried out on covalently coupled horseradish peroxidase, hemin, reconstituted horseradish peroxidase and blank glassy carbon paste electrodes. The cyclic voltammogram of native enzyme paste showed a clear oxidation peak at −50 mV and an increase in the oxidation currents in presence of the substrate was observed. In order to increase the electron transfer kinetics the redox center (hemin) of the enzyme was directly bound onto the electrode followed by reconstitution with apoperoxidase. Hemin bound paste showed a reduction peak at −275 mV and an oxidation peak at ca. −50 mV. Upon reconstitution with apoperoxidase a shift of 175 mV in the reduction peak and ca. 100 mV in the oxidation peak was observed. We have also studied the effect of electron donors like hydroquinone and p-aminobenzoic acid on the modified electrodes. It was observed that the native enzyme showed a clear increase in the electron transfer in the presence of electron donor whereas with the reconstituted enzyme paste the effect of the electron donor was not evident. Low frequency electrochemical impedance spectroscopy was carried out in the frequency range of 10 mHz to 1 kHz in order to study the kinetics of slow processes occurring at the electrode surface. The impedance data of the system in the supporting electrolyte was simulated to an equivalent circuit model, the modified Randles cell circuit for an irreversibly adsorbed species. The data in the presence of substrate was modeled to modified Randles cell circuit with Warburg impedance. The models show a reasonably good fit in the frequency region studied. The unmodified blank paste electrode showed more or less a constant phase angle behavior. From the simulated data the parameters values of the circuit used were obtained which were used to calculate the kinetic parameters.

Pair-preferences: a quantitative measure of regularities in protein sequences.

We present here the results obtained by applying several different methods to quantitatively measure regularities in protein sequences based on pair-preferences. We have studied the distribution of amino acid residues, singly as well as in pairs in a large data base and have attempted this task. We confirmed the existence of well-defined pair-preferences in proteins which were shown to be remarkably absent in simulated random sequences of similar amino acid distribution. The analysis of the sequences from the SWISS-PROT data base using simple statistical tests. Fourier analysis, fractal analysis and statistical thermodynamical tests were used to derive parameters to define a natural sequence. As a consequence of the existence of pair-preferences, parameters like fractal dimension (D), spectral exponent (beta), scaling parameter (H) and entropy (statistical) were found to be characteristic for natural sequences. For a reference state we chose a randomised state devoid of any pair-preference. The pair-preferences qualified well to be used as quantitative measures of regularities in protein sequences.