ABSTRACT
This paper describes the utilisation of methoxy-resorufin ether as an electrochemical probe for studying cytochrome P450 CYP6G1. Methoxy-resorufin ether is well established as a versatile substrate for cytochrome P450, as its demethylated product, resorufin, is a fluorophore. We show that in addition to these established properties, methoxy-resorufin ether also exhibits reversible two electron transfer on glassy carbon and edge plane graphite electrodes. Cyclic voltammetry measurements and differential pulse voltammetry measurements show that methoxy-resorufin ether can be easily detected at low concentrations (down to 200 nM) in a conventional three electrode electrochemical cell. These properties of methoxy-resorufin ether mean that it could be used as an electrochemical probe, to follow the rate of its demethylation by CYP6G1. We show that electrochemical measurements could discriminate between the enzyme activity of protein microsomes taken from two strains of Drosophila melanogaster (fruit fly).
Subject(s)
Cytochrome P-450 Enzyme System/analysis , Cytochrome P-450 Enzyme System/metabolism , Oxazines/chemistry , Animals , Carbon Monoxide/pharmacology , Cytochrome P-450 Enzyme System/chemistry , Drosophila melanogaster/classification , Drosophila melanogaster/cytology , Drosophila melanogaster/enzymology , Electrochemistry , Methylation , Microsomes/chemistry , Microsomes/metabolism , Molecular Structure , NADP/metabolism , Spectrometry, Fluorescence , Substrate Specificity , Time FactorsABSTRACT
The control of protein immobilisation and orientation at surfaces is of increasing relevance in biotechnological devices. These devices include reproducible biosensors, biofuel cells, and protein arrays, all of which require the immobilisation of protein/enzymatic sensing elements with retention of their activity. The control of protein orientation upon immobilisation is of importance in (1) facilitating equal access of analyte molecules to the protein that acts as a sensing element and (2) creating favourable interactions to stabilise the protein on the surface for long-term storage and usage of bio-devices. This review presents a comparison of a number of surface analysis techniques that have been widely used in the chemical analysis of surfaces that offer the potential to discriminate different orientations of the same protein on a conductive surface.
Subject(s)
Biosensing Techniques , Microchemistry/methods , Nanotechnology , Proteins/chemistry , Animals , Electrochemistry/methods , Surface PropertiesABSTRACT
A direct method has been developed for the quantitation of the amount of immobilised enzymes on biosensor surfaces. This quantity is of key importance in establishing the activity, kinetics and optimal immobilisation conditions in the construction of both amperometric and optical biosensors. Recombinant L-lactate dehydrogenase incorporating both a biosynthetically introduced radiolabel, 3H-leucine, and a hexahistidine peptide tag was immobilised on a poly(aniline) composite film and then quantitated by liquid scintillation counting. It was found that enzyme mass loading was proportional to the concentration of LDH in solution, and also depended on the morphology of the composite film. The LDH mass loading on the composite film doubled when a surface cysteine containing variant was used, possibly due to the covalent attachment of the cysteine to the diiminoquinoid rings of the poly(aniline).
Subject(s)
Aniline Compounds , Electrochemistry/methods , Enzymes, Immobilized/analysis , Equipment Design/methods , Isotope Labeling/methods , L-Lactate Dehydrogenase/analysis , Biosensing Techniques , Catalysis , Coated Materials, Biocompatible/chemistry , Enzyme Activation , Enzymes, Immobilized/chemistry , Isotope Labeling/instrumentation , L-Lactate Dehydrogenase/chemistry , Leucine/chemistry , Microscopy, Electron, Scanning , Recombinant Proteins/analysis , Recombinant Proteins/chemistry , Scintillation Counting/methods , Sensitivity and SpecificityABSTRACT
The immobilization of the mutants of L-lactate dehydrogenase (LDH) on poly(aniline) (PANi) composite films has been investigated. Mutants possessing peptide tags of varying charge and nucleophilicity were created to probe the nature of the interaction between the protein and PANi. These results are significant for the development of a 'generic' approach to the immobilization of enzymes and other proteins.
Subject(s)
Bioelectric Energy Sources , Electrodes , Enzymes, Immobilized/metabolism , L-Lactate Dehydrogenase/metabolism , Peptides/chemistry , Enzymes, Immobilized/chemistry , Enzymes, Immobilized/genetics , L-Lactate Dehydrogenase/chemistry , L-Lactate Dehydrogenase/genetics , MutagenesisABSTRACT
Immobilisation of enzymes is important for applications such as biosensors or biofuel cells. A poly(histidine) tag had been introduced on the C terminus of a lactate dehydrogenase enzyme. This mutant enzyme was then immobilised onto poly(aniline) (PANi)-poly(anion) composite films, PANi-poly(vinylsulfonate) (PVS) or PANi-poly(acrylate) (PAA). The NADH produced by the immobilised enzyme in the presence of beta-nicotinamide adenine dinucleotide (NAD(+)) and lactate is oxidised at the poly(aniline)-coated electrode at 0.05 to 0.1 V vs. saturated calomel electrode (SCE) at 35 degrees C.
