Ciarán Ó'Fágáin

Increased thermal and solvent tolerance of acetylated horseradish peroxidase

Abstract Thermal stability of horseradish peroxidase (HRP) has been enhanced by acetylation with acetic acid n -hydroxysuccinimide ester (AA-NHS) under mild conditions. The half-life at 65°C was increased fivefold. This modification has also resulted in greater tolerance of the water-miscible organic solvents, dimethylformanide and tetrahydrofuran, at 25°C and 60°C and of methanol at 60°C. Analysis of the free amino groups of HRP indicates that AA-NHS alters 3 of the HRP 6 lysine residues. This stabilization has been achieved by a simple chemical modification involving neither immobilization nor the use of cross-linking agents.

Stability characteristics of chemically-modified soluble trypsin

Abstract Bovine pancreatic trypsin was modified with acetic acid N -hydroxy-succinimide ester (AANHS) which neutralizes the positive charge on lysine residues. Approximately eight out of 14 lysine residues per trypsin molecule were modified. The AANHS-treated trypsin showed enhanced thermostability compared with the native between 30 and 70 °C. Tm values for native and AANHS trypsin were 46 °C and 51 °C, respectively. At 55 °C the modified trypsins half-life is doubled to 8.7 min versus 4.3 min for the native enzyme. AANHS trypsin exhibited a decreased rate of autolysis and also showed enhanced stability at 65 °C in aqueous mixtures of the following organic solvents: 1,4-dioxan, dimethylformamide, dimethylsulphoxide and acetonitrile.

The phylogeny of the mammalian heme peroxidases and the evolution of their diverse functions

BackgroundThe mammalian heme peroxidases (MHPs) are a medically important group of enzymes. Included in this group are myeloperoxidase, eosinophil peroxidase, lactoperoxidase, and thyroid peroxidase. These enzymes are associated with such diverse diseases as asthma, Alzheimers disease and inflammatory vascular disease. Despite much effort to elucidate a clearer understanding of the function of the 4 major groups of this multigene family, we still do not have a clear understanding of their relationships to each other.ResultsSufficient signal exists for the resolution of the evolutionary relationships of this family of enzymes. We demonstrate, using a root mean squared deviation statistic, how the removal of the fastest evolving sites aids in the minimisation of the effect of long branch attraction and the generation of a highly supported phylogeny. Based on this phylogeny we have pinpointed the amino acid positions that have most likely contributed to the diverse functions of these enzymes. Many of these residues are in close proximity to sites implicated in protein misfolding, loss of function or disease.ConclusionOur analysis of all available genomic sequence data for the MHPs from all available completed mammalian genomes, involved sophisticated methods of phylogeny reconstruction and data treatment. Our study has (i) fully resolved the phylogeny of the MHPs and the subsequent pattern of gene duplication, and (ii), we have detected amino acids under positive selection that have most likely contributed to the observed functional shifts in each type of MHP.

Modification of horseradish peroxidase with bifunctional n-hydroxysuccinimide esters: Effects on molecular stability

Abstract Horseradish peroxidase (HRP) has been chemically modified with the homobifunctional cross-linking reagents suberic acid n -hydroxysuccinimide ester (SA-NHS) and ethylene glycol bis-succinimidyl succinate (EG-NHS) yielding derivatives of native HRP with enhanced stability in a range of organic solvents. Modification did not cause any loss of activity. It has been shown that these modification reagents react specifically with the ϵ-amino groups of the six lysine residues of HRP. Stability increased with concentrations of EG-NHS up to a level of 4–5 mg ml−1. A marked difference was observed in the tryptophan fluorescence profiles of native and the EG-NHS peroxidases upon thermoinactivation at 65°C. Both modified peroxidases exhibited improved resistance to the denaturant guanidine hydrochloride.

Determination of glutathione at enzyme-modified and unmodified glassy carbon electrodes

The oxidation of the reduced form of glutathione (GSH) was found to occur at a decreased overpotential at a glassy carbon electrode when 1.2 mol dm–3 of dipotassium hydrogen phosphate were used as the supporting electrolyte. The resulting current for the oxidation peak of GSH varied linearly over the concentration range 0.16–2.4 mmol dm–3 GSH. The reaction between GSH and hydrogen peroxide catalysed by the enzyme GSH-peroxidase (GSH-PX) could be monitored using a glassy carbon electrode with GSH-PX immobilized under a layer of Nafion film.

Stability of alanine aminotransferase is enhanced by chemical modification

Abstract Alanine aminotransferase is a clinical diagnostic marker enzyme. It is often included in commercial control sera. The stabilization of soluble alanine aminotransferase catalytic activity by chemical modification with bis-imidates and with succinic anhydride is described. At 4°C, the bis-imidates-modified enzyme is predicted to be up to 43 times more stable, and the succinic anhydride-modified enzyme six times more stable, than the unmodified enzyme control. The degree of stability was estimated by an accelerated storage protocol with experimental data being analyzed by a dedicated computer program. The applicability of this methodology to studies of protein stability is briefly discussed.

Dye bleaching and phenol precipitation by phthalic anhydride‐modified horseradish peroxidase

The phenol precipitation and dye bleaching capabilities of phthalic anhydride–modified horseradish peroxidase C (PA–HRP) were compared with those of native HRP C and ethylene glycol- bis-(succinic acid N-hydroxysuccinimide ester)–modified HRP (EG–HRP) reported previously. Removal efficiency (percentage of phenol removed from solution under experimental conditions) was determined for native HRP and both modified forms. Removal efficiencies at 37 °C were very similar, with >95% removal in each case. Removal efficiencies were less at 70 °C overall (range 25–45%) but PA–and EG–HRP removed up to 50% more phenol than native HRP. The three HRP forms showed similar dye bleaching performance at 37 °C in the presence of H2O2 and accelerators (up to 86% colour removal). PA–and EG–HRP showed slightly greater bleaching abilities at 65 °C than native HRP for some of the dye/accelerator combinations tested. Modified HRPs performed better in 40% (v/v) mixtures of dioxane or dimethylformamide. © 2000 Society of Chemical Industry

Consensus mutagenesis reveals that non-helical regions influence thermal stability of horseradish peroxidase

The enzyme horseradish peroxidase has many uses in biotechnology but a stabilized derivative would have even wider applicability. To enhance thermal stability, we applied consensus mutagenesis (used successfully with other proteins) to recombinant horseradish peroxidase and generated five single-site mutants. Unexpectedly, these mutations had greater effects on steady-state kinetics than on thermal stability. Only two mutants (T102A, T110V) marginally exceeded the wild types thermal stability (4% and 10% gain in half-life at 50 degrees C respectively); the others (Q106R, Q107D, I180F) were less stable than wild type. Stability of a five-fold combination mutant matched that of Q106R, the least-stable single mutant. These results were perplexing: the Class III plant peroxidases display wide differences in thermal stability, yet the consensus mutations failed to reflect these natural variations. We examined the sequence content of Class III peroxidases to determine if there are identifiable molecular reasons for the stability differences observed. Bioinformatic analysis validated our choice of sites and mutations and generated an archetypal peroxidase sequence for comparison with extant sequences. It seems that both genetic variation and differences in protein stability are confined to non-helical regions due to the presence of a highly conserved alpha-helical structural scaffold in these enzymes.

The First EU ScienceOlympiad (EUSO): a modelfor science education

In April 2003 the first European Science Olympiad took place in Dublin City University in Ireland.In this competitionsecond level students were asked to carry out a number of tasks involving biology, physics and chemistry. Students had qualified initially in competitions in one of these subjects and were organised into integratedteams which worked together to complete the tasks. This paper examines the background to the competition,describes aspects of the biological input in developing the tasks and discusses the value of the approach andits potential benefits in improving both interest levels in science and the quality of science education.

Chemical derivatives of Pseudomonas aeruginosa elastase showing increased stability

Abstract Elastase from Pseudomonas aeruginosa has recently been used successfully for peptide synthesis. To improve its performance we attempted to increase its catalytic stability by chemical modification. Two distinct sorts of amino group-specific modifiers, dimethyl suberimidate and cyanuric chloride-activated polyethylene glycol (PEG), gave a two-fold increase in catalytic stability at 70°C and greater degrees of stabilization at lower temperatures. Suberimidate treatment seemed to act by intramolecular crosslinking, whereas the activated PEG gave rise to an elastase-PEG conjugate. The thermal transition (T m ) for suberimidate-treated elastase was unchanged from the native value of 72°C. PEG-conjugated elastase gave anomalous T m curves; therefore, a value could not be determined. The lack of correspondence of catalytic stabilization with increased T m values is discussed.