A study of the effects of varying temperature on the horseradish peroxidase enzyme
Peroxidase optimal temperature
The enzyme horseradish peroxidase HRP conjugate used for sensitive streptavidin-biotin interaction to allow flexible, rapid, and sensitive immuno-detection followed by Fast Red staining. The temperature dependence of the tertiary structure around the heme also affected the intrinsic tryptophan fluorescence emission spectrum of the enzyme. At low concentration of H2O2 2 mM , the first order rate of inactivation kobs was limited and fairly constant between pH 4. The observed effects of buffer concentration on catalytic rate and enzyme inactivation show that the desired increase in conversion rate by peroxidases is closely linked with undesirable inactivation of the enzyme. These findings will help in defining the optimal reaction conditions that preserve the activity of the peroxidase molecules. The H2O2 concentrations used in this study were significantly higher than that of the peroxidase, to ensure pseudo-first order inactivation conditions. Ionic strength dependent inactivation kinetics The kinetics of irreversible inactivation of horseradish peroxidase HRP by hydrogen peroxide was studied under pseudo first order conditions. Influence of pH on the thermal inactivation kinetics of horseradish peroxidase in aqueous solution. It is likely that low pH facilitates the generation of inactivating species from the excess H2O2 in the reaction mixture. Structural and conformational stability of horseradish peroxidase: effect of temperature and pH. Further investigations will look at the effects of different buffer ions on the catalytic and inactivation process. Secondly, Compound III might return to the ground state after catalyzing the oxidation of the surrounding protein, yielding an oxidized amino acid side chain group. Studies on horseradish peroxidase XI Nature of compounds I and II as determined from the kinetics of the oxidation of ferrocyanide. The extent and dynamics of suicide inactivation of peroxidases are sensitive to the presence and the nature of the donor substrate Valderrama et al.
Chattopadhyay K 1Mazumdar S. Generally, peroxidases are inactivated by extremes of pH, with inactivation more pronounced in the alkaline range than in acidic conditions Lemos et al.
IHC involved the conjugation of an antibody with an enzyme, e. This process also facilitates the conversion of substrates that are poor substrates for peroxidases since they can be oxidised via redox mediation with a primary substrate that is efficiently oxidised by the peroxidase. References Adediran SA The role of compound III in reversible and irreversible inactivation of lactoperoxidase.
The melting of the tertiary structure was found to be associated with a pK a of approximately 5, indicating that this phase possibly involves breaking of the hydrogen-bonding network of the heme pocket, keeping the heme moiety still inside it.
The observed effects of buffer concentration on catalytic rate and enzyme inactivation show that the desired increase in conversion rate by peroxidases is closely linked with undesirable inactivation of the enzyme.
The blots on the membrane were then incubated with the secondary antibody, horseradish peroxidase-conjugated anti-mouse IgG , Santa Cruz or horseradish peroxidase-conjugated anti-rabbit IgG , Santa Cruzfor 2 h at RT.
Dunford HB Biochemistry At 5 mM H2O2 concentration, the inactivation rate was higher in the acidic range compared to the reactions in which 2 mM were used 0.
based on 37 review