Structural Change and Catalytic Activity of Horseradish Peroxidase in Oxidative Polymerization of Phenol

Abstract

The effects of solvent and reaction conditions on the catalytic activity of horseradish peroxidase (HRP) were investigated for oxidative polymerization of phenol in water/organic mixtures using hydrogen peroxide as an oxidant. Also, the structural changes of HRP were investigated by CD and absorption spectroscopy in these solvents. The results suggest that the yield of phenol polymer (the conversion of phenol to polymer) is strongly affected by the reaction conditions due to the structural changes of HRP, that is, the changes in higher structure of the apo-protein and dissociation or decomposition of the prosthetic heme. Optimum solvent compositions for phenol polymerization depend on the nature of the organic solvents owing to different effects of the solvents on HRP structure. In addition to initial rapid changes, slower changes of HRP structure occur in water/organic solvents especially at high concentrations of organic solvents. In parallel with these structural changes, catalytic activity of HRP decreases with time in these solvents. At higher reaction temperatures, the yield of the polymer decreases, which is also ascribed to modification of HRP structure. It is known that hydrogen peroxide is an inhibitor of HRP, and the yield of phenol polymer is strongly dependent on the manner of addition of hydrogen peroxide to the reaction solutions. The polymer yield decreases significantly when hydrogen peroxide was added to the reaction solution in a large amount at once. This is probably due to inactivation of HRP by excess hydrogen peroxide. From the CD and absorption spectra, it is suggested that excess hydrogen peroxide causes not only decomposition of the prosthetic heme but also modification of the higher structure of HRP.

Experimental investigation of evanescence-based infrared biodetection technique for micro-total-analysis systems

“…3. Similarly, the absorbance value of ϳ0.02, as reported by Akita et al, 30 is the nearly the same value obtained when absorbance measurement experiments were carried out for H 2 O 2 taken independently at a 635-nm wavelength. Therefore, it is evident that absorbance is maximum when specimens are taken in 1:1 volumetric ratio for the given molar concentrations of the specimen.…”

“…The results obtained from the absorbance measurements obtained from the experiments were compared with the previously published results. 28,30 In the published results, the concentration Fig. 3.…”

Protein Modification with Amphiphilic Block Copoly(2-oxazoline)s as a New Platform for Enhanced Cellular Delivery

“…The Far-UV CD spectra of the conjugates revealed considerable decreases in the CD signals in the major bands at 215 nm and 225 nm and a shift of 225 nm band to 230 nm compared to the unmodified protein (Figure 4a). These CD changes were most pronounced for HRP-P(MeOx- b -BuOx) and are indicative of changes in the secondary structure of the HRP apo-protein (30–31). At the same time there was little if any changes of CD signal in the Near-UV-vis spectra of the conjugates (Figure 4b) suggesting that the tertiary structure of HRP apo-protein and the microenvironment of the prosthetic heme remained unaffected by the conjugation (30–31), even though the secondary structure of apo-protein was partially disrupted.…”

“…These CD changes were most pronounced for HRP-P(MeOx- b -BuOx) and are indicative of changes in the secondary structure of the HRP apo-protein (30–31). At the same time there was little if any changes of CD signal in the Near-UV-vis spectra of the conjugates (Figure 4b) suggesting that the tertiary structure of HRP apo-protein and the microenvironment of the prosthetic heme remained unaffected by the conjugation (30–31), even though the secondary structure of apo-protein was partially disrupted. The changes of the secondary structure may be ascribed to possible interactions between the block copolymer chain and α-helices on the surface of the apo-protein.…”

Microcantilevers Modified by Horseradish Peroxidase Intercalated Nano-Assembly for Hydrogen Peroxide Detection

“…c) Structure of the film. Recently, through circular dichroic (CD) and adsorption spectroscopic analysis, Akita 33 and Schmidt 30 reported that both the secondary and the tertiary structure of the HRP change upon the reaction with H2O2. It is well known that HRP can be oxidized by H2O2 to HRP compound I, followed by the fast oxidation to HRP compound II, and possibly to compound III.…”