Formation and properties of dimeric recombinant horseradish peroxidase in a system of reversed micelles

Abstract

Wild-type recombinant horseradish peroxidase purified and refolded from Escherichia coli inclusion bodies has been studied in the system of bis(2-ethylhexyl)sulphosuccinate sodium salt (Aerosol OT)-reversed micelles in octane. In contrast with native horseradish peroxidase the wild-type recombinant enzyme forms dimeric structures as judged by sedimentation analysis. Peroxidase substrates affect the equilibrium between monomeric and dimeric enzyme forms. The dependence of the catalytic activity of recombinant peroxidase on the degree of hydration of the surfactant exhibits two maxima with pyrogallol, o-phenylene- diamine, guaiacol and o-dianisidine, with different ratios of activities for the first and second maxima. The differences in activities of monomeric and dimeric forms of the recombinant horseradish peroxidase provide evidence for active-site screening in dimeric forms. This has been used to model a dimeric structure of recombinant horseradish peroxidase with the screened entrance to the active site. In the model structure obtained, three of eight glycosylation sites were screened. This might explain the absence of dimeric structures in native enzyme peroxidase. The system of reversed micelles provides, for the first time, evidence for the formation of dimeric structures by recombinant plant peroxidase with an altered substrate specificity compared with the native enzyme. Thus one can assume that haem-containing peroxidases in general are able to form dimeric structures.

Regulation of intracellular heme trafficking revealed by subcellular reporters

“…The equilibrium between monomeric and dimeric recombinant HRP is affected by the peroxidase substrates (45,46). By contrast, APX is not glycosylated and forms a noncovalent homodimer in a concentration-dependent manner (47,48).…”

Assembly of horseradish peroxidase within supported cationic bilayers

“…Indeed, hydrophobic microenvironments destabilized superactive conformations of HRP like those obtained on favorable interactions between hydrophilic HRP oligosaccharides and the similarly hydrophilic inner surface of reversed-micelles. 36 Furthermore, sequestration of the ABTS substrate, which is an anionic molecule, by the surface of the cationic bilayer (Table 1) would separate substrate and enzyme in different microenvironments, ABTS at the DODAB bilayer surface and HRP in the bilayer core. In fact, there are many examples from the literature reporting reduction of HRP activity on immobilization on solid surfaces or self-assembled amphiphiles such as reversed micelles, 36 lipids, 37 or micelle-forming surfactants such as stearic acid monolayers at the air-water interface 38 or polysaccharides such as chitosan.…”

“…36 Furthermore, sequestration of the ABTS substrate, which is an anionic molecule, by the surface of the cationic bilayer (Table 1) would separate substrate and enzyme in different microenvironments, ABTS at the DODAB bilayer surface and HRP in the bilayer core. In fact, there are many examples from the literature reporting reduction of HRP activity on immobilization on solid surfaces or self-assembled amphiphiles such as reversed micelles, 36 lipids, 37 or micelle-forming surfactants such as stearic acid monolayers at the air-water interface 38 or polysaccharides such as chitosan. 35 HRP was previously shown to adsorb irreversibly onto hybrid particles of poly(methylmethacrylate) (PMMA) and carboxymethylcellulose 39 with little (up to 20%) or no reduction of enzymatic activity in comparison with that observed for free HRP in solution.…”

Conjugation of Peroxidase from Brassica oleracea gongylodes for Use as a Label- Prospect of a Novel Enzyme Tag for Immunoassay Systems

“…Extensive work is been reported in literature on the glycosylation sites of HRP (Gazaryan et al, 1998;Veitch, 2003). The glycosyl residues of proteins are extensively exploited in immobilization and conjugation processes.…”