Acriflavine (3,6-diaminoacridine) is an anticeptic drug developed in 1912. Previous research has focused on investigation of the intercalating features of acriflavine, but little is known about its interaction with proteins. Drug-receptor interaction is of major interest in clinical science. The aim of the present study was to evaluate the ability of acriflavine to induce alterations in conformation and function of peroxidase, a critical enzyme in cell survival. Horseradish peroxidase C (HRPC) activity was determined by measuring H2O2-dependent oxidation of o-dianisidine at 460 nm using an extinction coefficient of 11.3 Mm-1 cm-1. Apparent Km and Vmax values were then calculated. The electronic absorption spectra were recorded for 300-700 nm. Both Kd and DG were calculated from changes in the absorbance of 403 nm. Intrinsic fluorescence was detected for the 297 nm excitation and the emission was recorded for 300-700 nm wavelengths. The Stern-Volmer constant and Hill coefficients were then obtained. All measurements were performed in 0.1 M citrate buffer, pH 4.0. Results indicated that acriflavine either stimulated or inhibited HRPC activity depending on concentration and preincubation time. The Drug-receptor complex formation occurred via binding of four molecules of acriflavine in two different binding sites on HRPC, and the heme environment became more polar. Finally, acriflavine quenched the only tryptophan residue of HRPC. The alterations in HRPC conformation identified in the present study, suggest that drugs that induce apoptosis could alter critical cell protein conformation and function.

Horseradish peroxidase was encapsulated in calcium alginate for the purpose of phenol removal. Considering enzyme encapsulation efficiency, retention activity and enzyme leakage of the capsules, the best gelation condition was found to be 1 % w/v of sodium alginate solution and 5.5 % w/v of calcium chloride hexahydrate. Upon immobilization, pH profile of enzyme activity changes as it shows higher value at basic and acidic solution. Besides, for each phenol concentration there would be an enzyme concentration which going further than this value has no significant effect on phenol removal. Investigation into time course of phenol removal for both encapsulated and free enzyme showed that encapsulated enzyme had nearly similar efficiency in comparison with the same concentration of free enzyme; however the capsules were reusable up to four cycles without any changes in their efficiency. The ratio of hydrogen peroxide/phenol at which highest phenol removal obtained, found to be dependent on initial phenol concentration and in the solution of 2 and 8 mM phenol it were 1.15 and 0.94 respectively.

Mercury is one of the three major environmental metal poisons, and mercuric chloride is a highly reactive compound which can harm cells by a variety of mechanisms including direct interaction with sulphydryl groups of proteins and enzymes, therefore affecting the enzymatic activity. This study focused on the effect of Hg++ on horseradish peroxidase (donor: hydrogen peroxide oxidoreductase, EC 1.11.1.7) (HRP) (Isoenzyme C) activity. In the presence of 88 mM hydrogen peroxide Km for o-dianisidine oxidation was 0.05 millimolar and Vmax was 8.5 μM.s-1. Incubation of the enzyme with 1 to 100 millimolar mercuric chloride for 5-20- and 60 min resulted in progressive inhibition of the enzymatic activity. At low Hg++ concentrations the inhibition was reversible by excess substrate, while at high Hg++ concentration the inhibition was not reversible. Results also indicated that the type of inhibition depended on the duration of incubation of the enzyme with metal ion and on the Hg++ concentration. So we could conclude that the type of inhibition changed from noncompetitive to mix with increased incubation time and increased metal concentration.

The present research discusses the structure stabilizing and protecting effects of Ni2+ against suicide-peroxide inactivation of horseradish peroxidase (HRP). Suicide inactivation of HRP by hydrogen peroxide (3 mM) was monitored by measuring change in the absorbance of the colored product (tetraguaiacol) of the catalytic reaction cycle at 470 nm. Progress curves of the catalytic reaction cycle were obtained at 27 oC, phosphate buffer (5 mM), pH 7.0. The corresponding kinetic parameters (e.g., initial enzyme activity (a o) and the apparent rate constant (ki) of suicide inactivation of HRP by peroxide) were evaluated using a kinetic equation derived in this study. Comparative activatory and inhibitory effects of Ni2+ on the kinetics of suicide-peroxide inactivation of HRP are discussed.

Background: Immunohistochemistry (IHC) is a practical technique that utilizes the specific binding between an antigen and antibody to detect and localize specific antigens in tissue and cells. The optimal sensitivity in IHC is of utmost importance to achieve reliable results even when antigens are present at low abundance on the samples. Here, a dextran polymer labeled with Horseradish Peroxidase (HRP) and an anti-body to improve the sensitivity of the IHC technique was synthesized. Methods: To this end, free thiol groups were introduced on sodium periodate-activated 30 kDa dextran using cystamine, followed by attachment of sulfo-MBS-activated goat anti-mouse antibody and sulfo-MBS-activated HRP to the activated dextran. Results: The production of Poly-HRP Antibody (PHA) was confirmed by the appearance of a new protein band exceeding 150 kDa on Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE). Additionally, Enzyme-Linked Immunosorbent Assay (ELISA) and IHC techniques were employed to characterize PHA’s functionality. The data demonstrated that PHA effectively detected target antigens in these assays. Conclusion: The newly synthesized PHA has the potential to provide a more sensitive platform for early detection of biomarkers in IHC. Further research is needed to evaluate its cost-effectiveness.