Optimizing the o-phenylenediamine assay for horseradish peroxidase: effects of phosphate and pH, substrate and enzyme concentrations, and stopping reagents.

Abstract

Horseradish peroxidase, assayed with o-phenylenediamine, is irreversibly inactivated when incubated in phosphate buffer, 100 mmol/L, at pH 5. The inactivation depends on both duration and incubation and phosphate concentration. Phosphate was the most potent inactivator and citrate the least potent of a series of buffers tested. The inactivation is not attributable to ionic strength per se or to Na+ or K+. The observed inactivation did not occur at high concentrations (2500 nmol/L, 0.1 g/L) of enzyme; however, this "protective" effect could not be reproduced by adding bovine serum albumin or a surfactant (Tween 20) to lower concentrations of enzyme. The inactivation was independent of commercial source of the enzyme or the kind of chromogenic assay used. On the basis of this information, we optimized the assay so that it gave eightfold greater absorbance values than those reported by others. The improved assay was sensitive to as little as 0.4 pmol/L (16 ng/L) of peroxidase, and was linear over the range of 0.4 to 5 pmol/L (16-200 ng/L).

Catalytic resonance scattering spectral determination of ultratrace horseradish peroxidase using rhodamine S

“…According to the procedure, 0.05 mL samples was added and the HRP content in the samples was determined and the results are given in Table 4. Table 4 showed that the results of the catalytic RS assay were agreement with that of the spectrophotometry (11). HRP has been widely used in water treatment (30).…”

“…Thus, it is important to develop a simple and sensitive method to detect its activity. Current methods for determination of HRP include voltammetry (1-3), chemiluminescence method (CL) (4)(5)(6)(7)(8), spectrophotometry (9)(10)(11)(12), fluorescence spectrometry (FS) (13,14) and flow injection analysis (FIA) (15,16). Using aniline as a substrate, the voltammetric method has high sensitivity.…”

Stabilization of Proteins by Nanoencapsulation in Sugar–Glass for Tissue Engineering and Drug Delivery Applications

“…Once shaken or vortexed, the samples were directly reconstituted with suitable buffer or lyophilized to remove the solvent from the system in the presence or absence of trehalose (0.012 mL of 23% trehalose in water) followed by reconstitution with buffer. Reconstituted proteins samples (protein–buffer and protein–SGnP samples with or without solvent treated) were used for their specific activity assay (see Supporting Information for details) 33, 34, 35. Catalytic activity was measured for reconstituted HRP/lipase samples.…”

Substrate specificity of the Chamaerops excelsa palm tree peroxidase. A steady-state kinetic study

“…Among the broad variety of organic and inorganic substrates of peroxidases, here four organic substrates were studied: the chromogenic substrates guaiacol (2-metoxiphenol) and ABTS, often used as reference substrates, and o-dianisidine and o-phenylendiamine, the latter three suitable for use in ELISA procedures that employ peroxidase conjugates [25][26][27][28].…”