2-2--azino-di-(3-ethylbenzothiazoline)-6-sulfonic-acid and thiocyanate

The oxidation of the pseudohalide thiocyanate (SCN(-)) by Euphorbia peroxidase, in the presence or absence of added calcium, is investigated. After incubation of the native enzyme with hydrogen peroxide, the formation of Compound I occurs and serves to catalyze the thiocyanate oxidation pathways. The addition of a stoichiometric amount of SCN(-) to Compound I leads to the native enzyme spectrum; this process clearly occurs via two electron transfers from pseudohalide to Compound I. In the presence of 10 mM calcium ions, the addition of a stoichiometric amount of SCN(-) to Compound I leads to the formation of Compound II that returns to the native enzyme after addition of a successive stoichiometric amount of SCN(-), indicating that the oxidation occurs via two consecutive one-electron transfer steps. Moreover, different reaction products can be detected when the enzyme-hydrogen peroxide-thiocyanate reaction is performed in the absence or presence of 10 mM Ca(2+) ions. The formation of hypothiocyanous acid is easy demonstrated in the absence of added calcium, whereas in the presence of this ion, CN(-) is formed as a reaction product that leads to the formation of an inactive species identified as the peroxidase-CN(-) complex. Thus, although monomeric, Euphorbia peroxidase is an allosteric enzyme, finely tuned by Ca(2+) ions. These ions either can enhance the catalytic efficiency of the enzyme toward some substrates or can regulate the ability of the enzyme to exploit different metabolic pathways toward the same substrate.

Topics: Benzothiazoles; Calcium; Cyanides; Dianisidine; Euphorbia; Hydrogen Peroxide; Hydrogen-Ion Concentration; Nuclear Magnetic Resonance, Biomolecular; Oxidation-Reduction; Peroxidase; Spectrophotometry; Sulfonic Acids; Thiocyanates

A sensitive assay for secretory peroxidase activity has been developed utilizing the fluorogenic substrate 2',7'-dichlorofluorescein in the presence of thiocyanate. The assay has been characterized using bovine lactoperoxidase and used to determine the peroxidase activities of salivas and extracts obtained from rat submandibular glands. Comparison of the 2',7'-dichlorofluorescein-thiocyanate assay and the commonly used 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) colorimetric assay indicates that the new assay is approx. 50-fold more sensitive. This has enabled measurement of peroxidase activities present in parasympathetic saliva samples which were beyond the detection limit of the colorimetric assay. Despite great differences in the peroxidase activities and protein concentrations of parasympathetic and sympathetic salivas and tissue extracts, the activities per unit protein were very similar. Unlike most other published methods, prior dialysis of samples to remove interference by endogenous thiocyanate is not required. The assay is therefore convenient and will be particularly useful for applications in which sample volume or peroxidase activity is low.

Topics: Animals; Benzothiazoles; Fluoresceins; Fluorescence; Fluorometry; Indicators and Reagents; Lactoperoxidase; Male; Peroxidases; Rats; Rats, Wistar; Saliva; Sensitivity and Specificity; Submandibular Gland; Sulfonic Acids; Thiocyanates

Because of the important biological functions of peroxidases, there is growing interest in the measurement of their concentrations in various secretions. At present, there is no standard method which allows for comparisons in reported activities. This report describes procedures which can be used to measure peroxidase enzyme concentrations by commonly employed assays. Regression equations have been determined which can be used to calculate concentrations of bovine lactoperoxidase (LPO), human salivary peroxidase (SPO), and human myeloperoxidase (MPO) from activities measured with the following donors: pyrogallol, guaiacol, 2,2'-azinobis(3-ethylbenzylthiazoline-6-sulfonic acid), and thiocyanate (SCN-). The peroxidation rates of these donors depend upon the concentrations of hydrogen peroxide (H2O2) used in the individual assays and thus, for accurate, reproducible results, these concentrations must be carefully controlled. The SCN- normally present in human saliva will reduce observed reaction rates by simple competition kinetics in the ABTS, guaiacol and pyrogallol assays and will increase the rates observed when Cl- is used as a donor in NBS assay for MPO. Therefore, SCN- must be removed from saliva samples prior to peroxidase activity determination by all assays except the thionitrobenzoic acid (NBS) assay. LPO cannot be used as a standard for either SPO or MPO because the specific activities of LPO, SPO, and MPO are significantly different.

Topics: Animals; Benzothiazoles; Binding, Competitive; Cattle; Humans; Hydrogen Peroxide; Hydrogen-Ion Concentration; Indicators and Reagents; Kinetics; Lactoperoxidase; Methods; Peroxidase; Regression Analysis; Saliva; Sulfonic Acids; Thiocyanates