A fluorometric assay of peroxidase activity utilizing 2′,7′-dichlorofluorescein with thiocyanate: application to the study of salivary secretion

https://doi.org/10.1016/0165-022X(94)90065-5Get rights and content

Abstract

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 at submandibular glands. Comparison of the 2′,7′-dichlorofluorescein-thiocyanate assay and the commonly used 2,2′-azinobis(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.

References (21)

There are more references available in the full text version of this article.

Cited by (16)

  • Effects of molecular weight of hyaluronic acid on its viscosity and enzymatic activities of lysozyme and peroxidase

    2018, Archives of Oral Biology
    Citation Excerpt :

    The experiments for measuring enzymatic activities of HEWL and salivary lysozyme were performed eight times in duplicate. Peroxidase activity was determined by the method described previously (Hannig et al., 2008, Hannig, Spies, Spitzmüller, & Hannig, 2010; Hannig, Spitzmüller et al., 2010; Proctor & Chan, 1994). In the presence of peroxidase and hydrogen peroxidase, fluorogenic 2′,7′-dichlorofluorescein (LDCF) is oxidized to fluorescent dichlorofluorescein (DCF).

  • Enzyme activities in parotid saliva of patients with the restrictive type of anorexia nervosa

    2017, Archives of Oral Biology
    Citation Excerpt :

    Extinction was read at γ = 405 nm at 25 °C. Peroxidase activity was determined as described in detail previously (Hannig et al., 2008; Proctor & Chan, 1994). In the presence of peroxidase and hydrogen peroxide, fluorogenic 2,7-dichlorofluorescein (LDCF) is converted to fluorescent dichlorofluorescein (DCF).

View all citing articles on Scopus
View full text