A fluorometric assay of peroxidase activity utilizing 2',7'-dichlorofluorescein with thiocyanate: application to the study of salivary secretion.
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.
Proctor
G B
GB
Secretory and Soft Tissue Research Unit, Rayne Institute, London, UK.
Chan
K M
KM
D000818:Animals; D052160:Benzothiazoles; D005452:Fluoresceins; D005453:Fluorescence; D005470:Fluorometry / Q000379:methods; D007202:Indicators and Reagents; D007784:Lactoperoxidase / Q000378:metabolism; D008297:Male; D010544:Peroxidases / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D012463:Saliva / Q000201:enzymology* / Q000378:metabolism*; D012680:Sensitivity and Specificity; D013363:Submandibular Gland / Q000378:metabolism*; D013451:Sulfonic Acids; D013861:Thiocyanates
D052160:Benzothiazoles; D005452:Fluoresceins; D007202:Indicators and Reagents; D013451:Sulfonic Acids; D013861:Thiocyanates; C002502:2,2'-azino-di-(3-ethylbenzothiazoline)-6-sulfonic acid; C037631:2',7'-dichlorofluorescein; D007784:Lactoperoxidase; D010544:Peroxidases; C031760:thiocyanate