A sensitive fluorimetric method with guaiacol as the hydrogen donor for peroxidase activity is described. The gist of this method is measurement of the fluorescence (excitation, 300 nm; emission, 340 nm) in cyclohexane of mainly a guaiacol dimer which forms in the early phase of the color formation. Optimum conditions of the reaction were compared for horseradish peroxidase and rat thyroid peroxidase preparations. The fluorescence intensity obtained by this method using an enzyme preparation from l/20 of a rat thyroid gland correlated well with the color development by the guaiacol method using the same preparation from whole glands of a rat.
Thyroid peroxidase plays an important role in biosynthesis of thyroid hormones ( l-3) and its activity increases after administration of TSH and decreases after administration of thyroid hormone (4,5). Therefore, determination of thyroid peroxidase activity in animal experiments gives important information on the state of the thyroid. For the assay, calorimetric methods using guaiacol or iodide have been used (4,6,7), but their sensitivity is insufficient in experiments with small animals. We established a fluorimetric guaiacol method which has a high sensitivity sufficient for measuring the thyroid peroxidase activity of small animals.
Methods
Chemicals. Guaiacol (Nakarai Chemicals, Japan) was purified by distillation at reduced pressure, enclosed in brown ampules, and stored in a refrigerator. Horseradish peroxidase (HRP'; Boehringer-Mannheim, Grade 1, freeze-dried), hydrogen peroxide (30%, Mitsubishi Gas Chemical, Japan), sodium