Kinetic characterization of thyroid peroxidase enzyme and its inhibition by autoantibodies

A guaiacol method was developed to measure thyroid peroxidase (TPO) activity and the inhibitory effect of anti-TPO antibodies using purified porcine TPO. The TPO preparation was characterized kinetically and identified by western-blotting technique. The K(M) for guaiacol determined in vitro was 5.6 x 10(-4) M. Investigating the effect of IgG's from 23 hypothyroid patients on TPO activity inhibition was detected in 15 cases with the guaiacol method. It was found that anti-TPO antibodies exerted a competitive inhibition TPO activity with respect to the substrate guaiacol. The inhibition is carried by IgGF(ab')2 fragment. We wanted to gain a method which is interpreted immunologically and can be well employed in the clinical practice.