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Improving thiaminase measurements in Great Lakes forage fish
1 Department of Chemistry, University of Missouri, 601 S. College Ave. Columbia, MO 65203
2 Biochemistry & Physiology Branch, Columbia Environmental Research Center, U.S. Geological Survey, Department of Interior, 4200 New Haven Rd., Columbia, MO 65201
Two new fluorescent molecules have been developed for thiaminase: one used to measure thiaminase activity and the other one used to inactivate this interesting enzyme. The thiaminase sensor was designed based on a PET mechanism. The internal quenching of the fluorophore (6Hex) by the attachment of a pyrimidine group makes it non-emissive. Under the action of thiaminase (representative by bisulfite), the free pyridine is released, which results in an increase in fluorescence. In addition to the thiaminase sensor, a red fluorescent, selective, irreversible inhibitor has also been synthesized. The inactivation of this interesting molecule has been confirmed by both plate assays and fluorescent experiences. Inactivation was found to go to completion if given sufficiently long incubation, and to be irreversible.