Tan A: a fluorogenic probe for thiaminase I activity
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Vitamin B1 plays important role in cell metabolism. Deficiency of vitamin B1 can cause ailments in living organism, including EMS (Early Mortality Syndrom) in the predatory fish salmon in the Great Lakes. Deficiency of vitamin B1 can be caused by excessive intake of thiaminase I, which is contained excessively in major forage fishes in the Great Lakes, is a transferase-type enzyme that can degrade vitamin B1 by SN(AE) mechanism. However, the occurrence of EMS in the Great Lakes has a year to year variation as large as 90%. In order to understand the pattern of EMS occurrence, a method for thiaminase I activity measurement is needed. A radiometric assay is accurate and has been used for over 30 years, however it is expensive and requires labs to be permit for radioactive material. Another assay is the 4-NTP colorimetric assay, but it suffers from accuracy problems. For these reasons, a fluorophorogenic thaimine analogue is presented as a fluorescent probe for Thiaminase activity. The emission of the fluorophore is quenched by photoinduced electron transfer (PET) to the N-substituted pyridinium portion of the probe. Action of the enzyme releases the free pyridine group causing a substantial increase in fluorescence and the measured thiaminase I activity (based on initial rates) was proportional to enzyme concentration up to 60 ng/assay.
2012 Freely available theses (MU)