Measuring Thiaminase Activity in Fish Extracts using Fluorescence Spectrophotometry.

Area of Science:

• Biochemistry
• Enzymology
• Analytical Chemistry

Background:

• Thiaminase enzymes degrade thiamine (vitamin B1).
• Measuring thiaminase activity in biological samples requires a sensitive and versatile assay.
• Existing assays may lack flexibility for diverse research needs.

Purpose of the Study:

• To develop a simple, sensitive, and versatile fluorescence-based assay for measuring thiaminase activity.
• To enable studies of thiaminase under various reaction conditions.
• To investigate thiaminase dynamics in aquatic ecosystems.

Main Methods:

• Developed a fluorescence spectrophotometry assay using a microplate reader.
• Measured thiaminase activity by quantifying thiamine destruction over time in fish tissue extracts.
• Optimized assay parameters including thiamine concentration, cosubstrate, and pH.
• Determined enzyme kinetics (Vmax, Km) and pH optimum.
• Compared results with a conventional assay.

Main Results:

• The assay successfully measured thiaminase activity in fish tissue extracts.
• Enzyme kinetics for Pacific herring thiaminase were determined (Michaelis-Menten kinetics).
• Positive cooperativity was observed for nicotinic acid as a cosubstrate.
• The pH optimum for rainbow smelt thiaminase was identified.
• First measurements of thiaminase activity in northern Bering Sea forage fishes were reported.

Conclusions:

• The developed fluorescence assay is a versatile tool for studying thiaminase activity.
• This assay facilitates research into factors causing thiamine deficiency in aquatic food webs.
• Provides crucial data for understanding thiaminase dynamics in the northern Bering Sea ecosystem.