Fluorometric-enzymatic lactate determination based on enzyme cytochrome b2 fluorescence

Area of Science:

• Biochemistry
• Analytical Chemistry
• Enzymology

Background:

• Lactate determination is crucial in various biological and food science applications.
• Existing methods may have limitations in sensitivity or specificity.
• Enzyme-based assays offer potential for high selectivity and sensitivity.

Purpose of the Study:

• To develop and validate a fluorometric-enzymatic procedure for accurate lactate quantification.
• To investigate the underlying principles of fluorescence changes during enzymatic lactate oxidation.
• To establish a reliable method for lactate determination in complex matrices like milk.

Main Methods:

• Utilized L-lactic dehydrogenase (cytochrome b2) for enzymatic lactate oxidation.
• Monitored changes in enzyme fluorescence intensity correlated with lactate concentration.
• Employed ferricyanide as an oxidizing agent and investigated its role in fluorescence quenching.
• Developed a mathematical model to linearize the analytical signal response.
• Validated the method using milk samples.

Main Results:

• Demonstrated an irreversible decrease in enzyme fluorescence intensity proportional to lactate concentration.
• Identified fluorescence quenching mechanisms including enzyme-ferricyanide complexation and inner filter effects.
• Established a linear relationship between fluorescence signal and lactate, cytochrome, and ferricyanide concentrations.
• Achieved precise and accurate lactate determination in milk samples.
• The method is effective for lactate concentrations ranging from 0.2 to 45 mg/L.

Conclusions:

• The developed fluorometric-enzymatic method provides a sensitive and specific approach for lactate determination.
• The mathematical model accurately accounts for signal variations, enhancing reliability.
• This procedure offers a valuable tool for routine lactate analysis in milk and potentially other biological samples.