Abstract :
Enzymes are Biological catalysts called enzymes that quicken metabolic reactions in living things. They play essential roles in metabolism, energy storage, and other biological processes. Enzymes can be isolated and used in commercial operations due to their catalytic properties. These enzymes act in low quantities without being consumed, making them highly efficient. UV-Vis spectroscopy is commonly employed to study enzyme kinetics and interactions, allowing real-time monitoring of enzymatic activity by measuring changes in absorbance at specific wavelengths. This technique is crucial for observing concentration changes of substrates and products during enzymatic reactions. The OPA spectrophotometric assay is a widely used method for measuring enzyme activity, particularly for proteinases. The assay involves reacting O-phthalaldehyde (OPA) with primary amines, forming a fluorescent adduct that can be measured at 340 nm to quantify proteolysis. The assay is efficient, rapid, and highly sensitive, making it suitable for various research and clinical applications. The NADH-linked enzyme assay utilizes the UV absorbance of NADH at 340 nm to track enzymatic reactions, especially those involving dehydrogenases like lactate or alcohol dehydrogenases. Changes in absorbance over time are used to determine reaction rates and enzyme activity. In addition, curve fitting is an essential technique in UV-Vis spectroscopy that helps interpret complex data. By fitting mathematical models to the experimental spectra, researchers can resolve overlapping peaks, perform quantitative analysis, and study reaction kinetics. Both the OPA and NADH-linked assays, alongside curve fitting, provide powerful tools for studying enzyme mechanisms and activities in diverse scientific fields.
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