Which reagent is essential in the coupling enzyme reaction for generating a colored product in the cholesterol oxidase method?

In the cholesterol oxidase method for cholesterol determination, the coupling enzyme reaction is critical for generating a colored product that can be measured spectrophotometrically. The correct reagent used in this process is 4-Aminoantipyrine.

4-Aminoantipyrine plays a pivotal role as it reacts with hydrogen peroxide (produced during the oxidation of cholesterol) in the presence of peroxidase. This reaction leads to the formation of a colored dye, which is quantifiable. The intensity of the color produced is directly proportional to the concentration of cholesterol in the sample, making it essential for the assay's sensitivity and specificity.

While cholesterol oxidase catalyzes the conversion of cholesterol to cholest-4-ene-3-one and produces hydrogen peroxide, it is 4-Aminoantipyrine that provides the necessary chemistry for color development, enabling the measurement of cholesterol levels in clinical samples.