In the described alkaline phosphatase assay, hydrolysis of the substrate yields phosphate and a colored product. What is this colored product?

In the described alkaline phosphatase assay, hydrolysis of the substrate yields phosphate and a colored product. What is this colored product?

• A. Phosphate
• B. 4-Nitrophenol
• C. Phenolphthalein
• D. Nitrite

Answer: (B)

This item tests how a common alkaline phosphatase colorimetric assay produces a visible color signal. In this assay, the enzyme cleaves the phosphate group from a substrate called p-nitrophenyl phosphate, releasing inorganic phosphate and p-nitrophenol. Under the alkaline conditions of the assay, p-nitrophenol loses a proton to form p-nitrophenolate, which has a distinct yellow color. That yellow product is what’s measured to quantify enzyme activity, typically by absorbance around 405 nm. The other options don’t fit because phosphate itself is colorless, phenolphthalein is not produced in this reaction, and nitrite is not a product of hydrolyzing this substrate.

This item tests how a common alkaline phosphatase colorimetric assay produces a visible color signal. In this assay, the enzyme cleaves the phosphate group from a substrate called p-nitrophenyl phosphate, releasing inorganic phosphate and p-nitrophenol. Under the alkaline conditions of the assay, p-nitrophenol loses a proton to form p-nitrophenolate, which has a distinct yellow color. That yellow product is what’s measured to quantify enzyme activity, typically by absorbance around 405 nm. The other options don’t fit because phosphate itself is colorless, phenolphthalein is not produced in this reaction, and nitrite is not a product of hydrolyzing this substrate.