In horseradish peroxidase (HRP) catalysis, which substance acts as the substrate?

• A. Naphthol-AS-phosphate
• B. Hydrogen peroxide
• C. 3,3'-diaminobenzidine
• D. Tetramethylbenzidine

Answer: (B)

In horseradish peroxidase (HRP) catalysis, hydrogen peroxide acts as the substrate that provides the oxidizing power to start the reaction. HRP interacts with hydrogen peroxide to form an activated intermediate, which then oxidizes a chromogenic substrate to produce the color. Substances like 3,3'-diaminobenzidine or tetramethylbenzidine are the chromogenic substrates that HRP oxidizes in the presence of hydrogen peroxide to yield color, but they are not the enzyme’s primary substrate in the catalytic cycle. Naphthol-AS-phosphate is used with alkaline phosphatase, not HRP. So the substance that acts as the substrate in HRP catalysis is hydrogen peroxide.