In the CK-coupled enzymatic method, which cofactor is used in the final reaction and measured by UV spectrophotometry?

In the CK-coupled enzymatic method, which cofactor is used in the final reaction and measured by UV spectrophotometry?

• A. NAD
• B. FAD
• C. FMN
• D. NADP

Answer: (A)

The final step in the CK-coupled assay is lactate dehydrogenase, which uses the NAD/NADH cofactor pair. Pyruvate accepts electrons from NADH to become lactate, converting NADH to NAD+. The spectrophotometric readout is the amount of NADH, which absorbs strongly at 340 nm; as NADH is consumed, the absorbance decreases. So the cofactor involved is NAD (as the NAD/NADH couple), and the UV signal comes from NADH. The other cofactors listed are not used in this final LDH step.

The final step in the CK-coupled assay is lactate dehydrogenase, which uses the NAD/NADH cofactor pair. Pyruvate accepts electrons from NADH to become lactate, converting NADH to NAD+. The spectrophotometric readout is the amount of NADH, which absorbs strongly at 340 nm; as NADH is consumed, the absorbance decreases. So the cofactor involved is NAD (as the NAD/NADH couple), and the UV signal comes from NADH. The other cofactors listed are not used in this final LDH step.