Q.54 The activity of lactate dehydrogenase can be measured by monitoring the following reaction:
Pyruvate + NADH → Lactate + NAD+
The molar extinction coefficient of NADH at 340 nm is 6220 M−1·cm−1.
NAD+ does not absorb at this wavelength. In an assay, 25 μL of a sample of enzyme
(containing 5 μg protein per mL) was added to a mixture of pyruvate and NADH
to give a total volume of 3 mL in a cuvette of 1 cm pathlength.
The rate of decrease in absorbance at 340 nm was0.14 min−1.
The specific activity of the enzyme will be __________ μmol·min−1·mg−1.
Final Specific Activity: 96 μmol·min-1·mg-1
Introduction
Lactate dehydrogenase (LDH) catalyzes the reversible conversion of pyruvate
to lactate using NADH as a cofactor. Because NADH absorbs strongly at
340 nm while NAD+ does not, LDH activity can be accurately
quantified by monitoring the decrease in absorbance using the
Beer–Lambert law.
Key Inputs
- ΔA340/min = 0.14
- εNADH = 6220 M-1·cm-1
- Pathlength (l) = 1 cm
- Total assay volume = 3 mL
- Enzyme volume added = 25 μL
- Protein concentration = 5 μg·mL-1
Calculation Steps
Step 1: Convert Absorbance Change to NADH Consumption Rate
Change in NADH concentration per minute:
Δ[NADH]/min = ΔA / (ε × l)
= 0.14 / 6220
= 2.252 × 10-5 M·min-1
Step 2: Convert to μmol NADH per Minute in Cuvette
Moles NADH/min = (2.252 × 10-5 mol·L-1·min-1) × 0.003 L
= 6.755 × 10-8 mol·min-1
= 0.0676 μmol·min-1
Step 3: Calculate Amount of Enzyme Protein Used
Protein added = 25 μL × 5 μg·mL-1
= 0.125 μg
= 1.25 × 10-4 mg
Step 4: Apply Dilution Factor
Dilution factor = Total volume / Enzyme volume
= 3000 μL / 25 μL
= 120
Activity in stock solution = 0.0676 μmol·min-1 × 120
= 8.11 μmol·min-1·mL-1
Step 5: Calculate Specific Activity
Specific activity = (8.11 μmol·min-1·mL-1) / (0.005 mg·mL-1)
= 96 μmol·min-1·mg-1
Correct Answer
Lactate dehydrogenase specific activity =
96 μmol·min-1·mg-1
Option Analysis
- ≈ 96: Correct; matches complete Beer–Lambert and dilution calculations.
- 50–60: Underestimates by ignoring dilution factor.
- >150: Overestimates due to incorrect protein mass handling.
- None: Invalid, as numerical solution is definitive.
Common Pitfalls
- Forgetting conversion from mol to μmol (×106)
- Ignoring assay dilution factor
- Incorrect protein unit conversion (μg ↔ mg)
Biotechnological Relevance
LDH specific activity measurements are essential for enzyme purification,
fermentation monitoring, and quality control in biochemical engineering.
Similar calculations are standardized in commercial LDH assay kits
and are frequently tested in competitive exams.
