This is an enzyme, lagunase.

Lagunase converts NAD+ and ethanol into aldehyde and NADH.

Lagunase needs two Zn2+ ions in order to function.

Design an assay for lagunase.

What will you measure?

What will you add to the mixture to start the rxn?

What will your data give you?

Question

This is an enzyme, lagunase.

Lagunase converts NAD+ and ethanol into aldehyde and NADH.

Lagunase needs two Zn2+ ions in order to function.

Design an assay for lagunase.

What will you measure?

What will you add to the mixture to start the rxn?

What will your data give you?

BlackTom AI · Accepted Answer

NADH formation is the convenient readout because reduced NADH has a strong, specific absorbance at 340 nm (ε340 ≈ 6220 M−1cm−1) while NAD+ does not. Therefore you measure the concentration change of NADH spectrophotometrically over time.

Relevant knowledge points:
- The enzyme reaction: ethanol + NAD+ → aldehyde + NADH. NADH accumulation reports reaction progress.
- Zn2+ is an essential cofactor for this enzyme, so assay buffer must supply Zn2+ (or use holo-enzyme).
- Initial rate measurements of NADH formation give enzyme activity (µmol product formed per minute), whereas specific activity requires separate protein concentration determination (units per mg).

Step-by-step reasoning and practical assay design:
1. Prepare assay buffer containing an appropriate concentration of NAD+ and Zn2+ (to ensure the enzyme is in its active, metal-bound form) and place it in the spectrophotometer cuvette. Add a known amount of lagunase so the enzyme concentration is fixed.
2. Start the reaction by adding ethanol (substrate) and immediately begin recording A340 vs time. Alternatively, you can pre-equilibrate everything except enzyme and start by adding lagunase — both are common approaches; the key is initiating substrate turnover and measuring NADH increase.
3. Convert absorbance change to concentration change using Beer–Lambert law: A = ε·c·l. For a 1 cm pathlength:
   - Rate (M/min) = (ΔA340/min) / 6220 M−1cm−1.
   - Convert to µmol/min: rate(µmol/min) = rate(M/min) × cuvette volume(L) × 10^6.
   - That value is enzyme activity (U = µmol/min).

Why the correct answer is correct:
- Measuring concentration change of NADH directly monitors product formation.
- The assay mixture must include NAD+, Zn2+, lagunase and ethanol (Zn2+ for activity and NAD+ + ethanol as reactants); starting the rxn involves adding the substrate or enzyme into that mixture.
- The raw rate gives activity (µmol/min). Specific activity would require measuring protein mass; since that extra step isn’t specified, “activity” is the correct output.

Why other options are incorrect (brief):
- “Absorbance of lagunase” is irrelevant: enzyme absorbance doesn’t reflect catalytic turnover.
- Mg2+ is wrong because lagunase specifically needs Zn2+.
- Options claiming “specific activity” are incorrect unless you also determine protein concentration to express rate per mg enzyme.

Confirming final answer: measure concentration change of NADH; assemble assay with NAD+, Zn2+, lagunase and ethanol (start by adding substrate or enzyme); your data yield enzyme activity — matching the provided answer.

This is an enzyme, lagunase. Lagunase converts NAD+ and ethanol into aldehyde and NADH. Lagunase needs two Zn2+ ions in order to function. Design an assay for lagunase. What will you measure? What will you add to the mixture to start the rxn? What will your data give you?单项选择题

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