What volume of ADH enzyme (in units of microlitres, µL) is added as the last reagent to the reaction mixture?

Question

BlackTom AI · Accepted Answer

Alcohol dehydrogenase (ADH) is an enzyme that catalyses the conversion of alcohols and is therefore added last to a prepared reaction mixture to start the reaction. Enzyme assay best practice: prepare buffer, substrate and cofactors first, equilibrate temperature, then add the enzyme in a small aliquot to initiate and time the reaction.

Step-by-step reasoning:
1. Relevant concepts: enzymes are catalysts and should be added last to start the reaction; enzyme stocks are typically concentrated so only a small volume is required; adding a small aliquot minimizes dilution of the substrate/cofactors and gives a clear start time.
2. Typical assay volumes: many cuvette or microplate assays have total reaction volumes in the range of ~200–1000 µL. Enzyme aliquots are normally small (often 1–10 µL, sometimes up to 20 µL) to avoid excessive dilution while providing sufficient activity.
3. Simple quantitative check: if total volume is 1 mL, adding 10 µL is 1% of the volume (minimal dilution). Adding 100 µL would be 10% — a large change in concentration of substrates and cofactors and an unusually large enzyme consumption. Similarly, 50 µL (5%) or 20 µL (2%) are progressively larger dilutions. Practical protocols therefore favor the smallest listed volume as the enzyme addition.
4. Why 10 µL is correct: it is the smallest plausible aliquot among the choices and aligns with standard lab practice of adding a concentrated enzyme last in a small volume to initiate the reaction.

Why the other choices are incorrect:
- 20 µL: larger than typical minimal enzyme aliquot and causes more dilution.
- 50 µL and 100 µL: unrealistically large for initiating an enzyme assay from a concentrated stock; would substantially alter reaction concentrations and waste enzyme.

Final confirmation: the correct answer is a. 10 µL, matching the provided answer.

What volume of ADH enzyme (in units of microlitres, µL) is added as the last reagent to the reaction mixture?单项选择题

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