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Q: What does H2 Biology Practical: Enzyme Kinetics (Catalase) Guide cover?
A: Run a syllabus-aligned catalase assay with serial dilutions, temperature control, initial rate estimation, and spreadsheet-backed graphs so MMO/PDO/ACE marks are predictable.TL;DR Treat enzyme practicals as controlled investigations: set a single independent variable,
keep conditions constant, record volumes/times consistently, and calculate initial rate
from the linear region. Use a clean serial dilution, control temperature, and graph rate vs
substrate concentration with error bars for ACE credit.1 Aim & overview (catalase + hydrogen peroxide) Investigate how substrate concentration affects catalase activity using the oxygen evolved.
Keep pH and temperature constant; measure initial rate via gas volume/time or pressure change.
Recommended timing: 60 minutes end-to-end with one variable across 5-6 levels.
2 Apparatus & reagents
,
).
Buffer (pH ~7), distilled water.
Measuring cylinders, syringes/pipettes, conical flasks, rubber bung + delivery tube.
Water bath set to
25 ∘ C \pu{25 ^\circ C} 25 ∘ C (or other chosen constant temperature).
Gas collection: inverted measuring cylinder or gas syringe (preferred for precision).
Stopwatch, marker pens, labels, ice/heat packs for quick temperature adjustments.
Safety: Wear goggles/gloves; handle hydrogen peroxide carefully; secure glassware; keep workstation dry.
3 Serial dilution plan (substrate concentration) Target 5-6 concentrations with equal volume per trial. Example (10 mL reaction mix):
Tube \(\ce{H2O2} \space (\pu{mL})\) Buffer/Water (\(\pu{mL}\))Notes A \(1.0\) \(9.0\) Highest concentration B \(0.8\) \(9.0\) C \(0.6\) \(9.0\) D \(0.4\) \(9.0\) E \(0.2\) \(9.8 \) Lowest concentration
Mix gently; prepare fresh substrate (
H X 2 O X 2 \ce{H2O2} H X 2 O X 2 decomposes over time).
Label clearly; pre-equilibrate all tubes to
25 ∘ C \pu{25 ^\circ C} 25 ∘ C in the water bath.
4 Method (MMO focus) Place the gas syringe (or inverted measuring cylinder setup) and ensure an airtight seal.
Pipette a fixed volume of catalase (e.g.,
2.0 m L \pu{2.0 mL} 2.0 mL ) into each conical flask; equilibrate at temperature.
Add the substrate mix (e.g.,
8.0 m L \pu{8.0 mL} 8.0 mL ) to start the reaction. Immediately bung the flask and start the stopwatch.
Record gas volume at consistent
10 s \pu{10 s} 10 s intervals for 1-2 minutes (initial phase).
Repeat for all concentrations; run at least one repeat for a key concentration to estimate variability.
Controls: a heat-denatured catalase tube or a zero-substrate tube to confirm baseline.
5 Data treatment (PDO) with spreadsheets Tabulate time (
s \pu{s} s ) and gas volume (
m L \pu{mL} mL ). Use the first
30 − 60 s \pu{30-60 s} 30 − 60 s where the trace is most linear.
Compute initial rate from slope (e.g., change in volume per second). Do not force the line through the origin unless justified.
Plot rate vs substrate concentration with axes labelled and units shown.
Add error bars (e.g., standard deviation across repeats) where available.
Optional: If concentration saturates the enzyme, note the plateau behaviour qualitatively.
Example rate statement: “Initial rate = 0.18 m L ⋅ s − 1 \pu{0.18 mL.s-1} 0.18 mL ⋅ s − 1 at 0.8 % H X 2 O X 2 0.8\% \space \ce{H2O2} 0.8% H X 2 O X 2 , 25 ∘ C \pu{25 ^\circ C} 25 ∘ C , pH 7.”
6 Evaluation (ACE): uncertainties and improvements Gas leakage or delays when bungs are fitted → pre-seat bungs; practise sealing to reduce dead time.
Temperature drift → use a water bath and work quickly; log actual readings for each run.
Biological variability (enzyme prep differences) → mix thoroughly; prepare a single batch for all runs.
Reading resolution on gas syringe or cylinder → choose appropriate scale; read at eye level (avoid parallax).
Control of independent variable → confirm fresh substrate and verify volumes with calibrated pipettes.
Use a gas pressure sensor for continuous data; export CSV to speed up slope calculation.
Standardise catalase concentration using optical density (optional extension for school labs with spectrometers).
7 60-minute exam-pace schedule Minute Action 0-5 Label tubes, set up water bath, leak-check gas collection 5-15 Prepare serial dilutions; pre-equilibrate 15-40 Run reactions at 5-6 concentrations; record volumes 40-50 Compute initial rates; draft graph 50-60 Write conclusion, limitations, and improvements (ACE)
8 Links & next steps
Assessment anchors (MMO/PDO/ACE) MMO (Manipulation, Measurement, Observation)
Correct serial dilution, accurate volumetric transfer (fixed total volume), airtight gas path, immediate timing on mixing.
Temperature/pH control maintained and logged; repeats at a key concentration.
Appropriate controls (heat‑denatured enzyme or zero substrate) included.
PDO (Presentation of Data and Observations)
Time (s) and volume (mL) tables with units and consistent s.f.; clear note of the initial linear window used.
Rate calculation shown (e.g., Δvolume/Δtime) with units; graph of rate vs concentration with axes/units; error bars where available.
Legend/caption explains conditions (temperature, pH, enzyme source).
ACE (Analysis, Conclusions, Evaluation)
Trend described (rate increases then plateaus) with biological reasoning (active site saturation).
Limitations discussed (leaks, H2O2 decomposition, temperature drift); improvements proposed (logger, standardised enzyme batches, more repeats around plateau).
References SEAB. (2024). Singapore-Cambridge GCE A-Level Biology (9729) Syllabus (for examination in 2026). Singapore Examinations and Assessment Board.
Campbell, N. A. et al. (2020). Campbell Biology (12th ed.). Pearson - enzyme catalysis laboratory practices.
H2 Biology Practical: Enzyme Kinetics (Catalase) Guide