What Affects Enzyme Activity (Practical)

Propose a method means set out a procedure someone else could follow and defend the choices in it. The marks live in the variables, the repetition and the risk management as much as in the steps themselves.

7 marks. Identify the three classes of variable, give a measurable dependent variable, set out the procedure, state repeats and the safety measure, and say what you would do with the data.

The independent variable is temperature, which I would set at five values across a suitable range, for example 10, 20, 37, 50 and 70 degrees Celsius, using thermostatically controlled water baths. The dependent variable is the rate of reaction, measured as the volume of oxygen gas produced in a fixed time. The controlled variables are the concentration and volume of hydrogen peroxide, the mass or concentration and source of the catalase, the pH of the solution, and the total time over which gas is collected.

Place a fixed volume of hydrogen peroxide solution of known concentration in a conical flask and, in a separate tube, the same mass of catalase source, for example a measured disc of potato tissue. Stand BOTH in the first water bath for five minutes so that each reaches the set temperature before they meet. Then tip the catalase into the flask, seal it with a bung connected by delivery tube to an inverted measuring cylinder or gas syringe, and start timing immediately. Record the volume of oxygen collected after exactly 60 seconds. Repeat the whole procedure at each of the remaining temperatures.

Perform three trials at each temperature and calculate a mean volume of oxygen, discarding any obvious outlier and noting why it was discarded. Repeating in this way reduces the effect of random error, such as small variations in the surface area of the potato discs or a slight delay in inserting the bung, and allows the consistency of the results to be judged.

Hydrogen peroxide is corrosive at the concentrations used, so safety glasses and gloves should be worn and any spillage rinsed immediately with water. The hot water bath at 70 degrees Celsius presents a scald hazard, so flasks should be handled with tongs or a heatproof glove. The reaction produces gas and the flask is sealed, so the bung must not be forced tightly enough to prevent the delivery tube venting.

Plot mean oxygen volume against temperature and draw a line of best fit. The expected pattern is a rise in rate up to an optimum near 37 degrees Celsius, as increasing kinetic energy produces more frequent enzyme-substrate collisions, followed by a sharp fall as the enzyme denatures and its active site is permanently distorted.

The investigation is valid because it changes only temperature while holding every other factor that affects catalase activity constant, and because the dependent variable, the volume of oxygen produced in a fixed time, is a direct measure of reaction rate rather than a proxy for it.

Assess means make a judgement of value, so the answer must end somewhere. Listing faults is not assessing; each fault has to be weighed as affecting validity, reliability or both, and then a verdict given.

5 marks. Define the two terms as you use them, judge validity, judge reliability, then state an overall verdict with the improvement that matters most.

An investigation is valid if it measures what it claims to measure, which requires that only the independent variable is changed and that the dependent variable genuinely reflects the quantity of interest. It is reliable if repeating it produces consistent results, which requires sufficient repetition to identify and reduce random error. The two are independent: an investigation can be reliably wrong.

Validity is compromised in several respects. Counting bubbles is a poor measure of reaction rate because bubbles vary in size, so equal counts need not represent equal volumes of oxygen; measuring gas volume with a syringe would be a direct measure. Only three pH values are used and they are widely spaced, so the optimum can only be located to within four pH units and the shape of the curve cannot be established. Several controlled variables are unstated — the surface area and age of the potato discs, the temperature of the solutions, and the concentration of hydrogen peroxide — and any of these varying between trials would change the rate independently of pH. There is also no mention of allowing solutions to reach a common temperature before mixing.

Reliability is very poor: each pH was tested once, so there is no way to identify an anomalous result, no mean can be calculated, and the contribution of random error to any single reading is unknown. A difference between two pH values could be entirely the product of one unusually thick potato disc. Three trials at each pH, with a mean taken and outliers noted, would be the minimum needed to make the results reliable.

Overall the investigation is neither valid nor reliable, and the conclusion it could support is correspondingly weak: it might indicate that catalase is more active near neutral pH, but it could not establish an optimum or the shape of the relationship. Of the improvements available, replacing bubble counting with a measured gas volume and repeating each pH three times are the two that matter most, since the first addresses the measurement itself and the second makes it possible to know whether any observed difference is real.