Biology 9700/51 — October/November 2014

Background Concept

In any experiment the independent variable is the one the experimenter deliberately changes between trials, while the dependent variable is the one that is measured to record the effect of that change. Every other factor (concentrations, volumes, temperature, pH) must be held constant so that any difference in the dependent variable can be attributed to the independent variable.

In this investigation the student is comparing two physical forms of the same enzyme, so the independent variable is the physical state of the enzyme. The measurement they take is the time taken for the methylene blue indicator to lose its colour, which is the dependent variable. Note that time (not rate) is what is actually measured; rate is then derived from it.

Understanding the Question

The student has set up a comparison between free and immobilised ethanol dehydrogenase. Everything else (NAD concentration, ethanol concentration, methylene blue concentration, enzyme concentration before immobilisation) has been fixed by the procedure described in the stem. The question asks you to name what is varied (independent) and what is measured (dependent).

Approach

Read the stem and identify: (1) the one factor that is deliberately different between the two trials — that is the independent variable; (2) the quantity that the student records with the stop clock — that is the dependent variable.

Step-by-Step Reasoning

• The two conditions being compared are "free enzyme" vs. "immobilised enzyme", so the independent variable is the type/state of the enzyme.
• The student uses a stop clock/timer to record when methylene blue turns colourless; this is a measurement of time. Time is the dependent variable.
• The mark scheme specifically rejects "rate" as a description of the dependent variable, because rate is a derived quantity (1/time) and the student does not directly measure rate.

Key Takeaways

• The independent variable is what you change; the dependent variable is what you measure.
• Time is a directly measured quantity; rate is derived from it.
• Vague phrasings such as "amount of enzyme" or "enzyme concentration" are wrong here because these were not varied — the same 1 mg dm⁻³ solution was used to make both preparations.

Common Mistakes

• Writing "amount of enzyme" — the concentration was the same; only the form (free vs immobilised) differed.
• Writing "rate of reaction" as the dependent variable — rejected by the mark scheme because rate is calculated, not measured.
• Writing "colour change" without specifying that it is the time taken for the colour to change that is being recorded.

Things to Be Careful About

• Use the CIE wording: "decolourise" (or "become colourless") for what methylene blue does; "time" must be explicit.
• The independent variable must refer to the form of the enzyme (free/immobilised), not to the enzyme itself.

Background Concept

Enzymes can be immobilised by trapping them inside an insoluble, porous matrix. One common CIE method uses sodium alginate: when a solution of sodium alginate is dropped into a solution of calcium chloride, the calcium ions replace the sodium ions and cross-link the alginate polymer, forming insoluble calcium alginate beads. Enzyme molecules inside the droplets become trapped in the gel lattice while substrates and products can still diffuse in and out, so the enzyme remains catalytically active and can be reused.

Understanding the Question

The stem says the student "immobilised" the ethanol dehydrogenase solution. You are asked to outline how this is done in enough detail that another person could repeat it. Three marks are available for the key procedural points.

Approach

Identify the three essential steps that the mark scheme rewards: (1) mixing with alginate, (2) contacting with calcium chloride, (3) a method of producing the droplets (beads).

Step-by-Step Reasoning

• Step 1 — Make a sodium alginate solution (typically 2–3 %) and mix it with the enzyme solution so that the enzyme is uniformly distributed.
• Step 2 — Prepare a calcium chloride solution (typically around 1–3 %) in a beaker.
• Step 3 — Draw the alginate/enzyme mixture into a syringe (or pipette) and squeeze/drop it into the calcium chloride. Each drop forms a bead as the calcium ions cross-link the alginate. After a few minutes the beads harden and can be collected and rinsed.

The mark scheme credits these three points and accepts Ca²⁺/calcium ions for calcium chloride and "syringe or pipette" (or even "dropper") for the method of forming beads. Specific concentrations are not required and are ignored if given.

Key Takeaways

• The standard CIE immobilisation technique is alginate beads set in calcium chloride.
• Only three key ideas are needed: alginate, calcium chloride, a method of producing droplets.
• The beads are porous: substrates/products can diffuse in and out, but the enzyme stays trapped.

Common Mistakes

• Writing "add calcium chloride to alginate" without specifying dropwise — the beads only form if the alginate enters the calcium chloride as discrete droplets.
• Omitting the mixing step and going straight to "add to calcium chloride".
• Suggesting a different method (e.g. adsorption on to a surface, entrapment in gelatine, covalent bonding) — these are valid but the question's wording "immobilise a 1 mg dm⁻³ solution" and the mark scheme both expect the alginate/calcium chloride route.
• Including glass beads as an immobilisation method — glass beads alone are not an immobilisation technique in this context.

Things to Be Careful About

• "Dropwise" / "using a syringe or pipette" is the credit-worthy detail that turns the method from "add to CaCl₂" into "form beads".
• Do not give specific concentrations — the mark scheme ignores them.

Background Concept

A control is a trial that is identical to the experimental trial except for the factor being tested; it tells you what would happen in the absence of the active enzyme. For an enzyme-catalysed reaction, the enzyme must be present and active for the reaction to proceed at a meaningful rate. Anything that destroys enzyme activity (boiling denatures the protein) or removes the enzyme entirely (water) acts as a control for any non-enzymic changes that might occur — for instance slow spontaneous oxidation of methylene blue by air, or a chemical reaction between ethanol and methylene blue that does not depend on the enzyme.

Understanding the Question

The student is measuring how quickly methylene blue decolourises in the presence of ethanol dehydrogenase. We need to know how much of that decolourisation is actually due to the enzyme and how much is due to other factors. A control answers that question by running everything else the same but with no active enzyme.

Approach

The mark scheme credits the simple idea of replacing the active enzyme with something that has no enzyme activity (boiled enzyme, denatured enzyme, water). One mark is enough.

Step-by-Step Reasoning

• A suitable control = same volume of liquid in place of the enzyme solution, but containing no active enzyme.
• Boiled enzyme satisfies this — boiling denatures the protein so it can no longer catalyse the reaction, but all the other components are still present.
• Water is the other accepted answer; it provides no enzyme at all.
• The mark scheme explicitly rejects "without enzyme" unqualified (because it is not specific enough), "glass beads", and "NAD/ethanol" as controls.

Key Takeaways

• The control must be identical to the experimental tube in every respect except the presence of active enzyme.
• Boiled enzyme and water are the two standard CIE-accepted controls for an enzyme experiment.
• The control allows the non-enzymic background rate (e.g. spontaneous decolourisation) to be measured and subtracted later.

Common Mistakes

• Writing "without enzyme" alone — this is too vague; the mark scheme requires the specific replacement (boiled enzyme or water).
• Writing "use glass beads" — glass beads are not a control here; they are sometimes used as an immobilisation matrix but they do not, by themselves, control the experiment.
• Writing "use a different concentration of enzyme" — that would be a different experimental variable, not a control.

Things to Be Careful About

• A control is not the same as a repeat; a control varies what is being tested, a repeat does not.
• Be specific about what replaces the enzyme (boiled enzyme or water).

Background Concept

Methylene blue is blue when oxidised and colourless when reduced. In this enzyme reaction NAD is reduced, and the reduced NAD then reduces methylene blue; the enzyme itself is not consumed. The faster the enzyme works, the quicker methylene blue is reduced and the sooner the blue colour disappears. Because the time taken is inversely proportional to the speed of the reaction, the rate is calculated as $1/t$.

A control tube (no active enzyme) is included because methylene blue can also decolourise slowly by other routes — for example by reaction with atmospheric oxygen or by slow spontaneous reduction by ethanol. Subtracting the control rate removes this background and leaves only the enzyme-catalysed component.

Understanding the Question

Table 1.1 gives four already-processed rates (with standard deviations), but the question is about how those rates were obtained. You must explain the two operations that turn the raw time readings into the figures in the table.

Approach

1. Convert time to rate by taking the reciprocal of the time taken for the indicator to turn colourless.
2. Subtract the corresponding control rate from the experimental rate so that only the enzyme-catalysed reaction is recorded.

Step-by-Step Reasoning

• For each tube, divide 1 by the time taken for the methylene blue to become colourless. This gives a rate in arbitrary units (the mark scheme accepts "arbitrary units").
• Do this for both the experimental tubes (free and immobilised enzyme + ethanol + NAD + methylene blue) and the control tubes (no active enzyme + ethanol + NAD + methylene blue).
• Subtract the control rate from the experimental rate for the free enzyme, and similarly for the immobilised enzyme. The two values that result are the true enzyme-catalysed rates, which is what is shown in Table 1.1.

A compact way to write this is:

The mark scheme's extra guidance gives exactly this algebraic form as an acceptable single-sentence answer: "$\frac{1}{\text{time experimental}} - \frac{1}{\text{time control}} = 2$" — meaning the control-corrected rate equals this difference.

Key Takeaways

• Time decolourised → rate by taking 1/time.
• Subtract the control to remove non-enzymic background.
• The two operations together give the true enzyme-catalysed rate.

Common Mistakes

• Writing rate as "1 ÷ time" for the experimental tube only and forgetting to subtract the control — the data in the table are already corrected for the control.
• Writing rate as "time⁻¹" without explaining the reciprocal step.
• Dividing instead by the control: the mark-scheme form is (1/t experimental) − (1/t control), not the other way round.

Things to Be Careful About

• The units in Table 1.1 are "arbitrary units", because 1/time has units of time⁻¹ (e.g. s⁻¹) but the absolute number depends on the volumes used. The mark scheme does not require a specific unit.
• Apply the correction separately to the free enzyme pair and to the immobilised enzyme pair; do not subtract controls across columns.

Background Concept

Standard deviation ($s$) is a statistic that summarises how far individual replicate measurements lie from their mean. A small $s$ means the replicates cluster tightly around the mean; a large $s$ means they are widely scattered. In practical terms, $s$ is a measure of repeatability/reliability of the data — the smaller it is, the more confidence you have that another repeat would give a similar value.

Understanding the Question

Table 1.1 gives rates with their standard deviations, e.g. $0.0333 \pm 0.0024$ for the free enzyme. The student wants to know what those "±" figures tell the reader about the quality of the investigation.

Approach

There are two distinct ideas to credit:

1. $s$ describes the spread of the values around the mean.
2. That spread is an indicator of reliability — and the small numerical values in the table (relative to the means) show the data are reliable.

Step-by-Step Reasoning

• Spread of the data: Standard deviation tells you how much individual repeat readings deviate from the mean; the larger the $s$, the more variable the repeats.
• Reliability: Reliability refers to whether you would get similar results if you repeated the experiment. A small $s$ implies high reliability because the repeats were tightly clustered.
• Concrete interpretation of Table 1.1: For the free enzyme, $s = 0.0024$ on a mean of $0.0333$ — the standard deviation is roughly 7 % of the mean. For the immobilised enzyme, $s = 0.0022$ on a mean of $0.0222$ — about 10 %. Both are small in absolute and relative terms, so the data can be regarded as reliable.

The mark scheme explicitly rejects "reliability of the mean" (you cannot assess the mean's reliability from $s$ alone — that is what the standard error does) and rejects "accuracy" or "validity". It also ignores "standard deviation is less than one" as a stand-alone point. It accepts quoted numerical support for the smallness of $s$.

Key Takeaways

• Standard deviation = spread of data about the mean.
• Small $s$ → reliable (repeated measurements agree closely).
• $s$ does not tell you about accuracy (closeness to the true value) or about the reliability of the mean.

Common Mistakes

• Writing "$s$ shows the reliability of the mean" — that describes the standard error of the mean, not the standard deviation.
• Writing "$s$ shows accuracy / validity" — $s$ cannot tell you how close the mean is to the true value.
• Writing "$s$ is small" without saying what small $s$ implies (spread of data; reliability).

Things to Be Careful About

• Quote a numerical value if you can — e.g. "$s = 0.0024$ is much smaller than the mean of $0.0333$" — to support the reliability point.