2cBiological molecules

Carbohydrates, proteins and lipids are all large molecules built from smaller basic units joined together. Learn the elements and the building units — these are straight recall marks.

The chemical elements

The only thing that separates proteins from the other two is nitrogen. If a question asks how you would tell a protein apart from a carbohydrate by its elements, the answer is nitrogen.

Carbohydrates and lipids contain exactly the same three elements — they differ in the proportions, not the elements. Lipids contain proportionally much less oxygen.

Large molecules from basic units

Carbohydrates

Simple sugars such as glucose are the basic unit. Many glucose molecules join in a long chain to make starch (the storage carbohydrate in plants) or glycogen (the storage carbohydrate in animals and fungi).

The chemical formula of glucose is C₆H₁₂O₆ — learn it. It is needed for the respiration and photosynthesis equations elsewhere in the course, and candidates regularly lose marks for not knowing it or for writing an unbalanced equation.

Proteins

A protein is a chain of amino acids joined together. There are about 20 different amino acids, and the order of amino acids in the chain determines the shape and therefore the job of the protein. The chain folds into a specific three-dimensional shape — this matters enormously for enzymes.

Lipids

One molecule of glycerol joined to three fatty acids makes one lipid molecule. Fats are solid at room temperature, oils are liquid — both are lipids.

Common mistake: writing that proteins are used for insulation or as an energy store. That is lipids. Proteins are for growth and repair (and for making enzymes). Examiners report seeing "proteins are used for insulation of energy" — a muddle of two different food groups.

Common mistake: plants need nitrate ions from the soil to make amino acids and proteins; humans get their nitrogen by eating protein. Do not say humans absorb or transport nitrates to make protein — examiners flag this confusion between plant mineral requirements and human nutrition.

Quick self-test

• Which food group contains nitrogen? Protein.
• Glycogen is built from what? Simple sugars / glucose.
• How many fatty acids per lipid molecule? Three, plus one glycerol.

Examiner tip. Do not swap the functions of the food groups. Lipids (not proteins) are the insulator and the energy store; protein is for growth and repair. And remember glucose is C₆H₁₂O₆ — a surprising number of candidates cannot recall this or balance equations that use it.

You must be able to describe all four tests: the reagent, any heating, and the colour change from and to. A colour change is only worth a mark if you give both the starting and finishing colour.

Preparing a solid food sample

Grind the food with a pestle and mortar, add distilled water, stir, and filter to get a clear solution. Test the filtrate.

The four tests

Points examiners look for

• With Benedict's, the amount of colour change depends on how much sugar is present, so the test is semi-quantitative: more glucose gives a colour further along towards brick-red.
• With biuret, state that you add sodium hydroxide first, then copper sulfate (or say "biuret reagent"). Do not heat it.
• With the ethanol emulsion test, ethanol is highly flammable — never use a naked flame nearby. Use a water bath, not a Bunsen, for the Benedict's test on the same bench.
• Wear eye protection throughout; sodium hydroxide is corrosive.

Safety and control

• Use the same volume of food solution and of reagent for each sample so results can be compared.
• Test a known positive (e.g. starch solution) and a negative control (distilled water) so you know the reagents are working.

Wording that scores

"Add Benedict's solution to the food sample and heat in a water bath at 80 °C. If glucose is present the solution turns from blue to brick-red."

That sentence pattern — reagent, condition, from-colour, to-colour — earns full marks every time.

Examiner tip. Examiners report that the biuret test for protein is usually described well, but marks are lost when candidates give only one colour ("it goes purple") without the starting colour, or forget the heating step for Benedict's. Always write "from blue to brick-red", not just "red".

What an enzyme is

An enzyme is a protein that acts as a biological catalyst: it speeds up the rate of a metabolic reaction and is not used up or changed in the reaction, so it can be used again and again.

Metabolic reactions are the chemical reactions happening inside living organisms — building large molecules up and breaking them down. Almost every one is controlled by an enzyme.

The active site

Each enzyme has a region called the active site, with a shape complementary to its substrate (the molecule it works on).

1. The substrate fits into the active site, forming an enzyme–substrate complex.
2. The reaction happens and the products are released.
3. The enzyme is unchanged and picks up another substrate molecule.

Because only one shape of substrate fits, enzymes are specific — one enzyme, one reaction.

Named enzymes worth learning

Vague answers such as "enzymes with no functions" or "enzymes break down food" score nothing. Name the enzyme and its substrate and product.

Common mistake: writing that amylase breaks starch down into glucose. It produces maltose; maltase then produces glucose. Also, spell maltase (the enzyme) and maltose (the sugar) correctly — examiners cannot credit the wrong word. The same goes for lipase and protease.

Increasing temperature: below the optimum

As temperature rises, enzyme and substrate molecules gain kinetic energy and move faster. There are more frequent successful collisions, so more enzyme–substrate complexes form and the rate of reaction increases.

Rate roughly doubles for every 10 °C rise, up to the optimum temperature (about 37 °C for most human enzymes).

Above the optimum: denaturing

Above the optimum the rate falls sharply:

1. The extra energy makes the enzyme molecule vibrate.
2. Bonds holding the protein in its three-dimensional shape break.
3. The shape of the active site changes.
4. The substrate no longer fits the active site — it is no longer complementary.
5. No enzyme–substrate complexes form, so the reaction stops. The enzyme is denatured, and this is permanent (irreversible).

Low temperature

At low temperatures the rate is very slow because molecules move slowly and collide rarely. The enzyme is not denatured — it is simply inactive, and activity returns when it is warmed again. This is why food is kept in a fridge.

Examiner tip. Enzymes are molecules, not organisms: they are denatured, never "killed" and they never "die". Living things such as bacteria and yeast are killed by high temperature, never "denatured". Examiners specifically report both errors. And do not stop at "the active site changes shape" — go on to say the substrate no longer fits, no enzyme–substrate complexes form, and the reaction stops.

The standard investigation uses amylase digesting starch, with iodine solution to detect when the starch has gone.

Method (amylase and starch)

1. Put a drop of iodine solution into each well of a spotting tile.
2. Place a test tube of starch solution and a test tube of amylase solution in a water bath at 20 °C for 5 minutes so both reach the water bath temperature.
3. Mix them, start a stopwatch, and return the tube to the water bath.
4. Every 30 seconds, remove one drop of the mixture with a pipette and add it to a well of iodine.
5. Record the time taken for the iodine to stay orange-brown — this is the point at which all the starch has been broken down.
6. Repeat the whole experiment at 10, 30, 40, 50 and 60 °C.

Rate of reaction = 1000 ÷ time in seconds. Plot rate against temperature.

Expected result

Rate increases up to an optimum (around 35–40 °C) and then falls sharply as the amylase is denatured.

Alternative: catalase and hydrogen peroxide

Add a piece of liver or potato to hydrogen peroxide and collect the oxygen produced in a gas syringe or by displacement of water. Measure the volume of gas in 60 seconds at each temperature.

Variables — get the language right

Reliable means the results can be trusted because they repeat consistently. To improve reliability, repeat each temperature three times, discard anomalous results and calculate a mean. Repeating is not the same as being accurate, and it is not the same as the dependent variable — examiners report candidates muddling these terms.

Safety

Wear eye protection. Iodine solution stains and irritates; hydrogen peroxide is an irritant. Use a thermostatically controlled water bath rather than heating over a flame.

If the question says "plan" or "devise"

Write a method: apparatus, the range of temperatures, what you measure and how, controls, repeats. Examiners report that many candidates instead write an essay describing how temperature affects enzymes — that is a different question and scores almost nothing on a planning question.

Examiner tip. Read the command word. "Plan an investigation" needs numbered practical steps with apparatus, a stated range of the independent variable, control variables and repeats — not a description of the theory. Also be precise with "reliable" (consistent on repeating) and "dependent variable" (what you measure).

Objective 2.14 (the pH practical) is marked B in the specification, which means it is examined in Separate Biology but not in Science (Double Award). Objective 2.13, the theory behind it, is for everyone.

Why pH matters

Every enzyme has an optimum pH at which its rate of reaction is highest.

Explaining the effect — the full chain

If the pH moves away from the optimum:

1. The excess H⁺ or OH⁻ ions interfere with the bonds holding the enzyme in its three-dimensional shape.
2. The shape of the active site changes.
3. The active site is no longer complementary to the substrate, so the substrate no longer fits.
4. Fewer (or no) enzyme–substrate complexes form.
5. The rate of reaction falls; at extreme pH the enzyme is denatured and the reaction stops completely.

A graph of rate against pH is a symmetrical peak centred on the optimum.

Common mistake: stopping at step 2. Examiners repeatedly report that candidates say "the active site changes shape" and then write nothing more. You must go on to the consequence for function — substrate cannot bind, no enzyme–substrate complex, reaction rate falls. That is where the marks are.

Practical: effect of pH on amylase

1. Put a drop of iodine solution in each well of a spotting tile.
2. Add 1 cm³ of amylase and 1 cm³ of a buffer solution at pH 3 to a test tube.
3. Add 2 cm³ of starch solution, mix and start a stopwatch.
4. Keep the tube in a water bath at a constant 35 °C.
5. Every 30 seconds transfer a drop of the mixture to a well of iodine.
6. Record the time taken for the iodine to remain orange-brown (all starch digested).
7. Repeat using buffer solutions at pH 5, 7, 9 and 11.

Rate = 1000 ÷ time (s). Plot rate against pH; the optimum is the pH giving the shortest time / highest rate.

Variables

• Independent variable: pH (set using buffer solutions — this is the key piece of apparatus).
• Dependent variable: time for the starch to be fully digested.
• Control variables: temperature (water bath), volume and concentration of amylase, volume and concentration of starch, size of drop, sampling interval.

Reliability and safety

Repeat each pH three times and calculate a mean, ignoring anomalies. Wear eye protection; buffers at pH 3 and pH 11 are irritants.

Two-mark answer template

"At pH 11 the rate is low because the change in pH alters the bonds holding the enzyme's shape, so the active site changes shape and the starch can no longer fit, so no enzyme–substrate complexes form."

Examiner tip. Know the difference between 'describe' and 'explain'. Describe the graph = say what happens (rate rises to a peak at pH 7 then falls). Explain = give the reason (active site changes shape, substrate no longer fits). Examiners report these two command words being confused more than any other, and a description scores nothing on an 'explain' question.