Biology › Practicals › Required practical 1: enzyme rate with trypsin and milk

Required practical 1: enzyme rate with trypsin and milk

Milk powder in water is cloudy because of its protein, casein. Trypsin hydrolyses casein to soluble peptides, so the suspension clears as the reaction runs, and a colorimeter turns that clearing into a falling absorbance. The gradient at the start of the curve is the initial rate, and repeating the run at several temperatures shows the rate rising to an optimum and collapsing above it.

The capability this develops

This guide is one investigation under capability P05: Measuring the rate of an enzyme-controlled reaction, which sets out the techniques the investigation belongs to, the calculations it carries and the errors that cost marks in all of them. The notes behind the biology are in Enzymes and metabolic control.

Where this sits on the specifications

This library maps a qualification to its own specification one numbered heading at a time, against a document whose sha256 is recorded, and publishes the result as a route map. These are the headings whose practical work this investigation serves, on four of the five qualifications mapped so far. Each links to the route map the row belongs to.

Some boards number their practical activities, and those lists were read here from the same specifications as the headings above. This investigation is numbered on them as:

• AQA 7401/7402 required practical 1
• OCR Biology A H020/H420 PAG 4
• Pearson Edexcel Biology A (Salters-Nuffield) 8BN0/9BN0 core practical 4
• Pearson Edexcel Biology A (Salters-Nuffield) 8BN0/9BN0 core practical 12
• Pearson Edexcel Biology B 8BI0/9BI0 core practical 1

The other qualifications above are matched by specification heading only, because none of them numbers an activity that is this investigation. Use your awarding organisation’s current specification and its practical handbook as the authority on which activities your course requires and how they are assessed.

The principle

Casein makes a milk-powder suspension cloudy. Trypsin hydrolyses it to soluble peptides, so the suspension clears and its absorbance falls.

Trypsin is a protease: it catalyses the hydrolysis of peptide bonds inside a polypeptide chain. The colorimeter shines light of one colour through the cuvette and reports how much fails to reach the detector. The cloudier the suspension, the higher the reading, so the gradient of absorbance against time measures how fast casein is being broken down. Absorbance has no unit, so the rate is in s⁻¹.

The gradient falls throughout a run, because the casein is being used up. Only the gradient at t = 0, the initial rate, reflects the conditions that were set. A gradient taken later also reflects how much substrate happened to be left.

Temperature acts on the rate in two opposite directions. Warmer molecules move faster, so trypsin and casein collide more often and more enzyme–substrate complexes form each second. Above the optimum, the hydrogen and ionic bonds that hold the tertiary structure break, the active site changes shape, and the enzyme is denatured. The first effect is gradual and the second is fast, which is why the curve rises slowly and falls steeply.

Initial rate

The rate of a reaction at the moment it starts, found as the gradient of the tangent to the progress curve at t = 0.

Temperature coefficient, Q10

The rate at a temperature 10 °C higher divided by the rate at the lower temperature.

Denaturation

A permanent change in the tertiary structure of a protein, which in an enzyme changes the shape of the active site.

Apparatus

• Skimmed milk powder suspension in pH 8 buffer, made up as one batch for the whole session
• Trypsin solution, 1%, and the same pH 8 buffer for diluting it
• A colorimeter, cuvettes with lids or film to cover them, and one filter used for every reading
• Water baths at 20, 25, 30, 35, 40, 50 and 60 °C, or one bath reset between runs, and a thermometer
• Syringes or graduated pipettes, 1 cm³ and 5 cm³
• Test tubes, a rack and a stopwatch
• A boiling water bath for the boiled-trypsin control
• Eye protection

Safety

Risk assessment. Enzymes can cause allergic reactions, especially as powders: avoid raising dust and wash off splashes. Anyone allergic to milk says so beforehand. Iodine solution stains skin and clothing. Wear eye protection, lift tubes from a hot bath with a holder, and keep cables clear of the water. Check the CLEAPSS Hazcard for each substance and your school's risk assessment before starting; where they differ from this page, follow them.

Variables

Independent variable

Temperature, set by the water bath and checked with a thermometer in a tube of water standing beside the reaction tubes. Seven values: 20, 25, 30, 35, 40, 50 and 60 °C. The 25 °C and 35 °C runs are there so that Q10 can be calculated from a pair 10 °C apart below the optimum.

Dependent variable

The initial rate of hydrolysis: the gradient of the tangent to the absorbance–time curve at t = 0, with the minus sign dropped, since absorbance falls and the rate is how fast it falls. The unit is s⁻¹, or absorbance units per second.

Control variables

• The volumes. 2.0 cm³ of milk suspension and 2.0 cm³ of trypsin solution in every run, measured by syringe.
• The concentrations. One stock of trypsin, diluted once to 0.5%, and one batch of milk suspension, stirred before each volume is drawn.
• pH. Both made up in the same pH 8 buffer, because hydrolysis can change the pH and the rate depends on it.
• The time in the bath. Ten minutes for every pair of tubes, at every temperature, because above the optimum the enzyme denatures while it waits.
• The colorimeter. One filter, and the instrument zeroed on the same blank, for every reading.

Method

Preparing the solutions and the colorimeter

1. Make the milk suspension and the trypsin solution in the same buffer, for example pH 8. Hydrolysis can change the pH; the buffer holds it constant.
2. Make the trypsin 0.5% by diluting 5 cm³ of the 1% stock to 10 cm³ with buffer, and make enough of it, and of the milk, for every run in the session.
3. Zero the colorimeter with buffer and trypsin but no milk, and keep one filter throughout. The reading is then the cloudiness of the milk alone.
4. Boil 3 cm³ of the trypsin in a water bath for 5 minutes and cool it to the test temperature. Set aside a tube of buffer to use in place of trypsin as well.
5. Boiled trypsin, cooled to the test temperature, shows that clearing needs active enzyme. Buffer in place of trypsin shows whether the milk clears or settles by itself.

Running one temperature

1. Stand the milk and the trypsin in separate tubes in the water bath for 10 minutes, and check each with a thermometer. Both start at the test temperature, so the reaction does too.
2. Mix fixed volumes by syringe, start the stopwatch as they mix, and read the absorbance every 30 s for 5 minutes. Close early readings fix the shape of the curve near t = 0, where the tangent is drawn.
3. Cover the cuvette and invert it once before each reading, so that settled milk does not pass for hydrolysed milk, and stand it back in the bath between readings.
4. Run the boiled-trypsin tube and the buffer-only tube in the same way at the same temperature.

Describe the drawing

A graph of product formed against time. The upper cyan curve rises steeply from the origin, bends over and flattens into a plateau. A dashed amber straight line touches it at the origin and runs above it everywhere else, marking the initial rate. A lower grey curve made with half the substrate has the same shape but levels off at a lower plateau.

The other temperatures, and the rate from each curve

1. Repeat the run three times at each temperature, with fresh solutions each time. Work through the temperatures in a random order, not from cold to hot, so that anything drifting through the session is not mistaken for the effect of temperature.
2. Plot absorbance against time for each run, draw a smooth curve, rule the tangent at t = 0 and take its gradient.
3. Take the mean of the repeats at each value. A repeat far from the others is an anomaly: look for a cause, such as a late start, leave it out of the mean and say so.
4. Without a colorimeter, keep the tube in the bath in front of a card marked with a cross, and time until the cross can be seen through the mixture.

Sample data

The rates below are idealised. They were computed from a model of the relationship rather than measured, so that the analysis can be worked without scatter in the way, and the 25 °C and 35 °C values are the ones the lesson works. Real readings scatter, and each value stands for the mean of three runs. None of it is a record of an experiment.

The 35 °C row comes from one curve. The absorbance fell from 0.80 at mixing to 0.68 at 30 s, 0.59 at 60 s, 0.47 at 120 s and 0.33 at 300 s. A tangent ruled at t = 0 passes through (0, 0.80) and (100, 0.35).

Gradient = (0.35 − 0.80) ÷ 100 = −0.0045 s⁻¹.

So the initial rate is 0.0045 s⁻¹, which is 4.5 × 10⁻³ s⁻¹ in the table. The mean rate over the whole run is (0.80 − 0.33) ÷ 300 = 0.0016 s⁻¹, about a third of the initial rate, because casein was being used up from the first second.

Q10 from 25 °C to 35 °C is 4.5 ÷ 2.1 = 2.1, and from 20 °C to 30 °C it is 3.1 ÷ 1.5 = 2.1. From 30 °C to 40 °C it is 5.9 ÷ 3.1 = 1.9.

The boiled-trypsin tube held its absorbance at 0.80 for the whole 5 minutes. The buffer-only tube fell by 0.02 as the milk settled, which is small beside the fall of 0.47 at 35 °C but is the reason the cuvette is inverted before each reading.

Reading the graph

Plot the initial rate on the vertical axis against temperature on the horizontal axis, and join the points with a smooth curve rather than straight lines. No temperature between two points was run, and a curve is the better estimate of what happens there.

The rise. From 20 °C to 40 °C the rate rises, roughly doubling for each 10 °C: Q10 is 2.1 over the two lower ranges. More kinetic energy means more frequent collisions between trypsin and casein, so more enzyme–substrate complexes form each second.

The optimum. The highest rate measured is at 40 °C. The true optimum lies between 35 °C and 50 °C, and these readings cannot place it more closely, because nothing between 40 °C and 50 °C was run. Runs at 42, 44, 46 and 48 °C would narrow it.

The fall. Above the optimum the rate drops from 5.9 × 10⁻³ s⁻¹ at 40 °C to 0.5 × 10⁻³ s⁻¹ at 60 °C. Hydrogen and ionic bonds holding the tertiary structure break, the active site changes shape and fewer complexes can form. Draw the curve asymmetric, with a gradual rise and a steep fall.

Q10 and where to take it. Q10 falls from 2.1 to 1.9 between the lower ranges and the 30 °C to 40 °C range, because some trypsin is already being denatured during the ten minutes at 40 °C. A Q10 is a statement about the kinetic effect alone, so it is taken from a pair of temperatures below the optimum.

Use temperatures below the optimum, where denaturation during the run is negligible.

Evaluation

Each of these is a real source of error in this investigation, and the middle column is the part an answer earns marks for: what it does to the result, and in which direction.

Questions

Each answer opens where you tap it.

Coverage maps are available for AQA 7401/7402, OCR Biology A H020/H420, Cambridge International 9700, Pearson Edexcel Biology A (Salters-Nuffield) 8BN0/9BN0 and Pearson Edexcel Biology B 8BI0/9BI0. Use the relevant route map to check what InkBiology covers, and your awarding organisation’s current specification as the final authority on examinable content.