International A Level Biology / Proteins and DNA

Core practical 4: measuring the initial rate of an enzyme reaction

Edexcel International A Level Biology (YBI11), chapter 5: Proteins and DNA, specification point 2.8. Free lesson.

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The initial rate is how fast an enzyme reaction proceeds at the start, measured before conditions in the flask have changed. You find it from the gradient of a tangent at time zero, then explain how substrate being used up makes the reaction slow down.

How to measure oxygen production

Catalase breaks down hydrogen peroxide into water and oxygen. The oxygen pushes the plunger of a gas syringe out, so you can measure how much is made. Trypsin digesting milk can also be followed with a colorimeter, because the milk clears as its protein is digested. Use a conical flask connected by a bung and delivery tube to a gas syringe. Add a set volume of hydrogen peroxide and buffer to the flask. The buffer keeps the pH steady.

How to calculate the initial rate

Each curve is steepest at the start, then becomes less steep and eventually flat. The initial rate is the gradient of the curve at time zero. Draw a straight tangent, which touches the curve at that point and follows its starting slope. Use a large triangle on the tangent, not on the curve. Gradient = change in oxygen volume ÷ change in time. The unit is cm³ s⁻¹, because volume is measured in cm³ and time in seconds. Use a ruler and read both scales carefully.

Why the curve levels off

At the start, plenty of hydrogen peroxide molecules collide with enzyme active sites, so many enzyme-substrate complexes form each second. This gives a steep curve. As the substrate is used up, collisions and complexes become less frequent, so the rate falls. When all the substrate is broken down, no more oxygen is made and the curve is flat. The enzyme is not used up. The initial rate is measured while the substrate concentration is still the value you set, before other conditions can change, such as the enzyme denaturing at a high temperature.

How to change one variable fairly

To test the effect of a variable, change that variable and keep the others steady. A control is different from a controlled variable: it is a run with water instead of enzyme, used for comparison. Temperature: use a water bath at five or more temperatures, or a thermostatic water bath to keep one temperature constant. pH: use buffer solutions at a range of pH values, or the same buffer in every flask. Enzyme concentration: dilute catalase to make a range, or use the same volume of the same catalase solution each time. Substrate concentration: dilute hydrogen peroxide to make a range, or…

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