WJEC Biology for AS Level Student Book: 2nd Edition

Practical exercises To determine the effect of temperature on trypsin activity Rationale Old-fashioned camera film has a light-sensitive layer of silver halide crystals held on an acetate strip by the protein gelatin. Exposed film is black. If exposed film is incubated in a trypsin solution, the gelatin is digested and the silver halide crystals detach from the acetate film, which is transparent. The time taken for the film to clear is a measure of the rate of reaction of the trypsin. Design Experimental factor Description Value Independent variable temperature 10°C, 30°C, 50°C, 70°C, 90°C Dependent variable time for acetate film to become clear seconds Controlled variables pH pH 7 volume of trypsin 25 cm 3 concentration of trypsin 5 g / 100 cm 3 dimensions of acetate film 10 mm × 30 mm Control boil and cool trypsin before use; see Study point Reliability calculate a mean time for 3 strips of film to clear at each temperature; see Study Point on p90 and Key term on p65 Hazard trypsin is a protein and is potentially allergenic so skin should be covered and eye protection worn; the proximity of water and electricity is a potential hazard and water above 60°C scalds Apparatus ▪ Trypsin at 5 g / 100 cm 3 made up in buffer pH 7 ▪ Exposed photographic film cut into rectangles 30 × 10 mm ▪ 5 × boiling tubes ▪ Water baths at 10ºC, 30ºC, 50ºC, 70ºC and 90ºC ▪ Forceps ▪ Timer Method 1. Place 25 cm 3 of each trypsin solution in a separate boiling tube. 2. Equilibrate the trypsin solution to temperature in each water bath, for 10 minutes. 3. Without removing the tubes from the water bath, submerge a 30 × 10 mm rectangle of exposed photographic film in each trypsin solution. 4. Observe the films in turn to determine whether or not the acetate film has cleared. 5. Record in seconds the time taken for the photographic strip to become completely transparent. Study point For a control in an enzyme experiment, the enzyme should be boiled for 5 minutes to denature it and then cooled before use. Using water is not a suitable control as a zero concentration is just another value of the enzyme concentration. Study point Many experiments require controlled temperature. Thermostatically controlled water baths are most suitable. Study point When measuring time, e.g. for a colour change, give readings to the nearest second. Human judgement and response are not fast enough to measure accurately to less than 1 s, so a timer graduated to 0.01 s may be more accurate, but it will not improve the accuracy of the readings. Theory check 1. Explain the term ‘active site’. 2. Why does increased temperature increase the rate of an enzyme- catalysed reaction? 3. Despite the answer to q2, why does the rate of an enzyme- catalysed reaction decrease above a certain temperature? 4. When the temperature-induced rate increase (in q2.) equals the rate decrease (in q3.), the reaction rate is at its maximum. This happens at the enzyme’s ‘optimum temperature’. Why do different enzymes have different optimum temperatures? 6 WJEC Biology for AS Level 84