B2 L5Amylase practical

• 01.1 line 1 (the beaker of water): water bath(1)
• 01.1 line 2 (the tile): spotting tile(1)
• 01.1 line 3 (the pipette): dropping pipette(1)
• 01.2 Blue-black(1)

01.1: 1 mark for each correct label. If more than one word from the box is written on one line, do not give that mark.
01.2: if more than one box is ticked, 0 marks.

Model answer01.1 1 water bath; 2 spotting tile; 3 dropping pipette. 01.2 Blue-black.

• 02.1 160(1)
• 02.2 210 (seconds)(1)
• 02.3 1000 ÷ 80(1)
• 02.3 = 12.5 (per second)(1)

02.1: (150 + 180 + 150) ÷ 3.
02.2: allow "pH 7, test 3".
02.3: allow 13. Correct answer with no working = 2 marks. do not accept 0.08 (the time divided by 1000) for the answer mark.

Model answer02.1 (150 + 180 + 150) ÷ 3 = 160 s. 02.2 210 s (pH 7, test 3). 02.3 1000 ÷ 80 = 12.5 per second.

• 03.1 (pH) 6(1)
• 03.2 pH 8 is further from the optimum (pH)(1)
• 03.2 the shape of the active site changes / the amylase is (partly) denatured(1)
• 03.2 so starch does not fit the active site (as well) / less starch is broken down per second(1)

03.2: max 3. allow "pH 6 is the optimum" for the first mark. do not accept "the enzyme is killed / dies" (that point scores 0; other points can still score). ignore "the enzyme does not work".

Model answer03.1 pH 6. 03.2 pH 8 is further from the optimum pH of 6. At pH 8 the shape of the active site of the amylase changes, so starch does not fit as well. Less starch is broken down per second, so the rate is slower.

Any two from:

• the mixture was only tested every 30 seconds, so the starch may have gone up to 30 s before the recorded time(1)
• the colour changes gradually / it is hard to judge when the iodine stays orange-brown / the end point is subjective(1)
• the stop clock may not have been started exactly when the starch was added(1)

max 2. ignore "human error" or "reaction time" with no explanation.

Model answerThe mixture was only tested every 30 seconds, so the starch may have gone before the recorded time. The colour changes little by little, so it is hard to judge exactly when the iodine stays orange-brown.

Level of response. Read your answer, choose the level that fits best, then pick your mark.

Indicative content

• put a drop of iodine solution into each well of a spotting tile
• measure a volume of amylase solution (for example 2 cm³) into a test tube with a syringe
• add a volume of pH 5 buffer solution (for example 1 cm³)
• measure a volume of starch solution (for example 2 cm³) into another test tube
• stand both tubes in a water bath at a set temperature (for example 35 °C) for a few minutes, so they reach that temperature
• add the starch to the amylase and buffer, mix, and start the stop clock
• every 30 seconds, put one drop of the mixture onto a new drop of iodine
• blue-black shows that starch is still there
• record the time when the iodine stays orange-brown (the end point)
• keep the same temperature, the same volumes, the same concentrations of amylase and starch and the same time between tests
• repeat (three times) and calculate a mean
• wear eye protection

A method that adds iodine to the test tube, or gives the end point as "when the iodine turns orange-brown", is not a correct end-point method: such an answer cannot reach Level 3.

Your mark:

Model answer (Level 3)Put a drop of iodine solution into each well of a spotting tile. Use a syringe to put 2 cm³ of amylase solution into a test tube and add 1 cm³ of pH 5 buffer solution. Put 2 cm³ of starch solution into another test tube. Stand both tubes in a water bath at 35 °C for 5 minutes. Add the starch to the amylase and buffer, mix, and start the stop clock. Every 30 seconds, use a dropping pipette to put one drop of the mixture onto a new drop of iodine. Stop the clock when the iodine stays orange-brown, and record the time. Keep the temperature, the volumes and the concentrations of amylase and starch the same, so it is a fair test. Repeat three times and calculate the mean. Wear eye protection.