Required practical 8: dehydrogenase activity in chloroplasts - AQA A-Level Biology (Paper 3) Exam Questions

Practise 15 exam-style questions on Required practical 8: dehydrogenase activity in chloroplasts for AQA A-Level Biology (Paper 3). Write your answer as you would in the exam and get it marked in seconds against the mark scheme, point by point, with the Study Helper on hand if you get stuck.

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Sample questions with model answers

Here are 4 of the questions in this topic, each with a model answer, an explanation of why it earns the marks and the mark scheme an examiner would use.

Question 1

2 marks

In one method for this practical, test tubes of DCPIP and chloroplast extract stand in a glass beaker of ice-cold water and are lit by a very bright mains lamp about 10 cm away.

Give two hazards of this set-up and a precaution to reduce the risk from each.

Model answer

1. Water is close to a mains lamp, so there is a risk of electric shock: keep the lamp, its cable and plug away from the water and dry your hands before touching the lamp.
2. The lamp is very bright and gets hot: do not look directly at it and do not touch it until it has cooled.

Explanation

A risk assessment identifies each hazard and a precaution that reduces the risk from that hazard. The specific hazards here come from combining water with mains electricity and from a very bright, hot lamp. Each precaution must match its hazard: 'wear goggles' does not reduce the risk of electric shock.

Mark scheme

1 mark
Water near the mains lamp / electrical equipment could cause an electric shock: keep water and wet hands away from the lamp and its plug / mop up spills / dry hands before touching the lamp
Max 2. 1 mark for each hazard with a precaution that reduces it. List rule: if more than two hazards are given, each wrong or contradictory extra answer cancels one correct answer.
1 mark
The very bright light can dazzle / cause a long-lasting afterimage: do not look directly at the lamp / point it away from people's eyes
1 mark
The lamp gets hot and could cause burns: do not touch the lamp / let it cool before moving it
1 mark
The glass beaker or test tubes could break and cause cuts: stand them on a stable surface away from the edge / handle with care and report breakages
Ignore 'DCPIP is harmful' unless a precaution is given (e.g. wear eye protection).

Question 2

3 marks

A student timed how long a mixture of DCPIP and chloroplast extract took to change from blue-green to green in bright light. She judged the end point by eye. Her five repeat times were:

48 s 55 s 42 s 60 s 51 s

Calculate the mean time and its uncertainty, using half the range of the repeat times as the uncertainty.

Suggest one change to her method that would reduce this uncertainty.

Model answer

Mean = (48 + 55 + 42 + 60 + 51) ÷ 5 = 256 ÷ 5 = 51.2 s
Uncertainty = (60 - 42) ÷ 2 = ±9 s
Mean time = 51 ± 9 s

She could use a colorimeter and time how long the mixture takes to reach a set absorbance, which removes the need to judge the colour by eye.

Explanation

When readings are repeated, the uncertainty can be estimated as half the range, so here the mean is 51 ± 9 s, a large uncertainty of about 18%. The main source of this uncertainty is judging the end point by eye, which is subjective. A colorimeter, or a colour standard to compare against, makes the end point objective; a more precise stopwatch would not help because the error is in the judgement, not the timer.

Mark scheme

1 mark
Mean = 51 s (accept 51.2 s); a correct value with no working gains this mark
Points 1 and 2 are calculations: a correct value with no working scores the mark for that value.
1 mark
Uncertainty = ±9 s ((60 - 42) ÷ 2)
Accept '51 ± 9 s' or '51.2 ± 9.0 s'. Do not accept ±18 s (the whole range).
1 mark
Use a colorimeter (e.g. time how long it takes to reach a set absorbance) OR compare the tube with a colour standard (extract without DCPIP) OR view the tubes against a white background
Any one of these. Ignore 'repeat more times' (this does not reduce the spread of the readings). Ignore 'use a more accurate stopwatch'.

Question 3

3 marks

A student used a colorimeter to follow the reduction of DCPIP by an illuminated chloroplast extract. DCPIP is blue when oxidised and colourless when reduced.

Describe how she should use the colorimeter to obtain valid absorbance readings.

Model answer

She should select a red filter, because blue DCPIP absorbs red light most strongly. She should set the colorimeter to zero absorbance with a cuvette containing chloroplast extract and water but no DCPIP, so that only the absorbance due to DCPIP is measured. She should hold the cuvettes by the ridged sides, put them in the same way round each time, mix the contents before each reading and take readings at fixed time intervals.

Explanation

A colorimeter measures how much light of one colour a solution absorbs. The filter should be the colour the solution absorbs most (red for a blue solution), giving the biggest change in reading. The blank must contain everything except the substance being measured, here the green chloroplasts, so the reading shows only the DCPIP. Fingerprints, bubbles, settling and inconsistent timing all add error.

Mark scheme

1 mark
Use a red / orange filter (the colour of light absorbed most by blue DCPIP)
Max 3. Do not accept a blue filter.
1 mark
Set the absorbance to zero using a blank containing chloroplast extract and water / buffer, without DCPIP
Accept 'calibrate with a cuvette of extract without DCPIP'. Accept 'zero with water' for this mark only if the answer says the extract's own absorbance is then subtracted.
1 mark
Use clean cuvettes / handle them by the ridged (frosted) sides / no fingerprints or bubbles on the clear sides
1 mark
Put the cuvette in the same way round each time, with the light passing through the clear (smooth) sides
1 mark
Mix / invert the cuvette before each reading (because chloroplasts settle)
1 mark
Take readings at fixed time intervals / at the same time after mixing
1 mark
Check the zero with the blank regularly / before each set of readings

Question 4

6 marks

Dehydrogenase enzymes are found in several organelles. A student wanted to show that the dehydrogenase activity that reduces DCPIP in the light is found in chloroplasts and not in other parts of leaf cells.

She had: lettuce leaves, ice-cold isolation medium, a blender, muslin, a refrigerated centrifuge that can spin at different speeds, test tubes, DCPIP solution, a lamp, aluminium foil and a colorimeter.

Describe how she could use this apparatus to obtain a chloroplast fraction, and to show that the light-dependent reduction of DCPIP happens in this fraction.

Model answer

Blend the lettuce leaves in ice-cold isolation medium and filter the mixture through muslin to remove debris. Centrifuge the filtrate at a low speed; the cell debris and nuclei form a pellet, which is removed. Centrifuge the supernatant at a higher speed so that the chloroplasts form a pellet. Resuspend this pellet in ice-cold isolation medium, and keep the remaining supernatant to test as well. Add 1 cm³ of each fraction to 5 cm³ of DCPIP solution in separate tubes, place the tubes at the same distance from the lamp at the same temperature, and measure the absorbance of each every minute with a colorimeter. Set up the same tubes wrapped in foil as dark controls. If the activity is in the chloroplasts, only the chloroplast fraction will reduce DCPIP in the light, with little change in the dark tubes or in the other fraction.

Explanation

Differential centrifugation separates organelles by size and density: large, dense debris and nuclei sediment at low speed, chloroplasts at a higher speed, and smaller organelles such as mitochondria at higher speeds still. Everything is kept ice-cold and in isolation medium to protect the organelles. Mitochondria also contain dehydrogenases, so comparing each fraction in the light and in the dark shows which fraction carries out light-dependent reduction of DCPIP.

Mark scheme

1 mark
Blend the leaves in ice-cold isolation medium
Max 6. Accept steps in any sensible order. Ignore exact centrifuge speeds. Accept 'isotonic buffer' for isolation medium.
1 mark
Filter (through muslin) to remove cell debris
1 mark
Centrifuge (the filtrate) at a low speed and keep the supernatant / remove the pellet of debris (and nuclei)
1 mark
Centrifuge the supernatant at a higher speed so that the chloroplasts form a pellet
Accept 'the chloroplasts are in the second pellet'.
1 mark
Resuspend the (chloroplast) pellet in (ice-cold) isolation medium
1 mark
Keep the supernatant / the other fractions to test as well (for comparison)
1 mark
Add the same volume of each fraction to the same volume of DCPIP
1 mark
Place the tubes in the light (at the same distance from the lamp and the same temperature) and measure the absorbance at intervals / the time taken for the blue colour to disappear
Accept 'use a colorimeter with a red filter'.
1 mark
Set up identical tubes wrapped in foil (in the dark) as controls
Accept 'a tube of DCPIP and isolation medium only'.
1 mark
The chloroplast fraction reduces DCPIP in the light but not in the dark / much faster than the other fractions
Accept 'only the chloroplast fraction shows a light-dependent decrease in absorbance'.

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