AS & A Level Biology - 9700 Paper 5 2015 Winter Zone 2

(a) The student investigated the effect of these two enzymes on the hydrolysis of this protein by incubating the protein separately with each of the enzymes.
• Each mixture of enzyme and protein was incubated at 35°C and at a pH of 7.6.
• At intervals of 5 minutes, samples of each mixture were removed using a capillary tube.
• The products of hydrolysis of each mixture were separated by chromatography using the same solvent.
• The products of hydrolysis were located by spraying the chromatogram with the same specific dye.
• The student continued sampling every 5 minutes and running chromatograms for each mixture until hydrolysis of the protein was completed by each enzyme.
(i) Identify the independent variable in this investigation.
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(ii) Identify three variables, other than the chromatography solvent and the specific dye, that the student has standardised in this investigation.
Describe how the student might have standardised two of these variables.
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(iii) Suggest how the student determined when the hydrolysis was complete.
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(b) Describe a method that the student could use to prepare and use chromatograms to compare the changes in the products of hydrolysis of the protein by the two different proteases over time.
Your method should be detailed enough for another person to follow.
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(c) Fig. 1.2 shows the appearance of the chromatograms at the times when hydrolysis by the different enzymes was complete.

The student concluded that:
1. The endoprotease worked faster than the exoprotease because fewer bonds were hydrolysed.
2. The products of hydrolysis of the exoprotease were all single amino acids giving more spots on the chromatogram.
3. The larger peptides released by the endoprotease have fewer charges and moved more slowly than single amino acids and small peptides.
4. Hydrolysing the protein with a mixture of endoprotease and exoprotease would give the same results as for the exoprotease more quickly.
State and explain whether each of these conclusions about the hydrolysis of protein is supported or not supported by the evidence in Fig. 1.2 and the information in the question about these two enzymes.
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(d) Using an internet search the student found that:
• electrophoresis can also be used to separate the products of enzyme hydrolysis of proteins
• a combination of electrophoresis and chromatography was used to study haemoglobin from people with sickle cell anaemia (SCA).
In an investigation, the haemoglobin from people with SCA and people without the disease was hydrolysed by an endoprotease. The resulting mixture was placed centrally on one side of chromatography paper and separated by electrophoresis.
Negatively charged peptides moved to the anode (+) and positively charged peptides moved to the cathode (−). The paper was turned through 90° and chromatography used to separate the mixture.
Fig. 1.3 shows the main stages of this experiment.

(i) Sickle cell haemoglobin and normal haemoglobin have a difference in amino acid sequence.
On Fig. 1.3, draw a circle around the spot in each chromatogram that shows that the two types of haemoglobin have different amino acid sequences. [1]
(ii) Explain your answer to (i).
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(a) (i) State the dependent variable in this investigation.
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(ii) Suggest what liquid should be used as the control.
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(b) (i) State what is meant by standard error.
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(ii) Identify the results from Table 2.1 which suggest that the difference in the numbers of aphids as a result of the insecticide treatment may be significant.
Give a reason for your answer.
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(c) Identify three other features of the results shown in Table 2.1.
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(d) A $t$-test was used to find out if the number of aphids in Group $A$ and in Group $B$, 72 hours after treatment with insecticide, differed significantly.
(i) State one reason why a $t$-test was used.
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(ii) State a null hypothesis for this test.
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(iii) The number of degrees of freedom for this statistical test is 48.
Describe how this is calculated.
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