A-LevelBiologyEnzymes9700/22 · May/June 2025 · Q610 Marks
Horseradish peroxidase (HRP) is an enzyme that can be extracted from the roots of the horseradish plant, Armoracia rusticana. HRP is used extensively in industry and technology. In the reaction catalysed by HRP, hydrogen peroxide (H₂O₂) is used to oxidise an organic substrate. This is summarised in Fig. 6.1. [Figure 6.1]
- (a)Inhibitors can have an effect on Vmax, the maximum rate of reaction, and Km, the Michaelis–Menten constant, of HRP. Fig. 6.2 shows the effect of substrate concentration on the rate of reaction of HRP. [Figure 6.2] Complete Fig. 6.2 by drawing a curve to show how the presence of a non-competitive inhibitor will affect the rate of reaction of HRP. Use the curve you have drawn to obtain an estimate of Km. Km =[2]
- (b)Scientists can design synthetic DNA nucleotide sequences to produce a synthetic HRP gene. These sequences will include a start codon and a stop codon so that translation of messenger RNA (mRNA) can occur. Explain what is meant by a start codon and a stop codon.[2]
- (c)(i)HRP is used in an immunological test known as a sandwich ELISA. One use of the test is to diagnose disease. Fig. 6.3 outlines the main steps in a sandwich ELISA in which a toxin released by a pathogen is detected in a sample of body fluid taken from a person who is ill. The test involves two types of monoclonal antibody that can bind to the toxin, a capture antibody and a detection antibody. [Figure 6.3] Complete Fig. 6.4 to show the situation in the test well after step 5. [Figure 6.4][1]
- (c)(ii)With reference to Fig. 6.3, describe one difference between the capture antibody and the detection antibody.[1]
- (c)(iii)Fig. 6.5 outlines the reaction catalysed by HRP in step 6. The organic substrate, TMB, is in excess and changes colour when it is oxidised. This indicates a positive test result. [Figure 6.5] A student suggested that: • a low concentration of toxin may by diagnosed as a negative result instead of a positive result • using a colorimeter after step 6 in the sandwich ELISA test would provide a quantitative result and help to avoid this error. With reference to Fig. 6.5, explain why using a colorimeter would provide a quantitative measurement for detection of a low concentration of the toxin and help to confirm a positive result.[4]
This question uses a diagram from the original paper, which isn't shown here. Open the full paper from the past papers archive to see it.
Mark scheme
Marking points from the official mark scheme (May/June 2025). The mark or mark type is shown in brackets.
- [1] curve drawn to the right of original, same shape and lower maximum rate of reaction, passing through plot of 0.055 to 0.06 mmol dm⁻³ on x-axis ; must start branching away before third plot point at 2au R S-shaped / sigmoid curve
- [1] Km = 0.055 to 0.06 mmol dm⁻³
- [1] (Start codon) triplet of / three, (RNA / DNA) bases / nucleotides (begins translation), qualified e.g. coding for, methionine / first amino acid first codon attaching to ribosome OR (codon to begin translation is) AUG (for RNA) or TAC (for DNA transcribed strand) OR ATG (for DNA non-transcribed strand)
- [1] (Stop codon) triplet of / three, (RNA / DNA) bases / nucleotides, (ending translation), qualified e.g. does not code for amino acid stops, chain elongation / addition of amino acids allows / AW, polypeptide to detach (from ribosome) recognised by releasing factors OR (codon to end translation) UAG UAA / UGA (for RNA) or ATC / ATT / ACT (for DNA transcribed strand) OR TAG / TAA / TGA for DNA non-transcribed strand
- [1] (each) detection antibody attached to a (different) toxin ; allow one antibody attached to two toxins (astride) A at least one detection antibody attached R if one detection antibody attached directly to immobilised antibody R if unattached detection antibody shown
- [1] bind, to different, locations / sites / AW, on toxin ; AW A bind different, epitopes / antigenic determinants OR detection antibody has, HRP / enzyme (attached) ; ora OR capture antibody is immobilised ; ora
- [1] colorimeter measures, absorption of light (by reaction mixture) ; A transmission of light (through reaction mixture)
- [1] low(er) intensity of / pale(r), colour gives low(er), (colorimeter) reading / measurement / absorbance ; ora for transmission A or idea that different intensities of colour, gives different absorbances
- [1] low(er), intensity of colour / absorbances, indicates low concentration ; A idea that different, intensities of colour / absorbances, indicate different concentrations
- [1] will get different absorbance readings for low toxin positive result compared with negative result ; AW A would get same reading as blank if negative result (positive does not give same reading as blank) A not relying on by-eye judgement (between colourless and very pale blue-green) A colorimeter not subjective A by-eye judgment may not detect a slight colour change (colourless to very pale blue-green)
- [1] ref. to use of, standards / calibration curve, to obtain actual concentrations
- [1] ref. to use of, stop / acid, so only initial rate of reaction occurs low concentration of HRP so low quantity of, TMB / substrate, oxidised A less products formed ora
- [1] low quantity of, oxidised TMB gives a, light(er) (blue-green) colour / minimal colour change ora
- [1] low concentration of toxin results in, a low quantity of detection antibody binding / most detection antibody washed away
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