A student was provided with two proteases that hydrolyse the amino acid chains of protein in different ways, producing mixtures of single amino acids and peptides of varying lengths:
The student used these enzymes to hydrolyse a protein formed by the linking of two polypeptides.
Fig. 1 shows the hydrolysis sites of these two enzymes on this protein.
Fig. 1
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 a pH of 7.6.
- At intervals of 5 minutes, a sample of each mixture was removed using a capillary tube.
- The products of hydrolysis within each sample were separated by chromatography using the same solvent.
- The products of hydrolysis were located by spraying the chromatogram with a specific dye that stains proteins, peptides and amino acids.
- The student continued sampling the mixtures every 5 minutes and running chromatograms for each sample until hydrolysis of the protein was completed by each enzyme.
(i)
Identify the independent variable in this investigation.
[1]
(ii)
Identify three variables, other than the chromatography solvent and the specific dye, that the student standardised in this investigation.
[1]
(iii)
Describe how the student could have standardised two of the variables you described in (a) (ii).
[2]
(iv)
Suggest how the student could have determined when hydrolysis of the protein was complete.
[1]