Enzyme Analysis - Protease

David Anthony (CID:01520961)
Data from experiment A: Construction of calibration curve

Volume of 1 mM p- Volume of water Concentration of Amount of p-NA
NA (ml) (ml) p-NA (mM) (µmole) Absorbance
0.0 2.0 0.0 0.0 0.000
0.4 1.6 0.2 0.4 0.685
0.8 1.2 0.4 0.8 1.397
1.2 0.8 0.6 1.2 2.065
1.6 0.4 0.8 1.6 2.900
2.0 0.0 1.0 2.0 3.540
Calibration curve of p-NA

4.0
y = 1.7715x
3.5
3.0
2.5
Absorbance
2.0
1.5
1.0
0.5
0.0
0.0 0.5 1.0 1.5 2.0 2.5
Amount of p-NA (µmole)
Exercise 1
Absorbance is given by the equation: , where A is the absorbance, is the molar
extinction coefficient (M-1 cm-1), c is the concentration (M) and l is the path length
(cm). As the graph has absorbance on the y-axis and concentration on the x-axis,
the gradient, m, would have the units µmole L -2. Therefore, to convert this to the
molar extinction constant, which has units mole L -1 cm-1, the value for the gradient
(1.7715) would have to be multiplied by 10 6, to convert µmoles to moles, volume in
litres, to convert L-2 to L-1, and divided by the path length in cm, to give cm -1. This
gives: M-1 cm-1.
Data from experiments B and C: Determination of the pH optimums of Trypsin and

Chymotrypsin
rate of p-NA
rate of p-NA production in
µmole of p-NA µmole of p-NA production in Chymotrypsin
Absorbance A430 of produced in produced in Trypsin solution solution (µmole of
pH of of Trypsin Chymotrypsin Trypsin Chymotrypsin (µmole of p-NA p-NA
solution solution solution solution solution produced/minute) produced/minute)
1 0.001 0.001 5.64E-04 5.64E-04 3.76E-05 3.76E-05
2 0.000 0.002 0.00E+00 1.13E-03 0.00E+00 7.53E-05
3 0.000 0.002 0.00E+00 1.13E-03 0.00E+00 7.53E-05
4 0.000 0.008 0.00E+00 4.52E-03 0.00E+00 3.01E-04
5 0.026 0.109 1.47E-02 6.15E-02 9.78E-04 4.10E-03
6 0.220 0.156 1.24E-01 8.81E-02 8.28E-03 5.87E-03
7 0.825 0.225 4.66E-01 1.27E-01 3.10E-02 8.47E-03
8 0.835 0.187 4.71E-01 1.06E-01 3.14E-02 7.04E-03
9 0.749 0.119 4.23E-01 6.72E-02 2.82E-02 4.48E-03
10 0.516 0.160 2.91E-01 9.03E-02 1.94E-02 6.02E-03
How pH affects the rate at which Trypsin and Chymotrypsin produce p-

3.5E-02
NA
ε = 3543 M-1 cm-1
Rate of p-NA production (µmole of p-NA produced
3.0E-02
Optimal pH range for

2.5E-02
Trypsin is 7-9 and for
chymotrypsin is 6-8. this
2.0E-02 is +/- 1 from the peak pH
/minute)
shown on the graph as

further testing would be
1.5E-02
needed to fin the exact
optimum point.
1.0E-02
5.0E-03
0.0E+00
1 2 3 4 5 6 7 8 9 10
pH of solution
rate of p-NA production in Trypsin solution
rate of p-NA production in Chymotrypsin solution
Data from experiment D: Determination of the substrate specificities of Trypsin and

Chymotrypsin
µmole of p- µmole of p- rate of p-NA
NA NA production with rate of p-NA production
Absorbance Absorbance produced produced BAPNA (µmole of p- with NSLPN (µmole of p-
Protease with BAPNA with NSLPN with BAPNA with NSLPN NA produced/minute) NA produced/minute)
Trypsin 0.678 0 3.83E-01 0.00E+00 2.55E-02 0.00E+00
Chymotrypsin 0.026 0.172 1.47E-02 9.71E-02 9.78E-04 6.47E-03
How substrate specificity of Trypsin and Chymotrypsin differ

3.0E-02
Rate of p-NA production (µmole of p-NA
2.5E-02
2.0E-02
produced/minute)
1.5E-02
1.0E-02
5.0E-03
0.0E+00
Trypsin Chymotrypsin
Type of protease
rate of p-NA production with BAPNA
rate of p-NA production with NSLPN
Exercise 2:
Benzene and p-NA both have aromatic rings, which causes them to absorb light at a lower
energy, and thus a higher wavelength, than non aromatic compounds. p-NA has an NO2 and
an NH2 group added to its ring meaning there are N atoms containing lone pairs, which can
add to the overall resonance of the structure. This puts it in a lower energy state than that of
Benzene which is purely a hydrocarbon ring. Because of this, it’s π to π* transition is lower
than that of Benzene’s meaning it absorbs less energy and thus a greater wavelength of light,
putting its absorption in the visible spectrum while Benzene’s is in the ultra violet.
Exercise 3:
The circled amide linkages between BAPNA and p-NA, or NSLPN and p-NA are analogous to
the peptide bond and would therefore be cleaved by the enzymes
Exercise 4:
My results from experiment three show that Trypsin has a high affinity for BAPNA, evolving a
relatively large amount of p-NA, and a very low affinity, or no affinity at all, for NSLPN.
Chymotrypsin however binds preferentially to NSLPN, though still has some affinity for
BAPNA.

Enzyme Analysis - Protease

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Enzyme Analysis - Protease

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David Anthony (CID:01520961)

Data from experiment A: Construction of calibration curve

Calibration curve of p-NA

Data from experiments B and C: Determination of the pH optimums of Trypsin and

How pH affects the rate at which Trypsin and Chymotrypsin produce p-

Optimal pH range for

shown on the graph as

Data from experiment D: Determination of the substrate specificities of Trypsin and

How substrate specificity of Trypsin and Chymotrypsin differ

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