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The aim of this experiment was to investigate the effect of enzyme concentration on rate of hydrolysis of 4-nitrophenyl acetate using varying concentrations of α-chymotrypsin. The product was 4-nitrophenol which, in solution, is yellow therefore allowing UV/Vis Spectroscopy to be carried out. The rate of the product and therefore the rate of reaction was also measured.
An enzyme can be defined as a protein molecule that is a biological catalyst. The basic function is to increase the rate of reaction by providing an alternative route, whilst remaining chemically unchanged at the end of the reaction. 1 Enzymes are quaternary structured proteins (globular proteins) and are abundant in nature. An enzymes' quaternary structure is predominantly governed by the intramolecular forces of the molecule. This means their structure is affected by altering conditions such as pH level and temperature and as a result can increase or decrease in reaction rate.
An important feature of an enzyme-catalysed reaction is the specificity; the active site is formed in such a way that is highly specific as to the molecule that can enter and bind the active site. The induced fit of the substrate and enzyme together results in stress applied to certain bonds of the substrates and the outcome of these stresses increase the potential energy of the enzyme-substrate complex. This causes the activation energy to be reduced, and the reaction proceeds finally releasing the products.2
All enzymes are also generally substrate specific. This means enzymes have optimum conditions such as pH level, and if placed in an environment far different, the enzyme could be denatured. It is for this reason a buffer solution was used to keep the enzyme in a constant pH level environment during the reaction.
The enzyme used in this experiment was α-chymotrypsin. α-chymotrypsin is a digestive enzyme that can perform proteolysis. It preferentially cleaves peptide amide bonds - generally of peptides that contain an aromatic ring in their side chain that fits into a 'hydrophobic pocket' (the S1 position) of the enzyme.3
The product of the reaction will be analysed using UV/Vis spectroscopy. It is an analytical technique used within the pharmaceutical industry used to quantitatively determine the content of solutions. The technique uses radiation in the wavelength range of 200-700nm to excite electrons in the bonds within the molecule.4 The difference between the incident radiation and the transmitted light energy can be measured and the values used to determine different factors such as pKa and change in drug concentration over time by using Beer-Lamberts law. The absorbance of a solution increases as attenuation of the beam increases. Absorbance is directly proportional to the path length, b, and the concentration, c, of the absorbing species.5 Beer's Law states that:
A = ε c l6
Where A= absorbance, ε = molar absorption coefficient, c = concentration of solution and l = path length.
The method used followed the printed instructions provided.
Absorbance data was collected as follows:
Rearranging Beer's law gives c=A/εl which means dividing each absorbance by 17,700 (=εl) will give the concentration of 4-nitropheolate:
A graph was then plotted to show the rate at which 4-nitropheolate was produced:
Once the graph above was plotted, there were no pre- or post-steady state regions to be removed.
Using the line equations for each experiment, the [E]0 values were obtained and plotted against the concentration of α-chymotrypsin in each experiment as follows:
Again, using the line equations for each experiment the rate of the reactions were obtained and plotted against the concentration of α-chymotrypsin:
Rearranging the equation V(max)=k(cat)[E]0, k(cat) of the experiment can be calculated. k(cat) is the turnover number - the number of substrate molecule each enzyme site converts to product per unit time, V(max) is the maximum enzyme velocity extrapolated out to very high concentrations of substrate7 and [E]0 is the total enzyme concentration.
k(cat) = V(max)/[E]0 8
k(cat) = (1.15x10-7)/(1.75x10-5)
k(cat) = 6.57x10-3 s-1
The k(cat) of α-chymotrypsin for this experiment was calculated to be 6.57x10-3s-1. The k(cat) of α-chymotrypsin is 0.14s-1.9 This means there is a deviation of 46.93% from the literature value.
Using the relative molecular mass of α-chymotrypsin is 24.8 x 103 and the gL-1 of the stock solution, the molar concentration of the stock solution can be calculated:
2.0g L-1/(24.8 x 103) = 8.06 x 10-5 M
In order to determine the purity of the enzyme used to prepare the stock solution, a comparison was made with the result from experiment 1. This means dividing the above value by 5 as experiment 1 had a dilution factor of 5. It can be predicted that the value for the experiment 1 should be lower than the stock solution value.
(8.06 x 10-5) / 5 = 1.61 x 10-5 M
The Y intercept of experiment 1 (figure 1) shows the value 6.6 x 10-6.
Thus, the percentage purity of the enzyme used to prepare the stock solution was:
(6.60 x 10-6 / 1.61 x 10-5) x 100 = 40.99%
Ordinarily samples of α-chymotrypsin crystallized three times have a purity of 75-90%, a good part of the difference being due to water of hydration. 10
An approximate percentage error can therefore be calculated:
(40.99 / 82.5) x 100 = 49.68%
This error could be due to inaccurate pipetting or failure to regulate the temperature of the reaction.
Figure 1 and 2 both show a linear relationship which concludes that as time and enzyme concentration go up, so does the production of 4-nitropheolate. From figure 3 it is clear that there is also a linear relationship between the reaction rate and the concentration of 4-nitropheolate. This is supporting evidence of active site titration; an enzyme-bound intermediate accumulated during the reaction of the enzyme with its substrate and a reaction product was concomitantly released.11
The percentage purity of the enzyme was calculated to be 40.99% which deviated approximately 49.68% away from literature values.