Substrate Concentration On Rate Using Uv Spectroscopy Biology Essay

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Introduction- In biochemistry an enzyme will bind with a substrate, during a chemical process the substrate will be converted to product. After this chemical reaction takes place the enzyme will dissociate from the product formed. This means that the enzyme has acted not as a chemical reactant but as a catalyst.

The binding of enzyme to substrate is a physical process (and no covalent bonds are formed). It states in collision theory the higher the concentration of substrate the higher the chances of collision and therefore a reaction1. This therefore implies that the higher the substrate concentration the quicker the rate of reaction will be; this is true until Vmax is reached where the rate is at its maximum velocity.

In this experiment a fixed concentration of the enzyme α-chymotrypsin was added to varying concentrations of the substrate 4-aminophenyl acetate, and the rate of the reaction was measured by Gensys UV spectrophotometry every 30 seconds. The product formed, 4-nitrophenol, emits a yellow colour. The intensity of this colour can be measured by a UV spectrophotometer, then using the beer lambert law (Fig. 2) and then rearranging this formula (Fig. 2) the concentration can be calculated.

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The concentrations were then plotted against time; the gradient of this plot represented the rate of the reaction. To linearize the data set the Michaelis-menten double reciprocal equation (Fig. 3) was used2, this means that the Km and Vmax can be calculated and evaluated.

(Fig. 3)

The Vmax is when all available enzyme has been bound the rate at maximum concentration has been established3. Km is half the substrate concentration when the rate reached is half of Vmax; it is an inverse measure of the substrates affinity towards the enzyme3. A small Km means a high affinity between the substrate and enzyme therefore Vmax will be reached quicker. These in-turn evaluate the change in substrate concentration on rate.

Method-

A practical lab script was provided stating the procedure to carry out the experiment. The entirety of the lab script was followed correctly, apart from the fact that the lab equipment used did not allow a static temperature of 25ËšC to be maintained. This means that the temperature of each substrate reaction carried out could have been at varying unknown temperatures.

Results-

The following results came from the UV spectroscopy that took place in the lab. In table one all of the absorbance results have been divided by 17,700(=εl) to convert the absorbance's into 4-nitrophenol concentrations.

Table : a table to show the concentration of 4-nitrophenol formed over time.

[4-nitrophenol] (moldm-3)

Time (s)

Run 1

Run 2

Run 3

Run 4

Run 5

0.00E+00

5.16E-05

3.89E-05

4.17E-05

3.55E-05

2.21E-05

3.00E+01

5.55E-05

4.18E-05

4.47E-05

3.92E-05

2.56E-05

6.00E+01

5.95E-05

4.47E-05

4.76E-05

4.19E-05

2.76E-05

9.00E+01

6.36E-05

4.77E-05

5.06E-05

4.44E-05

2.88E-05

1.20E+02

6.76E-05

5.07E-05

5.34E-05

4.68E-05

2.96E-05

1.50E+02

7.16E-05

5.37E-05

5.64E-05

4.90E-05

3.02E-05

1.80E+02

7.58E-05

5.68E-05

5.95E-05

5.11E-05

3.05E-05

2.10E+02

7.98E-05

5.99E-05

6.25E-05

5.34E-05

3.06E-05

2.40E+02

8.40E-05

6.32E-05

6.56E-05

5.53E-05

3.07E-05

2.70E+02

8.81E-05

6.64E-05

6.86E-05

5.70E-05

3.08E-05

3.00E+02

9.23E-05

6.98E-05

7.18E-05

5.86E-05

3.08E-05

3.30E+02

9.64E-05

7.31E-05

7.48E-05

5.99E-05

3.08E-05

3.60E+02

1.01E-04

7.64E-05

7.79E-05

6.11E-05

3.09E-05

3.90E+02

1.05E-04

7.96E-05

8.11E-05

6.20E-05

3.10E-05

4.20E+02

1.09E-04

8.29E-05

8.42E-05

6.27E-05

3.09E-05

4.50E+02

1.13E-04

8.63E-05

8.73E-05

6.31E-05

3.10E-05

4.80E+02

1.17E-04

8.97E-05

9.03E-05

6.34E-05

3.10E-05

5.10E+02

1.22E-04

9.31E-05

9.34E-05

6.36E-05

3.10E-05

5.40E+02

1.26E-04

9.66E-05

9.65E-05

6.37E-05

3.10E-05

To increase the accuracy of the results all of the data that was not included in either the pre- or post- steady state region were eliminated from the data set. This approximation of steady-state is allowed due to the fact that the intermediate formed is at low concentrations.

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Table 2: a table to show [4-nitrophenol] over time from the pre- and post- steady state region of the UV spectrophotometry.

[4-nitrophenol] (moldm-3)

Time (s)

Run 1

Run 2

Run 3

Run 4

Run 5

0.00E+00

5.16E-05

3.89E-05

4.17E-05

 

2.21E-05

3.00E+01

5.55E-05

4.18E-05

4.47E-05

 

2.56E-05

6.00E+01

5.95E-05

4.47E-05

4.76E-05

4.19E-05

2.76E-05

9.00E+01

6.36E-05

4.77E-05

5.06E-05

4.44E-05

2.88E-05

1.20E+02

6.76E-05

5.07E-05

5.34E-05

4.68E-05

2.96E-05

1.50E+02

7.16E-05

5.37E-05

5.64E-05

4.90E-05

 

1.80E+02

7.58E-05

5.68E-05

5.95E-05

5.11E-05

 

2.10E+02

7.98E-05

5.99E-05

6.25E-05

5.34E-05

 

2.40E+02

8.40E-05

6.32E-05

6.56E-05

5.53E-05

 

2.70E+02

8.81E-05

6.64E-05

6.86E-05

5.70E-05

 

3.00E+02

9.23E-05

6.98E-05

7.18E-05

5.86E-05

 

3.30E+02

9.64E-05

7.31E-05

7.48E-05

5.99E-05

 

3.60E+02

1.01E-04

7.64E-05

7.79E-05

 

 

3.90E+02

1.05E-04

7.96E-05

8.11E-05

 

 

4.20E+02

1.09E-04

8.29E-05

8.42E-05

 

 

4.50E+02

1.13E-04

8.63E-05

8.73E-05

 

 

4.80E+02

1.17E-04

8.97E-05

9.03E-05

 

 

5.10E+02

1.22E-04

9.31E-05

9.34E-05

 

 

5.40E+02

1.26E-04

9.66E-05

9.65E-05

 

 

From the data in table 2 a graph could be plotted of product formation, [4nitrophenol], against time, this data is shown in graph 1.

Graph 1: A graph to show [4-aminophenol] against time, excluding pre- and post- steady state data.

run

Gradient (rate) (Ms-1)

1

1.38x10-7

2

1.02x10-7

3

1.07x10-7

4

6.74x10-8

5

6.07x10-8

Graph 2 shows that the general trend of the data in table two is that over time the concentration of 4-nitrophenol increases. Also that as the substrate concentration is decreased the gradient and therefore rate is generally decreased.

After graph 2 was plotted the substrate concentrations were calculated in order to plot the Michaelis-menten double reciprocal graph. The substrate concentrations were calculated in the following way:

(Fig.4)

The substrate concentration for each run was calculated and the gradient of each run from graph 2 was collected, these results are shown in table 3.

run

[S] (M)

v (mol dm3s-1)

1

8.8398E-03

1.3800E-07

2

4.4199E-03

1.0200E-07

3

2.2099E-03

1.0700E-07

4

1.1050E-03

6.7400E-08

5

5.5249E-04

6.0700E-08

Table 3: a table to show the rate of reaction at substrate concentration.

In order to process these results the double-reciprocal Michaelis-Menten equation (Fig. 3) to therefore enable analysis and the discovery of Km and Vmax. This means that [S] and v must be reciprocated.

run

1/[s] (M-1)

1/v (mol-1dm3s)

1

1.1312E+02

7.2464E+06

2

2.2625E+02

9.8039E+06

3

4.5251E+02

9.3458E+06

4

9.0498E+02

1.4837E+07

5

1.8100E+03

1.6474E+07

Table 4: a table to show the reciprocals of substrate concentration and rate.

Using both reciprocals a double-reciprocal graph otherwise known as a Lineweaver-Burk plot can be made. This Lineweaver-Burk plot (Graph 2) is an interpretation of the Michaelis-Menten equation.

Graph 2: a graph to show the reciprocal of substrate concentration Vs. Rate of reaction.

The above graph (Graph 2) can be used to find Km and Vmax this is due to that fact that this graph used the Michaelis-Menten equation. Therefore the y-intercept is equal to 1/Vmax and the gradient is equal to Km/Vmax. Using these pieces of information Vmax and Km can be calculated (Fig. 5 & 6).

Below is the calculation of Vmax:

(Fig. 5)

Below is calculation of Km:

(Fig. 6)

1/Vmax

7.84E+06 mol-1dm3s

Km/Vmax

5271.7 M-1

Vmax

1.28E-07 moldm-3s-1

Km

6.72E-04 M

Table 5: The collected results from graph 2 and the calculated Vmax and Km, using Fig 5 & 6.

Discussion-

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The aim of this experiment was to assess how changing the substrate concentration affected the rate of reaction. The expected result is that as the substrate concentration increases so will the rate of reaction, this theory fits with the collision theory and the Michaelis-Menten equation. This is true until Vmax is reached, this is where all the substrate has been bound therefore the maximum rate has been achieved. In this set of data (table 2) the same is found to be true. It is very clear in graph 1 that as the substrate concentration is lowered in each run the gradient also decreases. Due to the fact that gradient is representative of rate it therefore proves that as substrate concentration is decreased so does the rate. Although run 3 is has a higher gradient than run indicating and error in either run 2 or 3. .

Km represents the substrates affinity towards the enzyme. A small Km means a high affinity; therefore Vmax will be reached quicker. In this experiment Km was found to be a small number (6.72x10-4 M) which means that α-chymotrypsin and 4-nitrophenyl acetate have a high affinity to each other; this means that the reaction between the two reactants is fast.

The technique used in this experiment was based on the rearrangement of the Michealis-Menten equation into the double reciprocal form. This is then used to plot a Lineweaver-Burk plot of 1/v against 1/[S]. the Lineweaver-Burk plot is commonly used to linearize data sets in kinetics, however more commonly used in today's kinetics is a non-linear least squares regression. The use of Michaelis-Menten assumes certain aspects of the data are true such as K-1>>K2, that the first reaction step is rapid and reversible and also that the enzyme substrate intermediate is formed in low and steady concentrations3. Michaelis-Menten is also used in steady state kinetics which means that all pre- and post- steady state data was not included. This decision to include certain sets of data was done at the experimenters' discretion. These assumptions can lead to the introduction of errors in the calculation of Km and Vmax. The Lineweaver-Burk plot can be used a guide to Km and Vmax; however it is not highly accurate. A disadvantage of the Lineweaver-Burk plot is that it places undue emphasis on lower concentration values, which are the higher values of 1/v and 1/[S] 2. The data collected at the lowest concentrations are the most inaccurate due there being the lowest amount of product formed, which means one slight inaccuracy can carry a huge carry on effect2. Another disadvantage of the Lineweaver-Burk plot is that extrapolation is used to determine Km, which introduces another area of error. One way around this is to use another graphical method to determine Km; one option is the Eadie-Hofstee plot which plots v against v/[S] 4, 5.

A source of error in the experimental procedure was that the UV spectrophotometry equipment could not regulate the temperature of the reaction. This means that each run could have been conducted at different unknown temperatures. Another source of error in the experimental procedure could have been the use of the pipettes. The pipettes have an error in volumes collected (±0.05cm3) but there may have been a chance of cross-contamination between substrate and enzyme.

In future experiments other graphical methods could be used to determine the Km and Vmax of the data set. Also the experiment could be repeated more than once do the Km and Vmax can be compared for accuracy.

Conclusion-

To conclude, it is clear from the evidence provided that a decrease in substrate concentration will decrease the rate of reaction. The aim of the experiment was to be able to analyse and record data that could be interpreted and used to determine Km and Vmax. In term of these aims the experiment has been has success as Vmax was found as 1.28E-07 moldm-3s-1 and Km was determined as 6.72E-04 M.

Other methods presented in the discussion could be used if further work was to be carried out on this experiment.