enzymes lab report

LSC-10034 The effect of temperature, pH, substrate

concentration and inhibitors on the enzyme activity.

Student number: 14003336 Word count: 3 804

Student number: 14003336

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Introduction

Aims:

1. To determine optimum wavelength.

2. To produce calibration graph.

3. To investigate the effect of the following factors on the enzyme activity:

pH,

substrate concentration,

inhibitor,

temperature.

Background information

Enzymes are biocatalysts that speed up the chemical reaction.

The reaction is (Watson, 2014):

Rate of reaction is the amount of product produced per unit of time. In this experiment it is

mol/min.

The rate of the enzyme reaction depends on various factors:

pH,

substrate concentration,

inhibitor,

temperature.

When the substrate concentration is low, molecules react slowly and there are fewer

collisions which result in low rate of reaction. However, when the concentration of substrate

increases, the solution becomes more saturated, the rate increases. At the beginning substrate

reacts very fast, but to a lesser and lesser and finally it reaches the maximum rate (

(Hames, Hooper, 2009). This happens because there is not enough enzyme to convert all the


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substrate at once, enzyme is being damaged, a reversible reaction starts, or pH deviates from its

optimum value. It can be solved by the addition of enzyme (Berg, Tymoczko, Stryer, 2012).

The relationship between the substrate concentration and velocity can be presented as a

Michaelis-Menten equation.

In order to calculate the rate of reaction, the following formula is used (Watson, 2014):

where is called the Michaelies constant and is the substrate concentration at which the rare

of reaction is equal to half maximum velocity. The does not change with substrate

concentration. It is affinity indicator. Low value means high affinity and weak substrate-

enzyme bonding.

The Michaelis-Menten graph has many limitations, one of them are inaccurate values due to

hyperbolic form. and can be calculated more accurately using the Lineweaver-Burk

equation. The example of the graph (Watson, 2014):


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Temperature and pH also affect enzymatic reactions. In both cases with the increase of

pH/ temperature, there is an increase in the rate of reaction, until it reaches peak value and

starts to denature. In case of temperature, enzyme get too much energy and the side chains

move, destroying the tertiary structure and changing the active site. Non-covalent interactions

are broken. The enzyme cannot work(Martini, Nath, Bartholomew, 2012).

With pH, even small change in pH can result in big difference in the rate of reaction. It happens

because, pH deviation causes changes in charges carried by ionisable side chains of amino

acids. This changes the intermolecular forces, tertiary structure is changed and protein

denatures (Hames, Hooper, 2009).

There are different types of inhibitors. They lower enzyme activity. One of them is

competitive inhibitor, it is reversible inhibitor which competes with substrate in order to bind to

enzymes. When it wins, the enzyme-inhibitor complex cannot react and the rate of reaction

decreases or falls to zero. With the increase of inhibitor, there is an increase in slope in the

Lineweaver-Burk equation and y-intercept remains unchanged. Competitive inhibitors do not

affect value. The increase in substrate concentration can overcome the inhibitor (Berg,

Tymoczko, Stryer, 2012).


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Data collection and presentation

Preliminary investigations

To determine optimum wavelength needed to determine the absorbance of product

(PNP), to both control (1 ml of distilled water) and sample (0.1 ml of 1mM PNP and 0.9 ml of

distilled water) tubes 2ml of diluting solution was added. The solution were mixed and next the

reaction was stopped by the addition of 3 ml of NaOH. Absorbance measurement was taken

using spectrophotometer at wavelength range of 340-500nm.

In order to determine the optimum wavelength, data from Table 1 were presented on graph.


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From the Graph 1, it can be determined that the wavelength gives the peak

absorbance and therefore is an optimum wavelength .

Absorbance depends on the concentration of the solution and the distance which the

light has to pass. The more saturated solution, the bigger absorbance value (Watson, 2014).

Therefore according to the Beer-Lambert Law, only concentrations that give the absorbance

value below 1.000 will be used in the next part of the experiment. To narrow PNP

concentration range, absorbance measurement of solutions containing different amounts of

1nM PNP were taken. The following results were obtained.

Table 2

Absorbance value obtained at different 1mM PNP volume using spectrophotometer.

Test tube number Amount of reagent (ml) Absorbance

1mM PNP Distilled water

1 0.000 1.000 0.000

2 0.010 0.990 0.026

3 0.050 0.950 0.172

4 0.100 0.900 0.745

5 0.250 0.750 1.111

6 0.500 0.500 1.770

7 0.750 0.250 2.664

8 0.900 0.100 3.053

9 1.000 0.000 2.276

It was observed that after 0.1M NaOH was added to each tube, colour change was

observed. First test tube was colourless, 2nd

had a slightly yellow colour which became more

saturated with the increase of PNP volume in the test tube. The last test tube (number 9) had a

dark yellow colour.

To determine the concentration that gives the absorbance 1.000, the volume of 1mM

PNP were converted into the concentration of PNP. Dilution equation was used (Watson,2014):

where C is the concentration and V is the volume. Therefore to calculate the concentration of

PNP in the test tube number 1, the following equation is used:


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Table 3

Concentration of 1 mM PNP calculated for different 1mM PNP volumes.

Test tube number Amount of reagent (ml) Absorbance Concentration of PNP

(mM) 1mM

PNP

Distilled

water

1 0.000 1.000 0.000 0.000

2 0.010 0.990 0.026 0.010

3 0.050 0.950 0.172 0.050

4 0.100 0.900 0.745 0.100

5 0.250 0.750 1.111 0.250

6 0.500 0.500 1.770 0.500

7 0.750 0.250 2.664 0.750

8 0.900 0.100 3.053 0.900

9 1.000 0.000 2.276 1.000

From the Graph 3, it can be determined that for the PNP concentration equal to

0.260mM, the absorbance is equal to 1.000. This means that the concentration range of 1mM

PNP, for which it is directly proportional to absorbance is 0.00-0.25ml of PNP. Hence for these

values absorbance readings were obtained.


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Table 4

The absorbance value at different volume of 1mM PNP.

Test tube

number

Volume of reagent (ml) Absorbance

1mM PNP Distilled water

1 0.000 1.000 0.000

2 0.030 0.970 0.094

3 0.060 0.940 0.214

4 0.090 0.910 0.302

5 0.120 0.880 0.391

6 0.150 0.850 0.474

7 0.180 0.820 0.589

8 0.210 0.790 0.713

9 0.250 0.750 0.793

To calculate the amount of PNP, the following equation is used (Watson, 2014):

where N is the amount of PNP, C is the concentration of PNP and V is the volume of PNP.

To calculate the amount of PNP in test tube number 3:

Table 5

The amount of PNP ( moles) at different volume of PNP reagent (ml).

Test tube

number

Volume of reagent (ml) Absorbance Amount of

PNP( moles) 1mM

PNP

Distilled water

1 0.000 1.000 0.000 0.000

2 0.030 0.970 0.094 0.030

3 0.060 0.940 0.214 0.060

4 0.090 0.910 0.302 0.090

5 0.120 0.880 0.391 0.120

6 0.150 0.850 0.474 0.150

7 0.180 0.820 0.589 0.180

8 0.210 0.790 0.713 0.210

9 0.250 0.750 0.793 0.250

Finally, the calibration graph was produced.


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Graph 3 shows that increase of PNP amount ( mol) is directly proportional to increase in

absorbance value. It is supported by high value of

Effect of substrate on enzyme activity

To understand the influence of substrate on enzyme activity, in this part of experiment,

different concentrations of substrate (PNPP) were used. Two sets of controlled and

experimental tubes were prepared. In both, the amount of citrate buffer (pH=4.8) and 10mM

PNPP were the same. The only difference was that each control tube had 1ml more of distilled

water. It was to equal the mass in experimental and control tubes, because 1.0 ml of enzyme

solution was added to each experimental tube.


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Table 6

The absorbance values at different amount of substrate (10mM PNPP).

Test

tube

Amount of reagent (ml) Absorbance

10mM

PNPP

Distilled water Citrate buffer

pH=4.8

1 0.010 0.990 1.000 0.087

3 0.015 0.985 1.000 0.120

5 0.025 0.975 1.000 0.135

7 0.050 0.950 1.000 0.179

9 0.100 0.900 1.000 0.234

11 0.200 0.800 1.000 0.293

13 0.500 0.500 1.000 0.329

15 1.000 0.000 1.000 0.364

In the test tubes 1-7, there is a higher increase in absorbance than in test tubes 11-15.

To calculate PNPP concentration ([PNPP]), dilution equation is used (Watson,2014):

where C is the concentration and V is the volume. Therefore to calculate the concentration of

PNP in the test tube number 3, the following equation is used:

Table 7

Concentration of PNPP at different 10mM PNPP volumes.

Test

tube

Amount of reagent (ml) Absorbance [PNPP]

(mM) 10mM

PNPP

Distilled water citrate buffer

pH=4.8

1 0.010 0.990 1.000 0.087 0.033

3 0.015 0.985 1.000 0.120 0.05

5 0.025 0.975 1.000 0.135 0.083

7 0.050 0.950 1.000 0.179 0.167

9 0.100 0.900 1.000 0.234 0.333

11 0.200 0.800 1.000 0.293 0.667

13 0.500 0.500 1.000 0.329 1.667

15 1.000 0.000 1.000 0.364 3.333

To calculate the amount of PNP produced, the calibration graph (Graph 4) is used. The

absorbance value for each tube is divided by the line gradient ( . To calculate the

rate of reaction, the amount of PNP is divided by time of reaction (15 minutes).


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Table 8

The amount of PNP produced and the rate of reaction in each tube.

Test

tube

Amount of reagent (ml)

PNP ( mole) rate

( mole/min) 10mM

PNPP

Distilled

water

citrate buffer

pH=4.8

1 0.010 0.990 1.000 0.281 0.019

3 0.015 0.985 1.000 0.388 0.026

5 0.025 0.975 1.000 0.437 0.029

7 0.050 0.950 1.000 0.579 0.039

9 0.100 0.900 1.000 0.757 0.05

11 0.200 0.800 1.000 0.948 0.063

13 0.500 0.500 1.000 1.064 0.071

15 1.000 0.000 1.000 1.177 0.078

From the Graph 4, it can be observed that at the beginning with the increase of substrate, there

is a sharp increase in the rate of reaction. However, with time the increase is slower and finally

at some PNPP concentration the rate of reaction does not increase anymore and the maximum


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rate ( is reached. The values of and can be determined from the Michaelis-

Menten equation.

In order to calculate the rate of reaction, we use the following formula (Watson, 2014):

Table 9

Theoretical values of rate of reaction at different PNPP concentration.

Theoretical [PNPP] (mM) Theoretical rate of reaction

0.034 0.012

0.056 0.018

0.087 0.024

0.170 0.037

0.360 0.051

0.764 0.063

1.460 0.070

1.780 0.072

2.570 0.074

2.900 0.075

3.400 0.076


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By comparing experimental and theoretical rate of reaction, it can be noticed that for both

curves are very similar.

However, to get more accurate values of and , the Michaelis-Menten equation must be

converted into linear Lineweaver-Burk plot. To do so, reciprocals of rate of reaction and PNPP

concentration were calculated.

Table 10

Values needed to plot the Lineweaver-Burk plot.

V

( mole/min)

[PNPP] (mM)

0.019 0.033 52.363 30.000

0.026 0.050 38.161 20.000

0.029 0.083 33.973 12.000

0.039 0.167 25.699 6.000

0.051 0.333 19.702 3.000

0.063 0.667 15.757 1.500

0.071 1.667 14.041 0.600

0.079 3.333 12.697 0.300

From this graph, much more accurate values of and can be obtained.


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The effect of inhibitor on the enzyme activity

In this part of the experiment, instead of adding normal citrate buffer, a phosphate-containing

buffer was added. This buffer will inhibit the enzymatic reaction. To determine the influence of

inhibitor on enzyme activity, solutions with different amount of 10mM PNPP were made and

the absorbance values were measured.

Table 11

Absorbance at different 10mM PNPP volumes in the presence of inhibitor.

Test

tube

Volume of reagent (ml) Absorbance

10mM

PNPP

Distilled water Phosphate –

containing citrate

buffer pH=4.8

1 0.010 0.990 1.000 0.017

3 0.050 0.950 1.000 0.030

5 0.100 0.900 1.000 0.095

7 0.200 0.800 1.000 0.141

9 0.500 0.500 1.000 0.199

11 1.000 0.000 1.000 0.206

Using the absorbance values and the calibration graph the amount of product and therefore the

rate of the reaction can be obtained.

Table 12

The amount of PNP produced and the rate of reaction in each tube in the presence of

inhibitor.

Test

tube

Amount of reagent (ml)

PNP ( mole) rate

( mole/min) 10mM

PNPP

Distilled

water

Phosphate-

containing citrate

buffer pH=4.8

1 0.010 0.990 1.000 0.055 0.004

3 0.050 0.950 1.000 0.097 0.006

5 0.100 0.900 1.000 0.307 0.020

7 0.200 0.800 1.000 0.456 0.030

9 0.500 0.500 1.000 0.644 0.043

11 1.000 0.000 1.000 0.666 0.044


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From the Table 12, it can be easily noticed that with an increase of 10mM PNPP volume, the

rate of the reaction increases from 0.004 mole/min to 0.044 mole/min.

Graph 7 shows the relationship between the concentration of PNPP and its rate of reaction. The

increase at the beginning is not as steep as in the Graph 4. However to get more accurate

values, as in previous part of the experiment, the concentration of PNPP, in solutions with

different PNPP volume and in the presence of inhibitor, was calculated.

Table 13

Concentration of PNPP at different 10mM PNPP volumes.

Test

tube

Amount of reagent (ml) Absorbance [PNPP]

(mM) 10mM

PNPP

Distilled water citrate buffer

pH=4.8

1 0.010 0.990 1.000 0.017 0.033

3 0.050 0.950 1.000 0.030 0.167

5 0.100 0.900 1.000 0.095 0.333

7 0.200 0.800 1.000 0.141 0.667

9 0.500 0.500 1.000 0.199 1.667

11 1.000 0.000 1.000 0.206 3.333

As previously, to get more accurate values of and , the Michaelis-Menten equation

must be converted into linear Lineweaver-Burk plot. Reciprocals of the rate of reaction and

PNPP concentration were calculated.


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Table 14

Values needed to make the Lineweaver-Burk plot.

V

(�mole/min)

[PNPP] (mM)

0.004 0.033 250.000 30.303

0.008 0.167 125.000 5.988

0.020 0.333 50.000 3.003

0.030 0.667 33.333 1.499

0.043 1.667 23.256 0.600

0.044 3.333 22.727 0.300


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From the Graph 8, values of and can be obtained.

Effects of temperature on enzyme activity

In order to determine the effect of temperature on enzyme activity, samples were kept

for 15 minutes at different temperatures (range: 5oC-75

oC) and after the addition of NaOH to

experimental test tubes, the absorbance was measured. The amount of citrate buffer and

substrate were constant.

Table 15

Absorbance value at different temperatures in control test tubes.

controls

test tuba number 2 4 6 8 10 12

5.0mM PNPP (ml) 1 1 1 1 1 1

Citrate buffer pH=4.8 (ml) 1 1 1 1 1 1

Distilled water (ml) 1 1 1 1 1 1

Incubation temp (oC) 5 20 25 37 50 75

Absorbance 0.082 0.092 0.092 0.099 0.166 1.896

Table 16

Absorbance value at different temperatures in experimental test tubes.

experimental

test tuba number 1 3 5 7 9 11

5.0mM PNPP (ml) 1 1 1 1 1 1

Citrate buffer pH=4.8 (ml) 1 1 1 1 1 1

Incubation temp (oC) 5 20 25 37 50 75

Absorbance 0.121 0.264 0.332 0.621 1.025 1.794

To calculate the optimum temperature, the difference in absorbance of the corresponding test

tubes must be calculated as well as PNP amount and the rate of reaction.


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Table 17

The amount of PNP and the rate of reaction.

corresponding test tubes Difference in

absorbance

Amount PNP

(�mole)

rate

(�mole/min)

1&2 0.039 0.126 0.008

3&4 0.172 0.556 0.032

5&6 0.240 0.776 0.052

7&8 0.522 1.688 0.113

9&10 0.859 2.778 0.185

11&12 0.003 0.010 0.001

Table 18

Temperature against the rate of reaction.

Corresponding test

tubes

Rate (�mole/min) Temperature

(oC)

1&2 0.008 5

3&4 0.032 20

5&6 0.052 25

7&8 0.113 37

9&10 0.185 50

11&12 0.001 75

With the increase in temperature there is an increase in the rate of reaction. The trend remains

until the optimum temperature when the highest rate is observed. With further increase in

temperature, the rate falls quickly. From the Graph 9, we can see that the peak value is reached

at temperature around 40.8oC. On the other hand the graph is not very accurate and therefore

the best way is to determine the optimum range. In this case, it is 40.4-50.2oC.


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Table 19

Values of log(V) and 1/temperature.

Corresponding

test tubes

Rate

(�mole/min)

Temperature

(o

log(v) 1/Temperature

(o

1&2 0.008 278.200 -2.097 0.0036

3&4 0.032 293.200 -1.495 0.0034

5&6 0.052 298.200 -1.284 0.0034

7&8 0.113 310.200 -0.947 0.0032

9&10 0.185 323.200 -0.733 0.0031

11&12 0.001 348.200 -3.000 0.0029

The effect of pH on enzyme activity.

To measure how the rate of reaction is affected by the pH, the absorbance was

measured. In this part of the experiment, the amount of substrate and citrate buffer were

constant. The independent variable are pH values of citrate buffer.


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Table 20

Absorbance value at different pH of 1.0ml citrate buffer.

Test tuba number 1 3 5 7 9 11 13

Enzyme solution (ml) 1.0 1.0 1.0 1.0 5.5 1.0 1.0

pH of 1.0ml Citrate

buffer

3.0 4.0 4.5 5.0 5.5 6.0 7.0

Absorbance 0.036 0.214 0.291 0.363 0.328 0.253 0.05

With the increase of pH, in the test tubes 1-7 there is an increase in absorbance. However in the

test tubes 9-13, a decrease in absorbance can be observed. From this data, at pH=5.0, the

highest absorbance was recorded. In order to determine the optimum pH range, the amount of

PNP and the rate of reaction were calculated.

Table 21

Rate of reaction at different pH of 1.0 ml citrate buffer.

Test tube pH of 1.0 ml

citrate buffer

Amount of PNP

(�mole)

Rate (�mole/min)

1 3.0 0.116 0.008

3 4.0 0.692 0.032

5 4.5 0.941 0.063

7 5.0 1.174 0.078

9 5.5 1.061 0.071

11 6.0 0.818 0.055

13 7.0 0.162 0.011


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The Graph 11 presents the relationship between pH of 1.0ml citrate buffer and its rate of

reaction. As in the case of temperature, with the increase in pH, the rate increases up to a

certain point where it reaches the highest value. With further increase in pH, the rate of reaction

decreases. From the curve, the optimum pH range for acid phosphate is between 4.8 and 5.3.

Discussion

In these experiments, it has been demonstrated that there is a variety of factors that have

a tremendous effect on the rate of the enzymatic reaction. Temperature, amount of substrate,

presence of inhibitor and pH were investigated.

In order to confirm that the increase in substrate concentration results in the increase in

the rate of reaction up to a certain point, rate of the reaction was determined for different

volumes of 10mM PNPP in the solution. The results presented in the Michaelis-Menten

equation confirmed that hypothesis. Using the Lineweaver-Burk plot, the and were

equal to and respectively. In the Michaelis-Menten equation,

and The difference in results obtained from the Graph

4 and 6, gives the evidence how the real values can be underestimated using the first graph. By

comparing experimental and theoretical rate of reaction, it can be noticed that for both

curves are very similar. This suggests that the experimental curve is correct with the theoretical

data and the results obtained are correct.

After reaching a maximal velocity, when more substrate (PNPP) is added, molecules

have to wait for free enzyme to undergo catalytic reaction (Berg, Tymoczko, Stryer, 2012).

Unlike the , is independent on the acid phosphatase concentration and therefore is an

excellent enzyme affinity indicator. A low suggests weak enzyme-substrate binding and

therefore a low substrate concentration may be enough to reach a maximum rate (Hames,

Hooper, 2009).

Secondly, the effects of the inhibitor presence on the enzyme activity were noticed.

Literature states that buffer will inhibit the enzymatic reaction (Martini, Nath, Bartholomew,

2012). To prove that, a constant amount (1ml) of phosphate-containing citrate buffer was added

to the solutions with different amount of 10mM PNPP and the absorbance values were

measured. At the beginning in the Michaelis-Menten equation, the increase in the rate is slower


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than in inhibitor free reaction, however with time the maximum velocity is reached, This

supports the hypothesis that the substrate increase overcome the acid phosphatase. From the

Graph 8, values of and were calculated.

Without Inhibitor With Inhibitor

By comparing the results for the reaction in the presence and without an inhibitor, it can

be noticed that there is a small decrease in in reaction with inhibitor and a bigger increase

in , equal to 0.145mM. This suggest that a phosphate-containing citrate buffer is an

competitive inhibitor.

The relationship between temperature and enzyme activity was determined by taking

the absorbance measurement for the solutions at different temperatures: 5, 20, 25, 37, 50, 75 oC.

The amount of substarte and citrate buffer were constant. The Table 18 shows that for the first

five sets of corresponding test tubes, there is an increase in the rate of reaction, reaching at 50 oC the velocity of 0.185 mole/min. In the last set, sharp decrease was noticed. This supports

the theoretical information that with the increase in temperature there is an increase in the rate

of reaction by increasing the thermal energy of the substrate molecules. It reaches the peak

value and then enzyme starts to denature, non-covalent interactions are disrupted and the

reaction rate rapidly falls down (Roberts, Reiss, 2000). Plotting the data (Graph 9), indicated

the optimum temperature range of 40.4-50.2oC for this reaction.

Calculating the values of log(V) and 1/temperature, presented the relationship between

the temperature and the reaction velocity in an linear pattern. The Graph 10 clearly shows

almost directly proportional dependence from which the optimum temperature can be more

accurately determined. On the other hand, in this experiment there are too big differences

between the following temperatures to safely determine the optimum value. From the same

graph, it can be observed that one result is separated from the others and does not follow the

linear pattern. This is a denatured enzyme and due to lost enzymatic activity it has a very low

rate of reaction ( ).

Finally, pH affects the rate of an enzyme- catalysed reaction by changing the charges

carried by ionisable side chains. This will result in disruption of tertiary structure of an enzyme

and finally in the denaturation. Even the small pH deviations lead to change in the rate of the

reaction (Martini, Nath, Bartholomew, 2012). As recorded in the Table 20, the change of pH from

4.0 to 4.5 resulted in pH change of 0.077. The results of the experiment shows that with the

increase of pH, in the test tubes 1-7 there is an increase in absorbance. However in the test


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tubes 9-13, a decrease in absorbance can be observed. As in case of temperature, the Graph 11

is not enough accurate to determine the optimum pH therefore, the optimum pH range for acid

phosphate is between 4.8 and 5.3.

The results of the experiment were correct with the information stated in literature.

However, conducting the same experiment for different types of enzymatic reaction and

comparing the results might give an interesting feedback.

References

1. Hames, D., Hooper, N. 2009. Biochemistry, 3rd

edn. Abingdon: Taylor & Francis

Group.

2. Kiskines, A.M.P., Klibanov, A.M. 1996. Enzymatic reactions in organic media.

Glasgow: Blackie Academic & Proffesional.

3. Martini, F.H., Nath, J.L., Bartholomew E.F. 2012. Fundaments of anatomy &

physiology, 10th

edn. Edinburgh: Pearson.

4. Roberts, M., Reiss, M. 2000. Advanced Biology, 2nd

edn. UK: Nelson.

5. Watson, D. 2014. Year 1Biochemistry and Biomedical ScienceProtocol Booklet

2014/14.Keele University.

enzymes lab report

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