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REACTIONS IN GLYCOLYSIS

Name

Student ID

Course Code

Date

: Kelvin

: 15WAR03018I

: RBS-2 Group 2

: 28 June 2016

Objective

1. To study the reversibility of an enzyme catalysed reaction in glycolysis

2. To determine the equilibrium constant of the reaction at different temperature

3. To construct a Gibb’s graph and observe the relationship with temperature

Materials

Beaker

Test tubes

Test tubes rack

Graduating cylinder

Pipette

Water bath

Spectrophotometer

2 mM glucose-6-phosphate

2 mM fructose-6-phosphate

Phosphoglucose isomerase

20% metaphosphoric acid

0.2 M tris-HCl buffers pH 8.0

1% resorcinol in 9% ethanol

28% HCl

Procedure

Part A

2 test tubes were prepared and labelled with G and F indicating glucose and fructose. 8.0 ml

of glucose-6-phosphate was pipetted into test tube G. 8.0 ml of fructose-6-phosphate was

pipetted into test tube F. Both test tubes were sat on a test tube rack at room temperature. 2.0

ml of phosphoglucose isomerase was then added into each test tube and mixed well. The time

recording was started during the addition. 7 sets of test tubes were prepared and labelled with

G and F according to the time intervals of sample extraction. 1.0 ml of sample was taken

from each tube at 5, 10, 20, 30, 45, 60 and 80 minute intervals. 1.0 ml of 20%

metaphosphoric acid was then added into each sample. All the samples excluding the first 2

test tubes were assayed using the Seliwanoff Assay.

Part B

2 test tubes were prepared and labelled with G and F indicating glucose and fructose. 0.5 ml

of glucose-6-phosphate was pipetted into the test tube G. 0.5 ml of enzyme was then added

into the same test tube. 0.5 ml of fructose-6-phosphate was pipetted into test tube F. 0.5 ml of

enzyme was then added into the same test tube. Both test tubes were left in a water bath at

55°C for 80 minute. 1.0 ml of 20% metaphosphoric acid was then added into each test tube

and assayed for fructose-6-phosphate using the Seliwanoff Assay.

Part C

7 test tubes were prepared and labelled with number 1 to 7. The sample for each test tube was

prepared following the table below:

Tube #

2 mM

2 mM

0.2 M Tris20%

% FructoseGlucose-6Fructose-6- HCl buffers Metaphosphori 6-Phosphate

Phosphate

Phosphate

pH 8.0

c acid

(ml)

(ml)

#1

0

0.5

0.5

1.0

100

#2

0.1

0.4

0.5

1.0

80

#3

0.2

0.3

0.5

1.0

60

#4

0.3

0.2

0.5

1.0

40

#5

0.4

0.1

0.5

1.0

20

#6

0.5

0

0.5

1.0

0

#7

0

0

0.5

1.0

0

All the 7 test tubes were then assayed using the Seliwanoff Assay for fructose-6-phosphate.

Seliwanoff Assay

2 ml of 1% resorcinol in 95% ethanol was added into each sample. 6 ml of 28% HCl was then

added into each sample. The samples were mixed well and left in a water bath at 80°C for 8minute sharp. The samples were then immediately cooled by placing them in cold water.

Each sample was poured into the cuvette to proceed with absorbance reading at 436 nm.

Spectrophotometer was calibrated using blank solution in test tube 7.

Results

Part A

Table 1

Time intervals (min)

5

10

20

30

45

60

80

Glucose-6-Phosphate

Absorbance

0.073

0.196

0.242

0.355

0.376

0.412

0.415

%fructose-6-phosphate

5.157

29.275

38.294

60.451

64.569

71.627

72.216

Sample calculation of %fructose-6-phosphate using the standard curve equation

**At 5 minute intervals
**

Abs = y = 0.073

y = 0.0051x + 0.0467

0.073 = 0.0051x + 0.0467

x = 5.157

At 10 minute intervals

Abs = y = 0.196

y = 0.0051x + 0.0467

0.196 = 0.0051x + 0.0467

x = 29.275

The same method is used to calculate the following %fructose-6-phosphate.

**From the graph above, the time where the concentration in equilibrium is 80 minute.
**

Therefore, the Ceq value for %fructose-6-phosphate is 72.216.

Table 2

Fructose-6-Phosphate

Absorbance

%fructose-6%glucose-6phosphate

phosphate

5

0.534

95.549

4.451

10

0.498

88.490

11.51

20

0.445

78.098

21.902

30

0.426

74.373

25.627

45

0.411

71.431

28.569

60

0.402

69.667

30.333

80

0.399

69.078

30.922

Sample calculation of %fructose-6-phosphate using the standard curve equation

Time intervals (min)

**At 5 minute intervals
**

Abs = y = 0.534

y = 0.0051x + 0.0467

0.534 = 0.0051x + 0.0467

x = 95.549

At 10 minute intervals

Abs = y = 0.498

y = 0.0051x + 0.0467

0.498 = 0.0051x + 0.0467

x = 88.490

The same method is used to calculate the following %fructose-6-phosphate. Then, by

deducting the %fructose-6-phosphate results from 100%, %glucose-6-phosphate can be

determined. For example, at 5 minute intervals, 100% - 95.549% = 4.451%.

**From the graph above, the time where the concentration in equilibrium is 80 minute.
**

Therefore, the Ceq value for %glucose-6-phosphate is 30.922.

Thus, we can proceed to the calculation of Keq as follow,

Ceq ( Fructose−6−Phosphate )

Keq( RT ° ) =

Ceq ( Glucose−6−Phosphate )

=

72.216

30.922

= 2.335

Calculation of ∆G

Assume room temperature at 27°C in this experiment.

Sample calculation of ∆G

∆G

= −2.303 × R × T × log ( K eq )

∆G

**= −2.303 ×1.987 × 300× log ( 2.335 )
**

= −505.591 cal mol

−1

Part B

Test #

**Absorbance (glucose-6Absorbance (fructose-6phosphate)
**

phosphate)

#1

0.143

0.249

#2

0.146

0.253

#3

0.140

0.250

3 test were done to obtain the average of the reading; the aim is to improve accuracy.

Glucose-6-Phosphate

Average Abs = 0.143

Abs = y = 0.143

y = 0.0051x + 0.0467

0.143 = 0.0051x + 0.0467

x = 18.882

The final Ceq for %fructose-6-phosphate is 18.882.

Fructose-6-Phosphate

Average Abs = 0.251

Abs = y = 0.251

y = 0.0051x + 0.0467

0.251 = 0.0051x + 0.0467

x = 40.059

The %fructose-6-phosphate is 40.059. Therefore, the Ceq for %glucose-6-phosphate is 100%

- 40.059% = 59.941.

Calculation of Keq

18.882

Keq(55 ° C )

= 59.941

= 0.315

Calculation of ∆G at 55°C

∆G

= −2.303 × R × T × log ( K eq )

= −2.303 ×1.987 × 328× log ( 0.315 )

−1

= 753.010 cal mol

**Calculation of ∆S using simultaneous equation method
**

∆H – (300) ∆S = -505.591

∆H – (328) ∆S = 753.010 (-)

28 ∆S = -1258.601

∆S

= -44.95 cal mol-1 K-1

Calculation of ∆H

Keq 2

−∆ H

1

1

−

log

=

2.303 × R T 2 T 1

Keq1

(

log

0.315

2.335

∆H

=

)

−∆ H

1

1

−

2.303 ×1.987 328 300

(

)

0.315

24600

= −log 2.335 × 2.303 ×1.987 ×− 7

−1

= −13991 cal mol

Graph for ∆G

From the linear equation obtained from the graph, we can prove that ∆S = -slope of the linear

equation which is -44.95. As for the intersect which is -13991 is equal to the ∆H calculated.

Part C

Table

Tube#

#1

#2

#3

#4

#5

#6

%fructose-6-phosphate

100

80

60

40

20

0

Absorbance

0.56

0.47

0.32

0.27

0.13

0.06

**The linear equation obtained from the graph is y = 0.0051x + 0.0467
**

Discussion

Glycolysis uses enzyme to catalyse the reaction in a reversible manner. In this case,

we were using phosphoglucose isomerase which convert glucose-6-phosphate into fructose6-phosphate. This reaction catalysed by the enzyme is reversible, meaning that the enzyme

catalysed both forward and back reaction. So, the rates of reactions will become equal or in a

state of equilibrium in terms of concentration. Thus, producing an equation such as

[ C ][ D ]

K eq is equilibrium constant where it will not be

K eq =

[ A ] [ B ] in A + B ↔ C + D.

affected in a presence of enzyme but depend on temperature. [1] Higher temperature

increases the rate of reaction in glycolysis conversion. Higher temperature favour reactant,

meaning that the equilibrium constant will be lower. Other than that, a test or assay called

Seliwanoff Assay was done to determine the fructose-6-phosphate. Seliwanoff Assay able to

distinguishes between aldose and ketose sugars. It able to differentiate them because of their

ketone/aldehyde group. In this test, a resorcinol reagent was used to react with the dehydrated

ketose to give pale coloured compound. [2]

In this experiment, we obtained data from the reading of absorbance in

spectrophotometer. From the absorbance obtained, we are able to construct a standard curve

in part C to get the linear equation. All the sample in section C experiment was used to

determine the % fructose-6-phosphate which will be constructed into a standard curve. The

equation obtained from the graph will be used for all the calculation of percent sugars in the

solution. The linear equation y = 0.0051x + 0.0467 is used to sub in the absorbance which

results in the value of x. The x value determined the percent concentration of sugars. For the

first set of data, which is glucose-6-phosphate reading, % fructose-6-phosphate is needed.

This is because we wanted to know how many percent of fructose-6-phosphate is formed

from the conversion of glucose-6-phosphate in the reaction. By using the % fructose-6phosphate with the time intervals for each intake of the sugars, a graph of glucose-6phosphate can be constructed. In this graph, we are going to determine the equilibrium

concentration of the reaction for fructose-6-phosphate. It is when the data in the graph

becoming constant and hence reached the equilibrium. The same goes to the second set of

data, where fructose-6-phosphate is the sample. Using the same method, we are able to

calculate the % fructose-6-phosphate. However, in this part, we need to know how many

percent the fructose-6-phosphate has been converted to glucose-6-phosphate. Thus, we have

to calculate the % glucose-6-phosphate by deducting 100% with % fructose-6-phosphate.

With this, we can construct another graph to determine the constant data and hence be able to

determine the equilibrium concentration of glucose-6-phosphate. These 2 equilibrium

concentration, C eq , obtained from glucose and fructose will be used to calculate the value

of equilibrium constant,

K eq .

**Next, after determining the equilibrium constant at room temperature, we can proceed
**

to the calculation of ∆G. the formula to calculate ∆G is provided in the laboratory manual,

which is ∆ G=−2.303 × R ×T ×log ( K eq ) . From this formula, we obtained -505.591 as the

∆G at room temperature. Then, we will proceed to part B where we obtained 3 sets of data for

glucose and fructose at 55°C. In this part, we were dealing with the sugars under higher

temperature which later is used to compare. We repeated 3 times of the experiment in order to

obtain a constant data by calculating the average from the 3 sets of data. Same methodology

of calculation is used in this part where the absorbance reading substituted into the y and

determine the x which is percent concentration of the sugars. No graph needed in this section

as we are able to obtain a constant data for equilibrium concentration. Then, the constant

equilibrium in this part is also determined. Hence, the ∆G value is obtained. With the

presence of both ∆G at room temperature and 55°C, entropy change, ∆S, can be determined.

From the equation ∆G = ∆H - T∆S, entropy change is determined by substituting the data in a

simultaneous equation to eliminate enthalpy change which is still an unknown. Enthalpy

change can be determined by comparing the equilibrium constant from both temperature,

room temperature and 55°C. To show and prove that the enthalpy change, entropy change and

free energy change is proportional to the temperature, a Gibb’s graph is constructed at

different temperature. From the graph, it is positive to assume that temperature affects the

reaction.

The results obtained from the experiment is positive results. However, there are some

deviation found in the results. It is due to some practical errors during the experiment. One of

suspected error is when transferring the chemical, graduated cylinder was used instead of

pipette. Measuring cylinder is not accurate compare to graduated pipette, it may affect the

outcome of the experiment. Another error is suspected to be an error in pipetting technique

where transferring different chemical using a single pipette without rinsing it. It may have

minor effect on the concentration of the sample. Other than that, during the absorbance

**reading procedure, cuvette was placed wrongly or the clear side of the cuvette accidentally
**

touched causes the visible light fail to pass through.

Conclusion

The objective of this experiment is to study the reversibility reaction in glycolysis by

the help of equilibrium constant and Gibb’s graph. From the results, equilibrium constant tells

us that glycolysis catalysed by an enzyme is a reversible reaction where after some time, the

net movement of reaction slowed down and stop. It is where the equilibrium constant

reached. Lastly, the effect of temperature on the reaction can be observed through the changes

in Gibb’s graph.

References

1. The laboratory Manual. (2016). The Reversibility of Enzyme Catalysed Reactions in

Glycolysis. [Accessed 1 Jul. 2016].

2. The Wall of Biochemistry. (2012). Carbohydrate’s Test. [online] Available at:

https://fulltimes.wordpress.com/carbohydrates-test/ [Accessed 1 Jul. 2016].

3. Clark, J. (2013). equilibrium constants and changing conditions. [online]

Chemguide.co.uk. Available at:

http://www.chemguide.co.uk/physical/equilibria/change.html [Accessed 1 Jul. 2016].

4. Pulido, K. and Chappell, C. (2013). Effect of Temperature on Equilibrium - Chemwiki.

**[online] Chemwiki.ucdavis.edu. Available at:
**

http://chemwiki.ucdavis.edu/Core/Physical_Chemistry/Equilibria/Le_Chatelier's_Principl

e/Effect_Of_Temperature_On_Equilibrium_Composition [Accessed 1 Jul. 2016].

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