TITLE OF THIS REPORT: Enzyme Kinetic of Acid Phosphate.
WRITTEN BY LAW JIA JUIN
WRITTEN BY LAW JIA JUIN
CONTENT OF THIS FULL REPORT
A. INTRODUCTION
B. AIMS AND OBJECTIVES
C. APPARATUS AND MATERIALS
D. PROCEDURES
E. RESULTS+EXPLANATIONS+CALCULATIONS +TABLES+ GRAPHS
F. DISCUSSION + ANSWERING TO QQUESTIONS
G. CONCLUSION
H. REFERENCES
PLEASE FOLLOW THIS BLOG IN ORDER TO UNLOCK THIS IMAGE/DIAGRAM/EQUATION/TABLE/GRAPH/ANSWER/EXPLANATION
A. INTRODUCTIONS
There are two types of phophatase enzyme, namely acid phosphatase and alkaline phosphatase. [1]Acid phosphatase works best in acidic medium, wherease alkaline phosphatase works best in alkaline medium. [1]Acid phosphatase is a body enzyme which plays an important role in catalyzing body’s chemical reactions. [1] Generally, it works by catalyzing the hydrolysis of phosphomonoesters. [1] It is found abundantly in men’s prostate glands. [1]Its’ physiological function is to liquefy the semen produced. [1]
Phophatase enzyme is found to be increased in those suffering from prostate cancers. [2] It is stored in lysosomes, these lysosomes will then fuse with endosomes, releasing the acid phosphatase. [2] Acid phosphatase works best in acidic medium, it works by eliminating the phosphate group from its’ substrate. This reaction is known as hydrolysis. [2]
PLEASE FOLLOW THIS BLOG IN ORDER TO UNLOCK THIS IMAGE/DIAGRAM/EQUATION/TABLE/GRAPH/ANSWER/EXPLANATION
<The diagram above shows us the hydrolysis of NPP by enzyme phosphatase. The product is 4-nitrophenol. This 4-nitrophenol can be converted into phenolate by reacting with NaOH. Phenolate has chromophore thus ,it can absorb UV-light >
From the results of this experiment, we are going to study on the properties of an acid phophatase. In our experiment, the 4-nitrophenyl phosphate (NPP) was used as a substrate. This NPP was then hydrolyzed by the acid phosphatase into 4-nitrophenol, which was further converted into phenolate by reacting it with NaOH. Phenolate was important in our experiment because it contains chromophore which can absorb UV-Vis light.
B. AIMS OF THIS EXPERIMENT
Part 1: To study the relationship among absorbance, concentration of product formed as well as time of reaction
Paet 2 : To study the optimal pH medium for acid phosphatase reaction. Also to study the relationship between reaction velocity and pH measured
Part 3: To study the effect of NaF on enzyme catalysed reactions.
C. APPARATUS, MATERIALS AND PROCEDURES
APPARATUS FOR PART 1: Test tubes, pipettes, suction bulb, micropipette, stop watch, electrical water bath, electronic shaker, thermometer, UV-Vis spectrometer, plastic cuvette
MATERIALS FOR PART 1: Buffer (pH 5.0), NPP (2.5 mM), diluted enzyme “ENZ” (2.5 mM), 1M NaOH, stickers for labelling
PROCEDURES FOR PART 1: Time linearity of hydrolysis
1. A test tube containing 2 ml buffer (pH 5.0) and 2 ml NPP (2.5 mM) was prepared.
2. This test tube was placed in a water bath (37oC) for 3 minutes.
3. The diluted enzyme “ENZ” was pre-prepared by the lab assistants.
4. 3 test tubes each containing 5.5 ml NaOH were prepared. The test tubes prepared were all labeled. First test tube labeled as “0 min”, second test tube labeled as “7.5min” and the third test tube labeled as “15 min”.
5. 1 ml of diluted enzyme “ENZ” was added to the reaction mixture (which remains in the water bath) using micropipette .At the same time, the stop watch is started. Immediately (0 min time), 0.5 ml of this mixture was transferred to the test tube of NaOH labeled “0 min” by my group mate using micropipette.
6. After exactly 7.5 and 15 mins of reaction time at 37oC, 0.5 ml samples of the reaction mixture was removed using micropipette and added into their corresponding NaOH test tubes labeled as “7.5mins” and “15mins” respectively. All the three test tubes were mixed thoroughly. During each time of transferring the physical appearances of reacting mixture was observed.
7. The absorbance of the 3 alkaline solutions were measured respectively at 400nm (Distilled water was used as blank)
Thus the gradient of the linear graph obtained, representing the initial rate of enzyme catalyzed reaction. This is because the amount of phenoate ion formed is proportional to the 4-nitrophenol formed.
APPARATUS FOR PART 2: Test tubes, pipettes, suction bulb, micropipette, stop watch, electrical water bath, electronic shaker, thermometer ,pH meter, UV-Vis spectrometer, plastic cuvette
MATERIALS FOR PART 2: Buffer (pH 5.0), NPP (2.5 mM), diluted enzyme “ENZ” (2.5 mM), 1M NaOH, stickers
PROCEDURES FOR PART 2:
Part 2: Time linearity of hydrolysis
Determination of optimal pH
1. 8 test tubes were prepared and labeled as pH3àpH8. Each test tube received 2 ml of buffer with one of the following pH values: 3.0, 4.0, 4.5, 5.0, 5.5, 6.0, 7.0, 8.0. Besides that, 2 ml of 2.5 mM NPP was also added into each tube. The mixture in each test tube was mixed thoroughly before been put into the 37oC water bath for 3 min.
2. Another set of 8 test tubes were used to hold 5.5ml of 0.1 M NaOH. And they were labeled as pH3à8
3. After 3 minutes, 1 ml of diluted acid phosphatase (“ENZ”) was added into every buffer test tubes using the micropipette (stop watch was started instantly).
4. The test tube with buffer pH 8 was shaken and 0.5 ml of its’ reaction mixture was immediately been transferred using micropipette to another test tube containing 5.5 ml of 0.1 M NaOH, labeled as pH 8. The remainder of the reaction mixture was retained. The other 7 test tubes(pH3àpH7) were remained in water bath.
5. Step 4 was repeated after 15 minutes for test tubes holding buffer solution of pH3à pH7.
6. The amount of 4-nitrophenol liberated from the ester at each pH were determined using UV-VIS spectrometer at wavelength 400nm
7. The remainders of the reaction mixtures were cooled down to room temperature. And their respective pH was re-measured using a pH meter fitted with a calibrated glass electrode.
8. A graph of reaction velocity (mol of 4-nitrophenol formed per minute in the reaction mixture) against the measured pH of the reaction mixture was plotted.
APPARATUS FOR PART 3: Test tubes, pipettes, suction bulb, micropipette, stop watch, electrical water bath, electronic shaker, thermometer, UV-Vis spectrometer, plastic cuvette
MATERIALS FOR PART 3: Buffer (pH 5.0), NPP (2.5 mM), diluted enzyme “ENZ” (2.5 mM), 1M NaOH, stickers for labelling
PROCEDURES FOR PART 3:
Part 3:Inhibition of hydrolysis
1.Test tubes series of 3 reaction mixtures were prepared. These tubes were labeled as F0, F0.5 and F1. 2 ml of buffer pH 5.0 and 2 ml of 2.5 mM NPP were added into each tube. To tube labeled as F0, 1 ml of buffer pH 5.0 was added; for tube F0.5 1 ml of 2.5 mM NaF was added; and for tube F1, 1 ml of 5.0 mM NaF was added.
2.The tubes were placed in the water bath (37oC) for 3 minutes.
3. The diluted enzyme “ENZ” was pre-prepared by the lab assistants.
4. 3 test tubes each containing 5.5 ml NaOH were prepared. They were labeled as F0, F0.5 and F1.
5. 1 ml of the diluted enzyme “ENZ” was added to each reaction mixtures (which remains in the water bath).
6.After exactly 7.5 mins of reaction time at 37oC, 0.5 ml samples of the reaction mixtures was removed using pipette and was added into the corresponding tubes of NaOH and they were all mixed properly.
7.The absorbances of the 3 alkaline solutions at 400nm against H2O as blank were determined.
D. RESULTS ,CALCULATIONS, TABLES AND GRAPHSfor part 1, 2, 3:
PART 1: Time linearity of hydrolysis
TEST TUBE at time
|
0 MINUTES
|
7.5 MINUTES
|
15 MINUTES
|
Absorbance at 400nm
|
0.007
|
0.047
|
0.082
|
Colour
|
clear
|
Slight yellowish
|
More yellowish
|
Table 1: Absorbances and concentrations of 4-nitrophenol obtained at different intervals.
PLEASE FOLLOW THIS BLOG IN ORDER TO UNLOCK THIS IMAGE/DIAGRAM/EQUATION/TABLE/GRAPH/ANSWER/EXPLANATION
GRAPH of Absorbance at 400nm versus time
The gradient of this linear graph will be representing the initial rate of enzymatic reaction.
This is because :
1. absorbance is proportional to concentration of the phenoate ion formed. (As suggested by Beer’s lambert Law).
2. Besides that, the amount of phenoate ion formed is proportional to the 4-nitrophenol formed.
The initial rate of reaction =(0.076-0.02)/(13.6-2.4)= 5x10-3molL-1min-1
PART 2: Determination of optimal pH
PLEASE FOLLOW THIS BLOG IN ORDER TO UNLOCK THIS IMAGE/DIAGRAM/EQUATION/TABLE/GRAPH/ANSWER/EXPLANATION
From the equation above we know that 1 mole of NPP react with 1 mole of enzyme to produce 1mole of 4-nitophenol, and 1mole of 4-nitophenol reacts with 1 mole of OH- to produce 1 mole of phenolate ion.
In UV-Vis spectrometer we are actually measuring the concentration of phenolate ion, but since we know that 1mole of 4-nitrophenol will produce 1 mole of phenolate when reacts with 1 mole of NaOH, then we can actually use the concentration of phenolate ion to find the concentration of 4-nitrophenol. Or in the other word we can say that the concentration of phenolate formed is proportional to the concentration of 4-nitrophenol produced.
1.The concentration (mol/L) of phenolate ion formed
= C (mol/L)
2.The concentration (mol/L) of 4-nitrophenol reacting with NaOH = The concentration (mol/L) of phenolate ion formed
= C (mol/L)
3.The actual concentration (mol/L) of 4-nitrophenol that are produced in 5mLof reacting solution (inside test tube of water bath) is equal to = C (mol/L) x F= D(mol/L)
Thus the actual concentration (mol/L) of 4-nitrophenol that are produced in 5mLof reacting solution (inside test tube of water bath) is equal to= C (mol/L) x 12= D(mol/L)
4.The actual concentration (µmol/ml) of 4-nitrophenol that are produced in 5mLof reacting solution (inside test tube of water bath) is equal to
= C (mol/L) x 12 x (1000000/1000)= C (mol/L) x 12 x 1000= D**(µmol/ml)
5.The amount (µmol) of 4-nitrophenol that are produced in 5mLof reacting solution (inside test tube of water bath) is equal to= D** x V= E
Therefore, the amount (µmol) of 4-nitrophenol that are produced in 5mLof reacting solution (inside test tube of water bath) is equal to= D** x 5= E
6.Thus to relate the concentration of phenoate ion, C (mol/L) to amount of 4-nitrophenol (µmol) in 5ml reacting solution, E:
1. Calculations for the concentration (mol/L)of 4-nitrophenol reacting with NaOH, C
PLEASE FOLLOW THIS BLOG IN ORDER TO UNLOCK THIS IMAGE/DIAGRAM/EQUATION/TABLE/GRAPH/ANSWER/EXPLANATION
pH 3 Test Tube pH 4 Test Tube
C = 
C = 
C =
= 5.52 x10-7 mol.L-1 = 2.32 X 10-6 mol.L-1
pH 4.5 Test Tube pH 5 Test Tube
C = 
C = 
C =
= 2.87 X 10-6 mol.L-1 = 3.04 X 10-6 mol.L-1
pH 5.5 Test Tube pH 6 Test Tube
C = 
C = 
C =
= 2.76 X 10-6 mol.L-1 = 2.48 X 10-6 mol.L-1
pH 7 Test Tube pH 8 Test Tube
C = 
C = 
C =
= 1.32 X 10-6 mol.L-1 = 2.76 X 10-7 mol.L-1
2. Calculations for the amount of 4-nitrophenol (µmol) in 5ml reacting solution, E
PLEASE FOLLOW THIS BLOG IN ORDER TO UNLOCK THIS IMAGE/DIAGRAM/EQUATION/TABLE/GRAPH/ANSWER/EXPLANATION
pH 3 Test Tube pH 4 Test Tube
E = 5.52x10-7x12x1000x5 E=2.32 X 10-6 x12x1000x5
=0.03312 =0.1392
pH 4.5 Test Tube pH 5 Test Tube
E = 2.87x10-6x12x1000x5 E=3.04 X 10-6 x12x1000x5
=0.1722 =0.1824
pH 5.5 Test Tube pH 6 Test Tube
E = 2.76x10-6x12x1000x5 E=2.48 X 10-6 x12x1000x5
=0.1656 =0.1488
pH 7 Test Tube pH 8 Test Tube
E = 1.32x10-6x12x1000x5 E=2.76 X 10-7 x12x1000x5
=0.00792 =0.01656
3.
PLEASE FOLLOW THIS BLOG IN ORDER TO UNLOCK THIS IMAGE/DIAGRAM/EQUATION/TABLE/GRAPH/ANSWER/EXPLANATION
pH 3 Test Tube pH 4 Test Tube
= 0.00228 
= 0.00928
pH 4.5 Test Tube pH 5 Test Tube
= 0.01148 
= 0.01216
pH 5.5 Test Tube pH 6 Test Tube
= 0.01104 
= 0.00992
pH 7 Test Tube pH 8 Test Tube
= 0.00528 
= 0.001104
Tables for part 2
pH of buffer solution
|
pH of buffer solution after cool
|
Absorbance at 400nm
|
Concentration C (mol/L)
|
4-nitrophenol (µmol) in 5ml reacting solution, E (µmol)
|
Velocity of reaction (µmol.min-1)
|
3.0
|
2.8
|
0.010
|
5.52 X 10-7
|
0.03312
|
0.02208
|
4.0
|
4.03
|
0.042
|
2.32 X 10-6
|
0.1392
|
0.00928
|
4.5
|
4.5
|
0.052
|
2.87 X 10-6
|
0.1722
|
0.01148
|
5.0
|
5.02
|
0.055
|
3.04 X 10-6
|
0.1824
|
0.01216
|
5.5
|
5.31
|
0.050
|
2.76 X 10-6
|
0.1656
|
0.01104
|
6.0
|
6.04
|
0.045
|
2.48 X 10-6
|
0.1488
|
0.00992
|
7.0
|
6.84
|
0.024
|
1.32 X 10-6
|
0.0792
|
0.00528
|
8.0
|
7.84
|
0.005
|
2.76 X 10-7
|
0.01656
|
0.001104
|
Table 2 : Absorbance, measured pH, calculated concentrations and velocity of 8 different test tubes containing buffers of different pH
PLEASE FOLLOW THIS BLOG IN ORDER TO UNLOCK THIS IMAGE/DIAGRAM/EQUATION/TABLE/GRAPH/ANSWER/EXPLANATION
GRAPH of Absorbance at 400nm versus measured pH of the reaction mixture
GRAPH of Reaction velocity against the measured pH of the reaction mixture
From the graph we can conclude that the pH for maximum reaction velocity of acid phosphatase is around pH 5(this is taken as optimum pH for the action of acid phosphatase)
PLEASE FOLLOW THIS BLOG IN ORDER TO UNLOCK THIS IMAGE/DIAGRAM/EQUATION/TABLE/GRAPH/ANSWER/EXPLANATION
PART 3: Inhibition of hydrolysis
1. Calculations for the concentration of 4-nitrophenol produced in 5ml of reacting solution
F0=(0.045/18120)x12x1000x5=0.149 μmol
F0.5 = (0.031/18120)x12x1000x5=0.1026 μmol
F1=(0.018/18120)x12x1000x5=0..060 μmol
2. PLEASE FOLLOW THIS BLOG IN ORDER TO UNLOCK THIS IMAGE/DIAGRAM/EQUATION/TABLE/GRAPH/ANSWER/EXPLANATION
F0=0.149/7.5=0.0199
F0.5 = 0.1026/7.5=0.01368
F1=0..060/7.5=0.008
Tube
|
Absorbance at 400nm
|
Concentration of 4-nitrophenol in 5ml of reacting solution
(μmol)
|
Velocity
(μmol min-1)
|
F0
|
0.045
|
0.149
|
0.0199
|
F0.5
|
0.031
|
0.1026
|
0.01368
|
F1
|
0.018
|
0.060
|
0.008
|
Table 3 : Absorbance, concentration and velocity of three tubes labeled F0, F0.5 and F1 which contain varying amounts and concentrations of sodium bromide, NaF
Each tubes contain 2ml of buffer pH5.0 and 2mM NPP then, for tube which labelled
F0 = add 1ml of buffer pH5.0
F0.5 = add 1ml of 2.5mM NaF
F1 =add 1ml of 5.0mM NaF
Time of reaction is 7.5minutes, at 37ºC
E. QUESTIONS AND ANSWERS:
1. During catalysis, is the product (4-nitrophenol) liberated from the substrate (NPP) linearly with time?
-Yes it is linear
-The gradient of this linear graph will be representing the initial rate of enzymatic reaction. [3]
This is because :
a. absorbance is proportional to concentration of the phenoate ion formed. (As suggested by Beer’s lambert Law).
b. Besides that, the amount of phenoate ion formed is proportional to the 4-nitrophenol formed.
-The graph shows a linear positive line. NPP is hydrolyzed to 4-nitrophenol by the help of acid phosphotase and then it will further turn into 4- nitrophenolate by addition of a OH- which will absorb lights at 400nm.
- The stronger the absorbance the higher the concentration of 4-nitrophenolate.
-As time pass by, the absorbance of the product increases, this shows that more and more 4- nitrophenolate is produced.
The initial rate of reaction =(0.076-0.02)/(13.6-2.4)= 5x10-3molL-1min
2. What is the pH optimum of the reaction catalysed by acid phosphatase?
- From the graph we can conclude that the pH for maximum reaction velocity of acid phosphatase is around pH 5(this is taken as optimum pH for the action of acid phosphatase)
-The optimum pH for the action of phophatase is pH 5.
-The graph shows us that the peak of the is the highest at the pH 5 and hence the absorbance is highest during this pH, this proves that the reaction is rapid and products are liberated more during pH 5.
- From the graph we can conclude that the pH for maximum reaction velocity of acid phosphatase is around pH 5(this is taken as optimum pH for the action of acid phosphatase)
3. What is the modulatory effect of sodium fluoride that you could observe on the reactions catalysed by acid phosphatase? Is this effect concentration dependent?
-There are different types of enzyme modulators, some act as inhibitor while some act as promoter.
-Sodium fluoride (NaF) is an enzyme inhibitor; therefore the acid phosphatase catalysed reaction will be reduce by sodium fluoride. [5]
-We can see from table 3, as the concentration of sodium fluoride increases, the reaction of acid phosphatase reduces which means that the inhibition activity increases.
-The velocity of reaction decreases as the concentration of NaF increases.
-So we conclude by saying that, the inhibition effect of NaF is concentration dependent.
F. DISCUSSION:
Part 1: time linearity of hydrolysis
From the graph obtained in part 1, we can say that there is a linear relationship between the product concentration and time of reaction. This means that as the time increases, the enzymatic reaction also increases. linearly But the substrate (NPP) can act as a limiting factor for this enzyme activity, hence the enzyme activity continue to increase before it reaches a certain point (where all the substrate have been used up) and stop, a plateau will be seen in the graph[3]
The gradient of the linear graph will be representing the initial rate of enzymatic reaction. [3]
This is because :
1. absorbance is proportional to concentration of the phenoate ion formed. (As suggested by Beer’s lambert Law). [3]
2. Besides that, the amount of phenoate ion formed is proportional to the 4-nitrophenol formed. [3]
The initial rate of reaction =(0.076-0.02)/(13.6-2.4)= 5x10-3molL-1min-1
Part 2: Determination of optimal pH
The enzyme activity is affected by pH of buffer used, this is because the surface charges of enzyme active site is affected by pH medium. [4]If the charge of enzyme’s active site is opposite to that of substrate, then the substrate can bind effectively to the enzyme active site (with the help of electrostatic forces), so enzyme reaction will be higher. [4]
On the other hand, if the charges of enzyme and substrate are similar, then the substrate will no longer bind to the enzyme due to the repulsion forces, and thus the enzyme activity will be low.
In addition to that, changes in pH condition will also lead to the unwanted conformation of enzyme active site, affecting the binding of substrate to enzyme. [4] From the experiment, the enzyme we used is acid phosphatase and the experiment also shows that the optimum pH of this enzyme is pH 5.
In a nut shell we can say that the enzymes work best at its optimum pH, not too high nor too low. In a highly acidic or basic condition, the conformation of active site together with the changes surface charges of the enzyme will alter the .enzyme activity.
Part 3: Inhibition of hydrolysis
Now let us discuss on NaF inhibitor. From the results obtained, the test tube labeled with F0.5 and F1 give a lower absorbance reading than F0. This is due to presence of NaF in the test tube F0.5 and F1. Thus, we can conclude that the NaF an enzyme inhibitor. NaF is a chemicals or moleculesd that alter, slow down, and even stop the action of enzymes. If the concentration of NaF is increased, the absorbance of the enzyme decreases. In this experiment, the inhibitor we used is sodium fluoride which is concentration dependent. We can see from the table 3 that when the concentration of sodium fluoride increases, the enzymatic reaction reduces. Inhibitor is a substrate which binds to enzyme and it will either cease or reduce the enzyme activity. There are 2 different types of inhibitor, one being the competitive inhibitor and another one is non-competitive inhibitor. [5]The competitive inhibitor acts by binding to the enzyme through the substrate binding site and hence the substrate is unable to bind with it. [5]But this is reversible, when the concentration of the substrate is increase; the binding of inhibitor to the enzyme will be replace by the substrate.[5]For the non-competitive inhibitor, the inhibitor binds to the allosteric side of the enzyme and changes the conformational of the enzyme and hence the substrates are not able to bind to the enzyme. This kind of inhibition is irreversible. [5]
G. CONCLUSION:
1. The enzymatic reaction of acid phosphatase is linear with time.
2. The optimum pH for the enzymatic reaction is pH 5.
3. Sodium fluoride is the inhibitor of the acid phosphatase enzyme.
H. REFERENCES:
I. Enzyme Phosphatase.[Online].[Cited on 2012, 14 March]. Available from URL:: www.phosphatase.edu/.../Lab%204%20-%20Enzyme%20Kinetics.doc
2. John K. Lee, Kazimierz Chrzan, Robert H. Witt .Clinical of Prostate Cancer. Vol. 22. United States: Academic Press.;1999.
3. Enzyme Kinetics.[Online].[Cited on 2012, 14 March]. Available from URL: www.clt.astate.edu/.../Lab%204%20-%20Enzyme%20Kinetics.doc
4. The Chemistry of enzyme. [Online]. [Cited on 2012, 12 March]. Available from URL: http://www.chemistryofenzyme..com/imeltcomplz.htm
5. Enzyme inhibitors. [Online] [updated: 2003 Cited on 2012, 12 March]Available from: http://www.elmhurst.edu/~chm/vchembook/573inhibit.html
hi.. thanks for uploading this but may i know how can i unlock the rest of the images, graphs etc? i have already followed this blog but the boxes are still blank.
ReplyDeleteYou may search for our products through the search bar on our website. If you would like to receive a copy of our product catalog, please contact us at info@alfa-chemistry.com. Kojic acid dipalmitate
ReplyDelete