Enzyme Assay

Student no :

Date : 23.11.2010

 

  

Enzyme assay

Aim
The aim of this experiment is to examine the hydrolysis of milk lipids by lipases at different temperatures.




Introduction 


Enzymes are a diverse and important class of proteins. Biologists refer to enzymes as biological catalysts because they increase the rate of chemical reactions in living cells. Each enzyme is capable of catalyzing a reaction between a very specific molecule or set of molecules. Molecules that an enzyme reacts with are called substrates (Campbell,2005).



The mammalian pancreas synthesises lipase enzyme, this enzyme hydrolyse lipids converting molecules of fat from milk to fatty acids and glycerol.(Jones,2007)
The major function of fat in an organism is energy storage, humans and other mammals stock their long term food reserves in adipose cells. Just born baby mammals need this fat molecules from milk to provide energy for their organism.(Campbell,2005)
Enzymes can catalyze very specific reactions for a given substrate with incredible precision and speed, in part because of the overall shape assumed by the amino acids that constitute the enzyme. The shape of the enzyme is very important on relationship between protein structure and function. All enzymes have a conformation that produces an active site – as shown in fig.1 - a pocket or groove in the enzyme where the substrate binds.


The active site of an enzyme is typically specific for only one substrate, because the overall three-dimensional conformation of the active site is designed to fit the molecular shape of the substrate as shown in fig.1. Substrate binding to the active site of an enzyme is only the first step toward catalyzing a reaction. An enzyme does not become part of the reactants or products. As shown in fig.1 after an enzyme has catalyzed a reaction, it releases its substrate and then the active site of the enzyme is available to bind to another fresh substrate and repeat this process. For most enzymes this process, known as the catalytic cycle of enzyme activity, can be repeated very rapidly as long as there is enough substrate for the enzyme to react on.
(Campbell,2005)


Fig. 1 The active site and catalytic cycle of an enzyme
Source : Campbell et al, Biology, 2005, 7^th edition, Pearson Education, Benjamin Cummings ,page 153.
The catalytic cycle of an enzyme is often described by the following equation:

E + S --> ES --> E + P

This cycle begins when an enzyme (E) binds to a substrate (S) to form an enzyme-substrate complex (ES). Enzyme-substrate complexes typically form as a result of weak bonds between amino acids in the active site and atoms of the substrate. Then the enzyme release products (P) and the cycle is ready to repeat. Enzyme-catalyzed reactions can be reversible or irreversible.

Although enzymes are absolutely essential for accelerating biochemical reactions, a number of conditions influence enzyme activity. Enzymes don't always operate at their maximal rate most enzymes demonstrate temperature optimums – a temperature at which enzyme activity is greatest. Varying temperature conditions typically affects the conformation of the enzyme, which in turn influences an enzyme's ability to bind to its substrate and catalyze a reaction.( Mathews, 2000)
Under high temperatures protein shape changes (unfold) it becomes less active or inactive; this process is called protein denaturation as shown in fig. 2. Enzymes in response to an increase in temperature can break bonds–such as hydrogen bonds, Van der Waals attractions.,causing it to lose its conformation and hence it`s ability to function.If the denatured protein remains dissolved,it can often renature when the temperature of environment is restored to normal.(Campbell, 2005).


Figure 2 Denaturation and renaturation of a protein
Source : Campbell et al, Biology, 2005, 7^th edition, Pearson Education, Benjamin Cummings ,page 85.


Lipase hydrolyse lipids converting molecules of fat from milk to fatty acids and glycerol.(Campbell,2005) as shown in fig 3. Examples of lipids found in milk are shown in fig.4. The catalytic cycle has following equation:
Lipase + Molecules of fat --> Enzyme complex --> Lipase + Fatty acids + Glycerol



Fig.3 Hydrolyse of fat molecule, one water molecule is removed for each fatty acid joined to the glycerol.(Campbell,2005)
Source : Campbell et al, Biology, 2005, 7^th edition, Pearson Education, Benjamin Cummings ,page 75.

 

Fig. 4. Example of lipids found in milk.
Source: http://www.colorado.edu/intphys/Class/IPHY3430-200/image/02-8.jpg














The role of bile salt in lipid hydrolysis.


As shown in the fig . 5, lipids tend to coalesce into larger droplets which reduces the surface area for reaction. The hydrophobic lipid in the diagram is only accessible to the water soluble lipases at the interface between lipid and water.To increase the access (increased surface area) the lipid droplet must be broken up.
Bile salts secreted from the liver have molecules with a combination of hydrophobic and (lipophilic) hydrophilic regions.
Bile salts break up the lipid droplet into many smaller droplets thereby increasing the surface area of lipase access.

Fig.5 The role of bile in lipid hydrolysis. a) Bile salts and fatty acids. ;b) The fatty acids. ;c) Protein Lipase ;d) The lipoprotein complex ;e) Glycerol

Source :Click4Biology: Option H, Digestion

http://click4biology.info/c4b/H/H2.htm (visited on 18.11.2010)

Apparatus and Procedure

 

Apparatus :

7 Test Tubes

3 Test tube rack

7 Test tube bung

3 ml 3% Lipase

7 ml 1% Bile salts

3 ml 0.1M Sodium carbonate

1 ml Universal Indicator

15 ml Whole Milk

4 ml Distilled Water

Ice Bath 0^oC

Room temperature

3 Thermometers

Glass Pipettes, capacity 5ml and 25 ml

Plastic pipetts

Pipettes filler

Glass rod

75 ml capacity beakers

Labels

3 Stop-clocks, Models : Fast Time and Quantum (Fig.6)
Fridge at 4^oC, Model Zanussi Electrolux , Serial no : 07596
Water Bath at 45^oC, Model Clifton, Serial no : MCOLL 07598
Water Bath at 37^oC, Model Clifton,Serial no : MCOLL 07599
Bunsen Burner , Model :IBS Fireboy Plus,Serian no: MCOLL 07329 (Fig.7)
Camera photo ,Model : Olympus µ 1000,Serial no: K28225447.




Fig.6 Stop-clocks used to measure the time of reaction

Photos source :Pictures taken in the Lab G12 on 16 nov 2010 with Olympus camera

 

Fig.7. Bunsen Burner used to boil the lipase in the test tube no. 3

 

Hazards:

Broken glass may cause injuries to skin and eyes.

1% Bile salts (sodium tauroglycocholate) may cause irritation to: eyes, skin.
0.1M Sodium carbonate may cause irritation to: eyes, skin.
3% Lipase is irritant.
Universal indicator solution is an irritant and the solution stains .
Milk may be allergic for some people
Lab coat must be worn.    
Safety goggles must be used.
Procedure:

1. All apparatus needed were brought to the lab bench, small quantities of solutions of Sodium Carbonate, Lipase, milk and Bile Salts were transferred from the big containers( bottles) into individual beakers and brought to the lab bench .
2. During the course of reaction all students were sharing use of : Ice bath 0⁰C, fridge 4⁰C,water bath at 45⁰C and water bath at 35⁰C,thermometers and buncen burner.
3. Using the pipette filler 15 ml of whole milk was measured into a clean pipette of 25 ml capacity then transferred into a 75 ml capacity beaker.
4. Using the pipette filler 1 ml of universal indicator was measured into a clean pipette of 5 ml capacity and then was transferred into the beaker with 15 ml milk.
5. Using the glass rod the solution was homogenised by stirring, then using a plastic pipette the solution of 0.1 M Sodium Carbonate was added into the beaker with milk , drop by drop, and still stirring with the glass rod until the solution colour was dark green.
6. Comparing the colour of the solution in the beaker with the pH colour spectrum from the universal indicator bottle the pH of the solution was noted as 7.8 ,as shown in fig.8
   
    
   Fig.8 Colour comparison between solution of milk and the pH colour spectrum.
   Photos source :Picture taken in the Lab G12 on 16 nov 2010 with Olympus camera
   
7. The test tubes were labelled from 1 to 7 and then put into the test tube rack(rack A labelled).
8. The test tubes were filled as follows:
9. Using the pipette filler,7 ml of Bile Salts were transferred from the beaker into a clean pipette. Each of 7 test tubes received 1 ml of Bile Salts.
10. Using the pipette filler, 3 ml of Lipase were transferred from the beaker into a clean pipette. The total of 3 ml of lipase from the pipette was distributed as follows: tube no.1 received none, all the rest of the tubes from number 2 to number 7 received each 0.5 ml of lipase.
11. Using the pipette filler, 4 ml of distilled water were transferred from the beaker into a clean pipette. The total of 4 ml were distributed to the test tubes as follows: tube no. 1 received 1 ml and the rest of the tubes from no. 2 to tube no. 7, received 0.5 ml each of distilled water.
12. Tube number 3 containing: 0.5 ml of water,0.5 ml of lipase and 1 ml of bile salts was taken to the bunsen burner and by activating the machine the hot flame was boiling the content of the tube in about 15 seconds.
13. By using the 2^nd (rack B) and the 3^rd (rack C) test tube racks the test tubes were distributed as follows:
14. tubes number 1, 2 and 3 were in the rack A;
    
15. tube number 4 was in the rack B;
    
16. tubes number 5, 6 and 7 were in rack C.
    
17. Each stop-clock was labelled as follows: clock A for rack A, clock B for rack B and clock C for rack C.
    
18. Rack A containing tube no.1 ,tube no. 2 and tube no.3 went into the water bath with the temperature of 37⁰ C. Using a plastic pipette of 1ml capacity, each tube received 2 ml solution of milk, indicator and sodium carbonate from the beaker. Clock A was set on and the counting started.
    
19. Rack B containing tube no. 4 went into the water bath with the temperature of 45⁰C. Using a plastic pipette of 1ml capacity, the tube received 2 ml solution of milk, indicator and sodium carbonate from the beaker. Clock B was set on and the counting started.
    
20. Rack C containing 1 thermometer, tube no. 5, tube no.6 and tube no. 7 was placed on the lab bench. Using a plastic pipette of 1ml capacity, each tubes received 2 ml solution of milk, indicator and sodium carbonate from the beaker. Clock C was set on and the counting started.
    
21. Tube 6 remained in the rack at the room temperature. Temperature in the room was taken by using the thermometer in the rack C,the value was recorded as 20⁰C.
    
22. Tube 5 was immediately placed into the fridge. Temperature in the fridge was recorded as 7⁰C from the thermometer situated in the fridge.
    
23. Tube 7 was immediately placed into the ice bath. Temperature was recorded as 0⁰C from the thermometer situated in the ice bath.
    
24. After organising the places and conditions of reaction the student started to monitories the time for reaction.
    
25. During the following ten minutes from the start of reactions the student was monitoring the changes in the colour of the content in the test tubes in all 7 tubes.
    
26. In tube no.2 was observed the change of the colour from dark green to yellow after 12 minutes from the start of reaction. The tube was situated in the bath water with the temperature of 37⁰C.Time for reaction was noted on the clock( A ) as 12 minutes and recorded in the lab book into the results table. Tube no. 2 was brought to the lab bench for recording of the pH value of the solution. The colour of the solution was compared with the pH colour spectrum and the match of colours was at the pH 6.5 domain.
    
27. In tube no.4 was observed the change of the colour from dark green to yellow after 18 minutes from the start of reaction. The tube was situated in the bath water with the temperature of 45⁰C.Time for reaction was noted on the stop clock( B ) as 18 minutes and recorded in the lab book into the results table. The clock (B) was stopped as the student finished the experiment taken place in that water bath and rack B was brought to the lab bench for reading the pH value. Colour comparison was matching at 6.5 pH value domain.
    
28. After 25 minutes from the start of the reaction tube no.1 and tube no.3 situated in the water bath of 37⁰C had no change in the colour, clock A was stoped and time was recorded in the lab book as 25 minutes time for reaction. Rack A was brought to the lab bench and the colour of the solutions from the test tubes was compared with the colour of initial solution, no change of colour was recorded during the reaction.
    
29. After 25 minutes from the start of the reaction the colour in the tube no.6 changed from dark green to yellow.Colour comparison between the content of the tube and the pH spectrum was matching at 6.5 pH value domain.
    
30. After 28 minutes from the start of the reaction the colour in the tube no.5 changed from dark green to yellow.Colour comparison between the content of the tube and the pH spectrum was matching at 6.5 pH value domain.
    
31. After 28 minutes from the start of the reaction the time was stoped on the clock for the reaction in tube no.7 as the entire time allocated to the hole experiment was coming to an end. The colour in the tube no.7 changed from dark green to light green.Colour comparison between the content of the tube and the pH spectrum was matching at 7 pH value domain.
    
32. Tubes were tested for smell, the following was observed: tube number 2,no. 4, no.5 and no.6 presented rancidity smell, tube number 1,no.3 and no.7 had no smell.
    
33. All the results were recorded in the lab book.
    
34. The apparatus was cleared and cleaned.
    
    
    
    Results
    
    
    
    
    
    
    During the experiment the results were recorded as shown in table below:

		Content of tube
Temperature oC	Tube no.	Water	Lipase	Bile salts	Milk , indicator , sodium carbonate	Initial Colour of solution	Colour after reaction	Reaction time minutes
37 (Water bath)	1	1 ml	none	1ml	2 ml	Dark green	Dark green	25
37 (Water bath)	2	0.5ml	0.5ml	1ml	2 ml	Dark green	yellow	12
37 (Water bath)	3	0.5ml	0.5ml Boiled	1ml	2 ml	Dark green	Dark green	25
45 (Water bath)	4	0.5ml	0.5ml	1ml	2 ml	Dark green	yellow	18
Fridge 70 C	5	0.5ml	0.5ml	1ml	2 ml	Dark green	yellow	28
Room temp 200C	6	0.5ml	0.5ml	1ml	2 ml	Dark green	yellow	25
00 C (Ice bath)	7	0.5ml	0.5ml	1ml	2 ml	Dark green	Light green	28

 

 

 

 

 Comparison of colours was recorder as follows:

Fig.9 Solution of milk, universal indicator and sodium carbonate in a 75 ml capacity beaker	Fig.10 Colour comparison between initial solution of milk and test tube no.1 Comparison was made after 25 minutes from addition of the solution of milk into the content of tube 1.Experiment was conducted in an environment of 370C (water bath).

Fig. 11 Colour comparison between initial solution of milk and test tube no.2 Comparison was made after 12 minutes from addition of the solution of milk into the content of tube 2. Experiment was conducted in an environment of 370C (water bath).	Fig.12 Colour comparison between initial solution of milk and test tube no.3 Comparison was made after 25 minutes from addition of the solution of milk into the content of tube 3. Experiment was conducted in an environment of 370C (water bath).

Fig.13 Colour comparison between initial solution of milk and test tube no.4 Comparison was made after 18 minutes from addition of the solution of milk into the content of tube 4. Experiment was conducted in an environment of 450C (water bath).	Fig.14 Colour comparison between initial solution of milk and test tube no.5 Comparison was made after 28 minutes from addition of the solution of milk into the content of tube 5. Experiment was conducted in an environment of 70C (fridge).

Fig.15 Colour comparison between initial solution of milk and test tube no.6 Comparison was made after 25 minutes from addition of the solution of milk into the content of tube 6. Experiment was conducted in an environment of 200C (room temperature).	Fig.16 Colour comparison between initial solution of milk and test tube no. 7 Comparison was made after 28 minutes from addition of the solution of milk into the content of tube 7. Experiment was conducted in an environment of 00C (ice bath).

Observations

• Reaction occurring or the speed of reaction depends on content of the tube and temperature of medium of reaction .
• Shown in table below is the change of colour, pH and the change of smell.

           

Discussion


The aim of this experiment was to examine the hydrolysis of milk lipids by lipases at different temperatures.
The activity of lipase was observed at different temperatures of the medium of reaction. Test tube no1. had no lipase ,therefore no reaction was expected and as observed in fig .10 no change in the colour of the solution ocured.
During the experiment, test tube no.2 was placed into a 37⁰C water bath and the reaction was observed as expected very quick. Lipase has an optimum temperature of 37⁰C for optimum activity. The reaction was a success and fatty acids were released by lipase in 12 minutes from the start of reaction.
Test tube no .3 had no reaction because the lipase was denatured by exposure of 65⁰C
Test tubes no 5, 6 and 7 had low temperatures in the medium of reaction therefore the reaction was very slow.
Test tube no.4 had high temperature of 45⁰C for the medium of reaction and this was above the optimum temperature for lipase therefore the time of reaction was longer (18 minutes) in comparison with tube 2 (12 min, optimum 37⁰C)
Activity lipase was observed to increase with increase of temperature, until an optimum was reached at 37⁰C. Above this point was observed the the activity of the enzyme decreased (47⁰C) until stoped as result of protein denaturation (boiled lipase) in test tube 3.
Comparison of colour was recorded in fig .17 and 18
The experiment was a success as the activity of lipase was observed increasing and decreasing with temperature fluctuations.




The table below represent the predictions of the student over the results of experiment .The prediction was taking place before the experiment started as an hypothesis for experiment.

Test tube no.	Predicted outcome		Recorded Results
	Reaction to occur between milk and lipase	Time to react	Reaction occurred between milk and lipase	Time to react
1 (no lipase) 370C	No Lipase is missing	-	no	After 25 min. No changes in colour
2 370C	yes	Faster than any other tubes 370C is the body temperature of human body	yes	12 minutes for reaction
3 370C (boiled lipase)	No Lipase is denaturated	-	no	After 25 min. No changes in colour
4 450C	yes	Slow reaction 450C Isn`t an optimum temperature for lipase	yes	18 minutes for reaction
5 70C	yes	Slow reaction 70C Isn`t an optimum temperature for lipase	yes	28 minutes for reaction
6 Room temperature 200C	yes	Slow reaction 200C Isn`t an optimum temperature for lipase	yes	25 minutes for reaction
7 00C Ice Bath	No Of maybe but very slow activity of enzyme	-   Extreme temperature	Yes	Reaction not complete colour was light green after 28 minutes of reaction.

Fig 17 Comparison in colour between milk without lipase and the test tubes after reaction.


















Fig 18. Comparison in colour between test tubes after reaction.






















Conclusion 


The aim of this experiment was to examine the hydrolysis of milk lipids by lipases at different temperatures. The results of the experiment showed that the performance of the enzyme lipase was affected by temperature. The activity of lipase was observed to increase with increase of temperature, until an optimum was reached at 37⁰C. Above this point was observed the the activity of the enzyme decreased (47⁰C) until stoped as result of protein denaturation (boiled lipase) in test tube 3.

Reference:

• Campbell et al, Biology, 2005, 7^th edition, Pearson Education, Inc., Benjamin Cummings, San Francisco. ISBN: 0-321-26984-5

• Click4Biology: Option H, Digestion
  http://click4biology.info/c4b/H/H2.htm (visited on 18.11.2010)
• Jones et al, Practical Skills in Biology, 2007, 4^th Edition, Pearson Education Ltd, Essex. ISBN: 978-0-13-175509-3
• Mathews et all, Biochemistry, 2000, 3^rd ed. Menlo Park, CA: Benjamin/Cummings
• University of Colorado, Introduction to Physiology
  
  http://www.colorado.edu/intphys/Class/IPHY3430-200/image/02-8.jpg
  (visited on 19.11.2010)

	Colour of content in the experiment tube		Smell of content in the experiment tube		pH of content in the experiment tube
	Before reaction of lipase with milk	After reaction of lipase with milk	Before reaction of lipase with milk	After reaction of lipase with milk	Before reaction of lipase with milk	After reaction of lipase with milk
Tube 1 No lipase = no reaction	Dark green	Dark green	No smell	No smell	7.8	7.8
Tube 2	Dark green	yellow	No smell	rancidity smell	7.8	6.5
Tube 3 Lipase boiled(denaturated) = no reaction	Dark green	Dark green	No smell	No smell	7.8	7.8
Tube 4	Dark green	yellow	No smell	rancidity smell	7.8	6.5
Tube 5	Dark green	yellow	No smell	rancidity smell	7.8	6.5
Tube 6	Dark green	yellow	No smell	rancidity smell	7.8	6.5
Tube 7	Dark green	Light green	No smell	No smell	7.8	7