Design Practice: Enzyme Action

Design practice: Enzyme action

Title: Difference in the action of the enzyme catalase from raw cow’s liver and liver boiled to two different times and temperatures

Introduction:

Enzymes are globular proteins that are found in very living cell. They work as a catalyst by speeding up chemical reactions without being altered themselves. Catalase is an enzyme found in the tissue cells of nearly all-living organisms. The function of this enzyme is to catalyze the decomposition of hydrogen peroxide to water and oxygen. The chemical reaction is as follows:

22222 + 2

As this is an exothermic reaction that releases 1960 kJ of heat, the amount of molecules that are being decomposed by the enzyme will be directly proportional to the amount of energy released as heat. Denaturation of enzyme varies for catalase enzymes from different organisms, and depends on several factors, such as pH, but in this practice we will look closer to temperature as a factor. The denaturation temperature depends on the composition of the protein (it's amino acid sequence). For most land mammals (cow, pig, human etc) the optimum temperature for any enzyme is likely to be between 36 Celsius and 39 Celsius (most often 37 Celsius). Mammalian catalase has an optimum temperature of about 37 Celsius.

The objective of this practice is to observe the difference in temperature in the reaction (to obtain the energy released) of catalase present in cow’s liver boiled at two different temperatures in decomposing hydrogen peroxide and comparing these reactions with the ideal reaction with raw liver.

Research question:

What is the difference in the action of the enzyme catalase from raw cow’s liver and boiled liver to different times and temperatures? What is the difference in the action of enzyme catalase from raw liver and liver boiled for 20 minutes that reached a temperature of 90 (±0.5 Celsius)? What is the difference if the liver is boiled for 10 minutes reaching a temperature of 75 (±0.5 Celsius)?

Hypothesis:

I suppose that catalase enzyme present in cow’s liver will suffer denaturalization reaching the temperature of 90 (±0.5 Celsius) being boiled for 20 minutes. The exposure of the liver to such temperature would probably change the shape of the active site of the catalase contained. So probably in this reaction there will no be a significative change of temperature. As the liver boiled at a temperature of 75 degrees (±0.5 Celsius) for 10 minutes is not plenty baked it might still be active sites that were not deformed by the action of the temperature. This reaction would still release some heat but there probably be a large difference from the raw liver.

Variables / Units/control method
Independent / Different points of baking cow’s liver / -Raw liver
-Baked in water 20 minutes at 90 (±0.5 Celsius)
-Baked in water 10 minutes at 75 (±0.5 Celsius)
Dependent / Temperature release as heat by the exothermic reaction / Measured using the temperature sensor of the Logger Pro 3
Controlled / External temperature / Covering the flask with aluminum paper, building a calorimeter with a foam glass and filling the inside with isolating materials.
Concentration of substrate / 50 ml of hydrogen peroxide at 1%
Concentration of the enzyme / 3 grams of cow’s liver

Materials:

2

·  Raw cow liver

·  Universal support

·  Aluminum paper

·  Double-perforated stopper

·  Data Logger software (Logger Pro 3)

·  Logger Pro 3 temperature sensor

·  Logger Pro 3 interphase

·  Computer

·  Triple beam balance (±0.50)

·  500 ml of Hydrogen peroxide (H2O2) at 1%

·  2 petri dishes

·  Dissection scissors

·  Knife

·  50 ml Burette (±10 sec)

·  Thermometer (±0.5 Celsius)

·  Big Beaker

·  Hot plate

·  Big foam glass

·  125 ml earlenmayer flask (5)

·  Latex gloves

6

Methodology:

Several trials were carried out in order to select the adequate quantities of substrate and the adequate quantity and concentration of solute. Finally the quantities were settled. And the experimentation was divided in three stages. The first stage was the trials testing raw liver; the second stage was developed with boiled liver for 20 minutes that reached a temperature if 90 (±0.5 Celsius). And the third stage was developed with the liver boiled for 10 minutes reaching a temperature of 75 (±0.5 Celsius).

1.  The universal support was set and, the Logger Pro 3 interphase was connected to the computer. The Logger Pro 3 was programmed for to record the temperature inside the calorimeter every second for a time of 300 seconds

2.  Using the triple beam balance (±0.50 mg) 3 grams of raw cow’s liver was weighted five times (for to have five repetitions). Weighted pieces of liver where placed in a petri dish.

3.  In the first trial a weighted piece of liver was placed inside a 125 ml Erlenmeyer flask () and it was covered with aluminum paper. Then the flask was placed inside a foam glass for to isolate the system from the surroundings.

4.  The double punched cork was placed at the top of the flask. In one of its holes was place the temperature sensor of the Logger Pro 3.

5.  In the universal support a 50 ml burette (±10 sec) was arranged and under the burette was place the foam glass with the flask inside of it. The burette was arranged for to fit into the left hole of the double punched cork. And the inside of the foam glass was full filled with aluminum paper and foam pieces also for to maintain the system isolated.

6.  Using latex gloves for precaution 50 ml of hydrogen peroxide (H2O2) at 1% were added to the burette (±10 sec).

7.  Then I proceed to open the key of the burette and left a tiny escape for air produced in the instant reaction just until all the hydrogen peroxide is out the burette. Immediately, start the software for to record the data.

8.  The same procedure was followed five times with the same amounts and quantities of the raw cow’s liver.

9.  Using a hot plate and a thermometer some liver was boiled with 400 ml of water in a beaker. First I boiled the liver that was going to be baked for 20 minutes at a temperature of 90 degrees (±0.5 Celsius).

10.  The same procedure as with raw liver was followed five times and all the data was recorded.

11.  Then the other quantity of liver was boiled for 10 minutes reaching a temperature of 75 degrees (±0.5 Celsius).

12.  The same procedure as with raw liver and liver boiled at 90 degrees (±0.5 Celsius) for 20 minutes was followed five times and all the data was recorded.

Picture 1. Set up Logger Pro 3 / Picture 2. Calorimeter

Data and observations:

In the following tables the temperature inside the calorimeter every 10 seconds is shown of every trial. Also a mean of the five trials was obtained. In the first table is recorded the temperature of inside the calorimeter when 3 grams of raw liver react with 50 ml of hydrogen peroxide at 1%. The second table shows the temperature of inside the calorimeter when 3 grams of liver baked in water at 75 (±0.5 Celsius) for 10 minutes react with 50 ml of hydrogen peroxide at 1%. And the third table shows the temperature of inside the calorimeter when 3 grams of liver baked at 90 (±0.5 Celsius) for 20 minutes react with 50 ml of hydrogen peroxide at 1%.

Table 1.

Shows data recorded for the temperature of the reaction of raw cow’s liver and hydrogen peroxide at 1% in the five trials

Time
In seconds / trial 1
temperature in Cº / trial2
temperature in Cº / trial 3
temperature in Cº / trial 4
temperature in Cº / trial 5
temperature in Cº / Mean
temperature in Cº
0 / 16.585 / 17.138 / 17.210 / 15.955 / 19.260 / 17.391
10 / 16.198 / 19.094 / 20.462 / 20.627 / 21.002 / 20.296
20 / 16.004 / 20.462 / 20.932 / 21.471 / 21.307 / 21.043
30 / 16.488 / 21.307 / 21.190 / 21.494 / 21.681 / 21.418
40 / 17.594 / 21.704 / 21.213 / 21.400 / 21.681 / 21.500
50 / 18.620 / 21.868 / 21.354 / 21.471 / 21.658 / 21.587
60 / 19.756 / 21.938 / 21.307 / 21.400 / 21.588 / 21.558
70 / 21.026 / 21.915 / 21.283 / 21.424 / 21.564 / 21.547
80 / 21.307 / 21.915 / 21.354 / 21.377 / 21.471 / 21.529
90 / 21.377 / 22.008 / 21.330 / 21.400 / 21.494 / 21.558
100 / 21.307 / 21.938 / 21.283 / 21.377 / 21.400 / 21.500
110 / 21.307 / 21.868 / 21.307 / 21.307 / 21.424 / 21.476
120 / 21.283 / 21.891 / 21.190 / 21.236 / 21.377 / 21.424
130 / 21.190 / 21.798 / 21.236 / 21.190 / 21.307 / 21.383
140 / 21.260 / 21.821 / 21.190 / 21.190 / 21.354 / 21.389
150 / 21.260 / 21.798 / 21.119 / 21.190 / 21.236 / 21.336
160 / 21.307 / 21.798 / 21.143 / 21.190 / 21.213 / 21.336
170 / 21.260 / 21.634 / 21.166 / 21.143 / 21.190 / 21.283
180 / 21.307 / 21.634 / 21.166 / 21.096 / 21.260 / 21.289
190 / 21.166 / 21.611 / 21.143 / 21.166 / 21.166 / 21.272
200 / 21.143 / 21.588 / 21.073 / 21.049 / 21.236 / 21.236
210 / 21.260 / 21.634 / 21.096 / 21.073 / 21.190 / 21.248
220 / 21.236 / 21.517 / 21.026 / 21.026 / 21.096 / 21.166
230 / 21.236 / 21.494 / 21.049 / 21.049 / 21.096 / 21.172
240 / 21.143 / 21.541 / 21.049 / 21.026 / 21.096 / 21.178
250 / 21.190 / 21.588 / 21.002 / 21.073 / 21.096 / 21.190
260 / 21.213 / 21.564 / 20.955 / 20.932 / 21.073 / 21.131
270 / 21.166 / 21.424 / 21.002 / 20.908 / 21.119 / 21.113
280 / 21.190 / 21.447 / 21.026 / 21.002 / 21.119 / 21.149
290 / 21.096 / 21.400 / 20.955 / 20.862 / 20.979 / 21.049
300 / 21.073 / 21.424 / 20.885 / 20.979 / 20.955 / 21.061

Table 2.

Shows data recorded for the temperature of the reaction of cow’s liver boiled for 10 minutes that reached a temperature of 75 Celsius and hydrogen peroxide at 1% in the five trials

Time
In seconds / trial 1
temperature
in Cº / trial 2
temperature
in Cº / trial 3
temperature
in Cº / Trial 4
temperature
in Cº / trial 5
temperature
in Cº / Mean
temperature
in Cº
0 / 14.364 / 13.845 / 14.315 / 15.249 / 15.517 / 14.658
10 / 14.315 / 14.018 / 14.414 / 15.444 / 15.566 / 14.751
20 / 14.266 / 14.117 / 14.438 / 15.468 / 15.566 / 14.771
30 / 14.340 / 14.068 / 14.414 / 15.493 / 15.590 / 14.781
40 / 14.364 / 14.068 / 14.438 / 15.371 / 15.566 / 14.761
50 / 14.191 / 14.142 / 14.438 / 15.468 / 15.566 / 14.761
60 / 14.340 / 14.093 / 14.315 / 15.493 / 15.566 / 14.761
70 / 14.216 / 14.167 / 14.414 / 15.468 / 15.566 / 14.766
80 / 14.315 / 14.142 / 14.463 / 15.517 / 15.542 / 14.796
90 / 14.241 / 14.093 / 14.463 / 15.517 / 15.663 / 14.795
100 / 14.191 / 14.142 / 14.389 / 15.420 / 15.663 / 14.761
110 / 14.266 / 14.142 / 14.463 / 15.298 / 15.639 / 14.761
120 / 14.241 / 14.216 / 14.537 / 15.468 / 15.542 / 14.801
130 / 14.266 / 14.191 / 14.438 / 15.420 / 15.615 / 14.786
140 / 14.340 / 14.117 / 14.463 / 15.395 / 15.663 / 14.796
150 / 14.266 / 14.216 / 14.438 / 15.444 / 15.566 / 14.786
160 / 14.216 / 14.216 / 14.512 / 15.493 / 15.566 / 14.801
170 / 14.191 / 14.191 / 14.414 / 15.395 / 15.663 / 14.771
180 / 14.290 / 14.167 / 14.488 / 15.517 / 15.639 / 14.820
190 / 14.315 / 14.241 / 14.512 / 15.420 / 15.639 / 14.825
200 / 14.241 / 14.216 / 14.488 / 15.493 / 15.615 / 14.810
210 / 14.241 / 14.241 / 14.562 / 15.517 / 15.615 / 14.835
220 / 14.340 / 14.290 / 14.488 / 15.493 / 15.639 / 14.850
230 / 14.315 / 14.241 / 14.438 / 15.444 / 15.615 / 14.811
240 / 14.266 / 14.167 / 14.512 / 15.468 / 15.590 / 14.801
250 / 14.315 / 14.241 / 14.512 / 15.468 / 15.688 / 14.845
260 / 14.389 / 14.266 / 14.512 / 15.420 / 15.663 / 14.850
270 / 14.117 / 14.191 / 14.463 / 15.493 / 15.615 / 14.776
280 / 14.266 / 14.266 / 14.537 / 15.420 / 15.688 / 14.835
290 / 14.290 / 14.216 / 14.438 / 15.444 / 15.712 / 14.820
300 / 14.241 / 14.241 / 14.537 / 15.444 / 15.639 / 14.820

Table 3.

Shows data recorded for the temperature of the reaction of cow’s liver boiled for 20 minutes that reached a temperature of 90 Celsius and hydrogen peroxide at 1% in the five trials

Time
In
seconds / Trial 1
temperature
in Cº / trial 2
temperature
in Cº / trial 3
temperature
in Cº / trial 4
temperature
in Cº / trial 5
temperature
in Cº / Mean
temperature
in Cº
0 / 14.881 / 13.795 / 14.216 / 14.266 / 13.770 / 14.186
10 / 14.463 / 13.969 / 14.167 / 14.315 / 13.820 / 14.147
20 / 14.438 / 13.894 / 14.241 / 14.315 / 13.870 / 14.152
30 / 14.463 / 13.993 / 14.241 / 14.290 / 13.845 / 14.166
40 / 14.414 / 13.944 / 14.241 / 14.340 / 13.919 / 14.172
50 / 14.389 / 14.018 / 14.241 / 14.290 / 13.870 / 14.162
60 / 14.389 / 13.944 / 14.290 / 14.290 / 13.870 / 14.157
70 / 14.512 / 14.018 / 14.216 / 14.266 / 13.944 / 14.191
80 / 14.438 / 14.018 / 14.315 / 14.266 / 13.944 / 14.196
90 / 14.438 / 13.993 / 14.290 / 14.315 / 13.870 / 14.181
100 / 14.463 / 13.993 / 14.315 / 14.315 / 13.969 / 14.211
110 / 14.463 / 14.018 / 14.266 / 14.315 / 13.919 / 14.196
120 / 14.463 / 13.993 / 14.241 / 14.290 / 13.894 / 14.176
130 / 14.414 / 13.969 / 14.340 / 14.315 / 13.944 / 14.196
140 / 14.389 / 13.969 / 14.290 / 14.364 / 13.894 / 14.181
150 / 14.414 / 13.993 / 14.266 / 14.315 / 13.919 / 14.181
160 / 14.438 / 13.969 / 14.290 / 14.389 / 13.969 / 14.211
170 / 14.414 / 13.993 / 14.340 / 14.340 / 14.0187 / 14.221
180 / 14.389 / 13.993 / 14.315 / 14.241 / 13.969 / 14.181
190 / 14.364 / 13.993 / 14.315 / 14.315 / 13.969 / 14.191
200 / 14.512 / 14.043 / 14.340 / 14.340 / 14.018 / 14.251
210 / 14.463 / 13.944 / 14.315 / 14.364 / 13.993 / 14.216
220 / 14.463 / 14.043 / 14.389 / 14.266 / 14.018 / 14.236
230 / 14.488 / 13.969 / 14.340 / 14.290 / 13.944 / 14.206
240 / 14.463 / 13.993 / 14.364 / 14.315 / 14.043 / 14.236
250 / 14.438 / 14.068 / 14.364 / 14.216 / 14.093 / 14.236
260 / 14.488 / 13.993 / 14.315 / 14.290 / 13.993 / 14.216
270 / 14.537 / 13.993 / 14.340 / 14.364 / 14.043 / 14.256
280 / 14.463 / 13.969 / 14.340 / 14.315 / 14.068 / 14.231
290 / 14.414 / 14.068 / 14.340 / 14.389 / 14.043 / 14.251
300 / 14.414 / 14.018 / 14.389 / 14.340 / 14.068 / 14.246

Data Processing: