Temperature's Effect Against the Irreversible Denaturation of Peroxidase

Abstract:

Enzyme-catalyzed hydrolysis reaction occurs when an enzyme cleaves glycosidic linkage where a substrate binds to active site forming an enzyme-substrate complex. By adding water to the enzyme-substrate complex, products are release. One of the main factor that effect enzyme-catalyzed reactions is temperature. After an enzyme reaches an optimal temperature, the enzyme will result in irreversible denaturation. The irreversible denaturation causes the protein to loose its function making it inactive to operate properly.

Methods:

In order to investigate the effects of temperature on the activity of enzyme-catalyzed reactions, we made fifteen tubes that contained reaction buffer, hydrogen peroxide, turnip extract, and the dye. These reagents were placed in large bottles and were labeled with a sharpie. We gathered fifteen small test tubes for testing and three large test tubes to fill it with stock solutions needed to carry out the experiment. The large test tubes were filled with buffer, dye, and hydrogen peroxide. Each test tube made contrasted in the amounts of solutions used. The odd numbered tubes contained 1.0 ml of turnip extract and 4.0 ml of reaction buffer. The even numbered tubes contained 1.0 ml dye and 2.0 ml of hydrogen peroxide.

Table1. Volumes of Reagents Used (Dini 2004):

Reaction Buffer Dye Hydrogen Peroxide Turnip Extract
Even numbered tubes: 0.0 1.0 ml 2.0 ml 0.0
Odd numbered tubes: 4.0 ml 0.0 ml 0.0 ml 1.0 ml

We dispensed each fluid into the small test tubes by using the correct amounts on the given chart. The test tubes were tested for temperature by placing it in hot water baths at various temperatures. Test tubes 2 & 3 were placed in a beaker at room temperature, 22°C. The rest of the test tubes were placed in hot bathes with a waiting period of ten minute interval. We placed test tubes 4 & 5 in 50°C, 6 & 7 at 40°C, 10 & 11 in 60°C, 12 & 13 in 70°C, and 14 & 15 in 80°C. We placed a thermometer on the test tubes to verify the mixtures reached the expected temperatures.

While the test tubes were equilibrating in the hot water baths, we tested tube number 1, the control one for the absorbance reading. This was done by using the new version of spectrophotometer. This device was used by turning the dial to 3 to attain the wavelength at 500 nanometers. After the initial reading of test tube one was found, we tested the other set of test tubes in a similar procedure. We poured the contents of tube 2 into 3, wiped the test tube 3 with a kim-wipe, and inverted it several times to mix the contents. After preparing the test tube 3 in this manner, we took the absorbance reading. The absorbance reading was done by using the same procedure that was used for testing the absorbance reading of test tube 1. The other set of test tubes were tested and analyzed in the same procedure.
Introduction:
Peroxidase, like any enzyme, performs best at its optimal temperature. The function of peroxidase is to catalyze the conversion of toxic metabolic wastes called peroxides into the harmless products water and oxygen. If the enzyme is heated at too high of a temperature, then the enzyme will denature. Sometimes when an enzyme denatures, and is put back at room temperature, the enzyme will regain its function and break down the peroxides. To determine if an enzyme is actively breaking down the peroxides, the sample containing the peroxidase is ran through a spectrophotometer. The sample will either show enzyme activity or no enzyme activity. The more activity shown, the higher the concentration of the enzyme present, and the higher the rate at which the product molecules will appear. If there is no activity, then the enzyme was irreversibly denatured and has totally lost its function.
Our experiment was to determine the temperature at which the enzyme, peroxidase, was reversibly denatured. Each trial was done within seconds after removing the mixture from the heat considering the temperature would change relatively fast if left out too long, thus altering our data. If the environment of the enzyme is too acidic or too basic, the enzyme will be irreversibly denatured, making the shape of the enzyme change and its function lost. The temperatures used to determine enzyme activity varied from 22°C to 80°C, with a 10°C interval.
Results:

The peroxidase enzyme was observed over a wide range of temperature by measuring the absorbance readings of each reaction test tubes. There seemed to be an apparent trend in the data to help indicate optimal enzymatic temperature as well as the temperature at which the irreversible denaturing of peroxidase occurs.

Starting at a temperature of 22°C and working at 10°C intervals from 30°C and optimal temperature at which peroxidase produced O2 was observed at 30°C (Table 2). As the temperature was increased the production of O2 from peroxidase and H202 was decreased until an infinitesimal amount was detected at 70°C - 80°C. When investing which temperature would demonstrate an irreversible denaturation, the absorbance readings indicated that at 60°C the enzyme could not renature to its effective conformation. As the temperature increased there was no absorbance reading (Table 3).

Table 2.Temperature Effects on Absorbance over Time of Peroxidase Time 0
(20 Sec) Time 1
(40 sec) Time 2
(60 sec) Time 3
(80 sec) Time 4
(100 sec) Time 5
(120 sec) Time 6
(140 sec) Time 7
(160 sec)
22°C
(Tube 2 & 3) 0.101 0.265 0.405 0.540 0.660 0.780 1.140 1.310
30°C
(Tube 4 & 5) 0.012 0.290 0.580 0.801 1.030 1.200 1.340 1.480
40°C
(Tube 6 & 7) 0.090 0.129 0.187 0.210 0.289 0.305 0.329 0.338
50°C
(Tube 8 & 9) 0.045 0.064 0.083 0.100 0.114 0.129 0.142 0.155
60°C
(Tube 10 & 11) 0.008 0.009 0.009 0.009 0.009 0.009 0.009 0.009
70°C
(Tube 12 & 13) 0.001 0.001 0.001 0.002 0.002 0.002 0.002 0.002
80°C
(Tube 14 & 15) 0.001 0.002 0.001 0.001 0.002 0.001 0.001 0.001

Table 3.Effect of Temperature on Absorbance over Time of Peroxidase when Heated and Cooled to Room Temperature Time 0
(20 Sec) Time 1
(40 sec) Time 2
(60 sec) Time 3
(80 sec) Time 4
(100 sec) Time 5
(120 sec) Time 6
(140 sec) Time 7
(160 sec)
22°C
(Tube 2 & 3) 0.027 0.057 0.068 0.089 0.097 0.114 0.145 0.150
30°C
(Tube 4 & 5) 0.029 0.062 0.078 0.093 0.131 0.149 0.155 0.164
40°C
(Tube 6 & 7) 0.028 0.058 0.089 0.095 0.124 0.134 0.148 0.160
50°C
(Tube 8 & 9) 0.022 0.048 0.070 0.092 0.111 0.128 0.146 0.162
60°C
(Tube 10 & 11) 0.002 0.003 0.005 0.006 0.006 0.006 0.006 0.006
70°C
(Tube 12 & 13) 0.001 0.001 0.001 0.001 0.001 0.001 0.001 0.001
80°C
(Tube 14 & 15) 0.001 0.001 0.001 0.001 0.001 0.001 0.001 0.001

Discussion:

The absorbance reading at 60°C helped to support our hypothesis that a high temperature would cause the irreversible denaturation of the enzyme peroxidase. As the heating of the enzyme occurred, the enzyme was unable to renature causing it to become ineffective. Assuming that the dye, guaiacol, oxidized at a constant rate, the brown coloration would be uniformed with the solution. An interesting discovery was seen in the decreased production of 02 between 50°C-60°C. A very small reading of absorbance was picked up by the spectrophotometer at 70°C & 80°C. This may have been the result of a select few enzymes that had adapted to function at a higher temperature then the enzymes that had been irreversibly denatured at 60°C.
To extend to further study of this enzyme a more precise measurement of temperature could be determined so a more optimal temperature for irreversible denaturation. We noticed error could be put towards the human error of heating up the test tubes. While heating some of the enzymes were not heated at an even pace so there was very little uniformity.

Acknowledgement:
We with like to greatly show our gratitude towards, David Rodriguez & Michelle, for providing the knowledge and materials for this independent investigation. Also we would like to thank Michael Dini for his superior knowledge in the field of Biology as well as the Biology Department.
Literature Cited:
Dini, M.L. 1996-2004. Laboratory Manual for Biology I (BIOL 1403). Stipes Publishing L.L.C., Champaign, IL.
Campbell, N. 1999. Biology, 6th Edition. Benjamin/Cummings, Redwood City, CA.

Cited: Dini, M.L. 1996-2004. Laboratory Manual for Biology I (BIOL 1403). Stipes Publishing L.L.C., Champaign, IL. Campbell, N. 1999. Biology, 6th Edition. Benjamin/Cummings, Redwood City, CA.