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Temperature'S Effect Against The Irreversible Denaturation Of Peroxidase

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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.


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, 22oC. 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 50oC, 6 & 7 at 40oC, 10 & 11 in 60oC, 12 & 13 in 70oC, and 14 & 15 in 80oC. 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.


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 22oC to 80oC, with a 10oC interval.


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 22oC and working at 10oC intervals from 30oC and optimal temperature at which peroxidase produced O2 was observed at 30oC (Table 2). As the temperature was increased the production of O2 from peroxidase and H202 was decreased until an infinitesimal amount was detected at 70oC - 80oC. When investing which temperature would demonstrate an irreversible denaturation, the absorbance readings indicated that at 60oC 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)


(Tube 2 & 3) 0.101 0.265 0.405 0.540 0.660 0.780 1.140 1.310


(Tube 4 & 5) 0.012 0.290 0.580 0.801 1.030 1.200 1.340 1.480



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