Results of Temperature Affecting Peroxidase Activity Rate
Dr. James Martiney
The purpose of this experiment was to gauge the effect that temperature had on enzyme activity and the substrate concentration. The rate of enzyme activity had a direct correlation to amount of concentration of the enzyme or substrate. If the concentration of substrate was halved, the enzyme activity rate was also halved. If the concentration of substrate was doubled, the enzyme activity rate was also doubled. The use of environments with a fixed temperature was used to aid enzyme activation rates. The group tested the absorbance levels of the enzymes for twenty seconds intervals out of a total two minutes. Using a spectrophotometer, results showed that enzyme activity rate had a correlation to increased temperature until the incubation chamber of temperature of 60 °C caused denaturation in the protein and showed a lower activation rate than the coldest temperature measured (5 °C). The independent variables in this experiment were temperature, concentration of substrate, and concentration of enzyme. The only dependent variable was the reaction rate.
Enzymes are macromolecules called proteins. Enzymes act as catalysts within living cells, thus speeding up biochemical reactions by lowering the activation energy required. Each individual enzyme has a structure that correlates to a specific function and an area of activation called the activation site. An activation site is a concave shape until a substrate combines with it. Within this specific experiment there were many enzymes within the horseradish, but the group can conclude that peroxidase was the only enzyme used because only peroxidase enzyme can have a specific reaction with the substrate in the reaction. This is because the definition of substrate is “a substance or layer that underlies something, or on which some process occurs, in particular” the key word being “particular”.
The perfect analogy to explain this process is called the Lock and Key. Imagine you get off from work and attempt put your car keys into your 2003 Cadillac CTS. As your try to insert your key you notice it stops halfway. Although your car is a 2003 Cadillac CTS the car you’re attempting to unlock is not yours. An everyday example is when rennin (enzyme) is added to liquid milk proteins (substrate) that produce coagulated milk solids known as curd (product). The main takeaway from these examples above is that only a compatible enzyme can work with a substrate. In this experiment the enzyme used was and extract of horseradish called peroxidase which is acquired by homogenization in buffer. These broken cells release many enzymes including peroxidase. To test for peroxidase the group had to mix the extract with the compound guaiacol. Guiacol is normally colorless but becomes brown after oxidation. Peroxidase Combined with 25 ml of 0.1M Citrate Phosphate buffer and the substrate hydrogen peroxide (H2O2) showed a chemical reaction.
There are many factors that can increase enzyme activation rate, but in this lab the primary focus was on temperature. Similarly to people, enzymes work optimally in a desirable temperature. Heat is used to increase the kinetic energy of the surrounding molecules which result in a greater number of collisions between molecules causing reactions to happen quicker. In colloquial terms a higher temperature can increase reaction rates while a lower temperature will decrease reaction rates. Because enzymes are proteins they are affected by denaturation which causes a protein’s structure to breakdown.
Temperature has a measurable effect on enzyme activity rates within the spectrophotometer. As temperature decreases so does activity rates. As temperature increases activity rates increases. This holds true until the high enough temperature creates an unsatisfactory for an enzyme and causes denaturation. Enzymes are similar to humans in this case. Human beings work optimal at 34° C (body temperature), but a lower or high body temperature can affect output negatively.
Description of experiment:
The production of this experiment began with the extraction of peroxidase from a horseradish, 25 mL 0.1M cirate-phosphate buffer, and a blender jar. The group then cut and weighed 1.0 gram of the horseradish and placed it in the blender for twenty seconds. After it was well blended, a double layer cheesecloth was used as a cover, then the contents (except for the chunks of horseradish were poured into a beaker (except for the chunks of horseradish). For the next steps the group labeled the enzyme concentration and a graduated cylinder with the word “buffer” that held citrate-phosphate ph 5 and then used two pump dispensers of hydrogen peroxide and guaiacol solutions into 9 labeled test tubes. Test tubes labeled #2 and #3 were placed in an incubation chamber of 5° C. Test tubes labeled #4 and #5 were placed in incubation chambers of 25°C. Test tubes labeled #6 and #7 were placed in incubation chambers of 34°C. The final test tubes labeled #8 and #9 were placed in an incubation chamber of 60°C. Once the test tubes reached an incubation time of ten minutes they were put through the spectrophotometer to calculate absorbance rate. Before calculating the absorbance rate the spectrophotometer was set to a wavelength of 500. Ian began to write down absorbance rate for twenty second intervals as soon as the enzyme and substrate were mixed for a total time of two minutes. After writing the absorbance rate for each twenty second interval.
Results: Table and Graph for Effect of enzyme concentration on the rate of reaction
Concentration 20 secs 40 sec 60 sec 80 sec 100 sec 120 sec
0.25 dil .291 .362 .445 .512 .588 .666
0.5 dil .483 .670 .849 1.015 1.220 1.380
1.0 dil .925 1.280 1.666 1.999 1.999 1.99
2.0 dil 1.999 1.999 1.999 1.999 1.999 1.999
Table and Graph of Effect of varying Temperature on rate of reaction:
Temperature 0 sec 20 sec 40 sec 60 sec 80 sec 100 sec 120 sec
5°C 0.075 0.17 0.27 0.388 0.522 0.688
25°C 0.288 0.47 0.688 0.91 1.115 1.4
34°C 0..558 0.811 1.618 1.86 1.999 1.999
60°C 0.058 0.123 0.225 0.356 0.518 0.74
Graph showing rate of change slope
These graphs show a direct correlation between temperature and the enzyme activity rate. The only outlier of this is when a protein is in an area where temperature is around 60 °C. High temperatures result in enzyme denaturation resulting in a protein losing its structure. The incubation chamber that showed the highest enzyme activity was at 34° C. Oddly enzyme activity at 5°C occurred at a faster overall rate than 60°C.
Temperature has a measurable effect on enzyme activity rates within the spectrophotometer. As temperature decreases so does activity rates.. Enzymes are similar to humans in this case. Human beings work optimal at 34° C (body temperature), but a lower or high body temperature can affect output negatively. Both tables from the spectrophotometer analysis shows that all temperatures and a steady increase in enzyme activity this can be seen that all slopes were going upwards. And this is concrete evidence that even though temperatures can speed up reaction rates, High temperatures can also adversely affect a protein by destroying its structure.