NAME Jonathon Slattery

Prac session Wed 9-11

TITLE Effects of temperature, Enzyme Concentration and Enzyme Inhibitor on Peroxidase (enzyme) converting Hydrogen Peroxide to Oxygen

INTRODUCTION (with HYPOTHESIS) Biochemical reactions are the basis of life on Earth. These reactions are either exergonic (release energy) or endergonic (store energy). Some examples of biochemical reactions are cellular respiration (food molecules been oxidised to release energy), photosynthesis and protein synthesis. Most chemical reactions require energy to get them to the transition stage. This is known as activation energy. This activation energy is the barrier which stops Chemical reactions from occurring spontaneously(Activation Energy). Organic catalysts also known as enzymes speed up the process of chemical reactions and lower the activation energy required for the transitional stage to take place. An enzyme is a 3 dimensional protein that has a particular shape which depends on the type of reaction that it is occurring in. The enzyme binds reactants to its active site forming an enzyme substrate complex. When the new product is formed the enzyme is free and ready for the next reaction. This write up focuses on enzymes, in particular peroxidase and how particular factors influence its efficiency .The enzyme peroxidase is found in turnips and potatoes in large quantities. This particular enzyme is made up of 296 amino acids(Mazza and Welinder 1980) and its purpose is to quickly break down hydrogen peroxide which is produced by some metabolic reactions within the cell. Hydrogen peroxide can be harmful to the cell if it is not broken down fast and thus peroxidase breaks it down to dehydrate and oxygen(Cohen and Hochstein 1963). Heat, enzyme concentration and the presence of enzyme inhibitor were all used as tests to see how they influence enzyme efficiency. To measure oxygen produced guaicol which turns brown when oxidised oxygen is present, was used been read with a spectrometer. Hypothesis: The dependant variable for this experiment is the amount of light that is absorbed by the spectrometer. The independent variable is the heat and enzyme

concentration variation and if enzyme inhibitor is present or not. In this experiment it is predicted that the enzyme solution with 2ml of enzyme peroxidase of have the highest absorbance rate in the spectrometer (high conversion of oxygen) whilst the solutions with an enzyme inhibitor and high temperature will have the lowest absorbance rate (low conversion of oxygen). The enzyme concentration with 1 ml of enzyme will be between the 2ml enzyme solution and the 0.5 ml enzyme solution in oxygen produced. MATERIALS & METHODS In this experiment a spectrometer, boiling bath, test tubes, test rack, enzyme peroxidase, guaicol and H202 are required. Before preparation of assays took place the spectrophotometer was adjusted to a 470nm wave length and absorbance was adjusted to 0 (100% transmission). To prepare assay A1 1ml of peroxidase was put in a test tube with 4 mls of water. In another test tube 0.1 ml of guaicol, 0.2 ml of H202 and 4.7 ml of water was put in it. These test tubes were then mixed and a timer was started. The solution was put into the spectrometer and results were recorded At 30 second intervals. This continued until 150 seconds. To prepare assay A2 0.5ml of enzyme peroxidase was put in a test tube with 4.5 ml of water. In the other test tube 0.1ml of guaicol, 0.3ml of H202 and 4.7 ml of water were poured in. When they were mixed the same recording process that occurred with A1 took place. To prepare assay A3 2.0ml of enzyme peroxidase was put in a tube with 3ml of water. In the other test tube 0.1ml of guaicol, 0.2ml of H202 and 4.7 ml of water were poured in. When these test tubes were mixed the same recording process took place as A1. To prepare assay B two test tubes were made up using the same method as assay A1. However then these test tubes were placed in a 90 degree Celsius hot water bath for 15 minutes before mixing took place. When 15 minutes had passed these test tube assays were mixed together and then recording methods in the spectrometer occurred every 30 seconds using the same method as with the assays A1, A2 and A3. To prepare assay C one test tube was prepared with 1.0 ml of enzyme peroxidase, 3ml of water and 1 ml of 10% hydroxylamine (enzyme inhibitor). The other test tube was prepared with 0.1 ml guaicol, 0.2ml H202 and 4.7 ml water. These test tubes were then mixed together and results were recorded using recording methods that were used for previous assays.

RESULTS As shown in graph 1 solution A3 (double enzyme concentration) had the highest rate of light absorbance in the spectrometer whilst solution B (enzyme heated at 90 degrees for 15 minutes) has the lowest rate of light absorbance. A3s absorbance rate increased at a fast rate until 90 seconds and then its rate began to slow. A3 reached an absorbance level of 1.819 in the time period of 150 seconds. As indicated by graph 1 solution A1 (normal enzyme concentration) had the next highest rate of absorbance. The solution increased at a steady rate from 0.037 at zero seconds to 0.93 at 150 seconds. Solution A2 (half enzyme concentration) also increased at a relatively even rate beginning at 0.061 at zero seconds and increasing to 0.577 after 150 seconds. Solution B and C (enzyme inhibitor) had very similar absorbance rates, with both solutions absorbance amount staying relatively constant from 0 seconds to 150 seconds. Solution B started at 0.03 and increased to 0.031 after 150 seconds. Solution C started at 0.015 at zero seconds and increased to 0.036 after 150 seconds.

Sphectrompeter Recordings of the Five Assays

2 1.8 1.6 1.4 Axis Title 1.2 1 0.8 0.6 0.4 0.2 0 0 30 60 90 120 150 Time (seconds) 1ml enzyme concentration 0.5ml enzyme concentration 3ml enzyme concentration enzyme heated at 90 celcius Enzyme inhibitor

Figure 1. DISCUSSION The results of this experiment were predicted successfully before the experiment took place and therefore the hypothesis can be accepted. The three solutions which had different enzyme concentrations had noticeably different reaction rates. The enzyme solution which was heated and the enzyme solution which also contained enzyme inhibitor had a very low conversion rate to oxygen from peroxidase in comparison to A1, A2 and A3. The solution with the highest enzyme concentration (3A) contained double the enzyme concentration of A1 and four times that of A2 and had the highest presence of oxidised guaicol thus indicating the highest rate of enzyme activity. This high rate of enzyme activity can be explained due to more enzymes been readily available to form an enzyme/substrate complex with the hydrogen peroxide(Aragon and Sols 1991). This resulted in a higher rate of oxygen been produced over the time period of 150 seconds. As shown by graph 1 the enzymes reacted with the hydrogen peroxide at a very fast rate up until 90 seconds and then the reaction rate began to slow. This can be explained due to the running out of substrate which meant that some enzymes had no substrate to react with. The Enzyme solution that contained 1ml of peroxidase had a lower reaction rate than that of A3 however it was higher than A2. This was to be expected as A1 did not have as many enzymes readily available to form enzyme/substrate complexes as the enzyme solution A3. Therefore it had a lower reaction rate than A3 in the time period of 150 seconds. However it would be expected that the reaction rate of A1 would continue to rise at a steady rate until it reaches the oxygen levels of A1. The enzyme solution with a low amount of peroxidase was much slower to react in comparison to the other two solutions. This is due to a low amount of enzymes been available to break down the hydrogen peroxide. However over time the amount of oxygen produced would be equal to that of the higher enzyme concentrations. The enzyme solution that was heated at 90 degrees Celsius had a very low reaction rate with little to none oxygen been produced. This can be explained do the denaturing of the enzyme complexes. The denaturing results in the change in shape

of the enzyme which can mean that the substrate does not fit into the enzyme complex anymore and thus the reaction is unable to take place. At 90 degrees Celsius the enzymes would be completely denatured(McLellan and Robinson 1981) and this explains why very little oxygen was produced (next to none). The enzyme inhibitor in solution C also resulted in very little oxygen been produced. The enzyme inhibitor used was the molecule hydroxylamine which has a chemical structure of NH2OH(Hydroxylamine - Compound Summary). Hydroxylamine has a very similar chemical structure to hydrogen peroxide which has a chemical structure of H202(Structure and Bonding:The Hydrogen Peroxide Molecule) .This similar structure resulted in competition for the active site of the enzyme peroxidase or in the case of this experiment resulted in very little hydrogen peroxide been reacted to oxygen. This Experiment could have been improved by doing the experiment multiple times to improve the reliability of the results. Other experiments could take place to expand on these results include testing how environmental factors such as heat and pH influence other common enzymes instead of peroxidase. Possible sources of error in this experiment are that the room temperature that the experiment took place in was much colder/warmer than usual thus influencing the results of the experiment. CONCLUSION Overall this experiment was a success with the enzyme efficiency been clearly influenced by the factors that were tested for in this experiment. This experiment showed that heat and enzyme inhibitors can completely stop enzymes from working and thus reactions cease, and that enzyme concentration has a significant influence on the speed in which reactions take place. Therefore in an organism temperature and pH levels have to be regulated so that enzymes can perform at their peak, which thus allows for vital biochemical reactions to take place at speed. REFERENCES

Activation Energy. Available from http://chemed.chem.purdue.edu/genchem/topicreview/bp/ch22/activate.html. Aragon, JJ, and A Sols. 1991. Regulation of enzyme activity in the cell: effect of enzyme concentration. The FASEB Journal 5 (14):2945-2950.

Cohen, Gerald, and Paul Hochstein. 1963. Glutathione Peroxidase: The Primary Agent for the Elimination of Hydrogen Peroxide in Erythrocytes*. Biochemistry 2 (6):1420-1428. Hydroxylamine - Compound Summary. Available from http://pubchem.ncbi.nlm.nih.gov/summary/summary.cgi?cid=787. Mazza, Gilbert, and Karen Gjesing Welinder. 1980. Covalent Structure of Turnip Peroxidase 7. European Journal of Biochemistry 108 (2):481-489. McLellan, Kathryn M., and David S. Robinson. 1981. The effect of heat on cabbage and Brussels sprout peroxidase enzymes. Food Chemistry 7 (4):257-266. Structure and Bonding:The Hydrogen Peroxide Molecule. Available from http://academic.pgcc.edu/~ssinex/struc_bond/H2O2_molecule.htm.

CRITERIA
TITLE -2

EXCELLENT
Concise statement that linked to the experiment (variables, measurements, and subject matter etc.) Explains the reason for undertaking the experiment Clear concise and discussion of background information that support the reason for the experiment Background information is from reputable sources and is cited correctly Clear concise statement explaining the dependent and independent variables that are to be tested and the expected outcome Relates directly to the experiment All procedures (including statistical methods) used are clearly identified and written in a logical order Procedures are written in past tense Does not contain lists Procedures can be repeated easily by a reader unfamiliar with the activity Results are presented in a clear and logical manner using tables and/or graphs Written information about the results clearly refers to relevant tables and figures Units are given and correct for all data sets Graphs are clearly labelled with title, axes, units and data legend

SEMESTER 1, 2012- MARKING RUBRIC SATISFACTORY NEEDS IMPROVEMENT

Missing one key part of the title Missing 2 key parts of the title

UNSATISFACTORY
No title present

INTRODUCTION

Max - 6

Explains the reason for undertaking the experiment Background information that supports the reason for the experiment is given Background information is mostly from reputable sources and is cited correctly A statement of the expected outcome Variables are not clearly stated Relates directly to the experiment Three of the requirements for Excellent met

Explanation of reasoning behind the experiment is limited or irrelevant Background information is limited or not relevant Sources used for the background information are inappropriate

There is no reason provided for undertaking the experiment No background information is provided

HYPOTHESIS Max - 3

Statement that is not experimentally testable Does not relate directly to the experiment One or two of the requirements for Excellent met

No hypothesis is provided

MATERIALS & METHODS

Procedures not listed or are incomplete

Max - 5

RESULTS DATA & ANALYSIS

Max - 8

Results are presented in a clear and logical manner using tables and/or graphs Written information about the results refers to relevant tables and figures Units are given and correct for all data sets Graphs are labelled with most of the required components

Data poorly presented Information incomplete with many missing components of graphs and/or tables Units missing Written information is not clearly presented or does not relate clearly to tables and figures

Data is not presented, incomplete and/or inaccurate Presented data has many missing components

DISCUSSION

Logically structured explanation of results supported by cited references Logically structured and supported argument for the acceptance or dismissal of the hypothesis Detailed discussion of possible sources of error and the possible effect of these errors on the outcome Concise statement that links the outcomes to the purpose of the experiment References are sourced from suitable sources References are presented in Chicago format in text and in the reference list Report formatted as required Report submitted into Blackboard as requested Three of the requirements for Excellent met

Discussion not supported by appropriate references Acceptance or dismissal of the hypothesis unclear Discussion of possible errors limited or missing Conclusion present but fails to provide a link outcomes to the purpose of the experiment One or two of the requirements for Excellent met

Discussion is missing, or Discussion does not reflect the experiment and its outcomes Conclusion is missing Reference list is missing Report not submitted into Blackboard

Max - 15 CONCLUSION Max - 3 REFERENCES Max - 8

Highlight where on the rubric you believe you have accomplished for each section and give yourself a final mark

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