Introduction
Enzymes are proteins that act as catalysts within living cells. Catalyst increase the rate at which chemical reactions occur without being consumed or permanently altered themselves. A chemical reaction is a process that converts one or more substances (known as reagents, reactants, or substrates) to another type of substance (the product). As a catalyst, an enzyme can facilitate the same chemical reaction over and over again.
Like all proteins, enzymes are composed of one or more long chains of interconnected amino acids. Each enzyme possesses a unique sequence of amino acids that causes it to fold into a characteristic shape. An enzyme 's amino acid sequence is determined by a specific gene in the cell 's nucleus. This
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Them I pour extract into a conical centrifuge tube and centrifuge the extract for 5 for 2 minutes. Them I decant the supernatant into a clean test tube and then put the tube in ice. This extraction I will use as a source of peroxidase for experiments B, C,D and E
PartB
Effect of Enzyme Concentration on Activity
Tube ml Buffer (PBS) Ml OPD ml Hydrogen Peroxide ml Enzyme (Potato extract) Absorbency
1 (Blank) 3.3 0.1 0.1 0 0
2 3.3 0.1 0 0.1 .572
3 3.2 0.1 0.1 0.1 .885
4 3.1 0.1 0.1 0.2 .905
5 3.0 0.1 0.1 0.3 1.020
Material
Test tubes and test tube rack, spectrophotometer tubes or cuvettes, spectrophotometer, phosphate buffer solution ( pH7.0 ), hydrogen peroxide solution, OPD, vortexes, enzyme extract from potato, graduate pipette pump and micropipette and micropipette tips
Procedure
I number five test tubes 1-5 them I load all five test tubes with the buffer, hydrogen peroxide, and OPD in the order listed above. Them I add the enzyme to each test tube sequentially with a 2 minute interval because the moment I add the enzyme, the enzymatic reaction starts. Them I vortex all the tubes gently when all contents have been added, after each tube was incubate for 5 minutes at room temperature. Them I start to measure the absorbency ( A) at 410 mm of each tube #1 as a blank to calibrate the spectrophotometer them I start to record my results in the table above. To conclude the graph has a bell curve on an absorbency vs amount of enzyme added. This
The preparation for the experiment started by gathering the solutions of enzyme Peroxidase, substrate hydrogen peroxide, the indicator guaiacol and distilled water. Two small spectrometer tubes and three large test tubes with numbered labels. In addition, one test tube rack, one pipet pump and a box of kimwipes were also gathered. Before the experiment, the spectrometer must be set up to use by flipping the power switch to on. Following, the machine was warmed up for 10 minutes and the filter lever was moved to the left. In addition, I set the wavelength to 500 nm with the wavelength control knob. Before the experiment, I had to create the blank solution by pipetting 0.1 ml of guaiacol, 1.0 ml of turnip extract and 8.9 ml water into tube #1. Following the creation of the blank, a control 2% solution was created.
The type of peroxidase is used is called turnip peroxidase. Turnip peroxidase is made up of Guaiacol and hydrogen peroxide. The reactants to the product are turnip peroxidase or called tertraguaiacol and water. The color of the react is brown. In the experiment was conducted there were baseline experiment, temperature, pH, 10X substrate, Inhibitor, and half the amount of enzyme.
12.Stir then pour 2.5ml of the enzyme mixture into one of the test tubes not allowing any water from the saucepan into the test tube.
9. If your biochemical test requires that a reagent is added, which tool do you need to use? (1 pt)
These results shown from this experiment led us to conclude that enzymes work best at certain pH rates. For this particular enzyme, pH 7 worked best. When compared to high levels of pH, the lower levels worked better. The wrong level of pH can denature enzymes; therefore finding the right level is essential. The independent variable was the amount of pH, and the dependent being the rate of oxygen. The results are reliable as they are reinforced by the fact that enzymes typically work best at neutral pH
C. Pour about one quarter of the first unknown packet into the first cup and add
Enzymes are biological catalysts, which accelerate the speed of chemical reactions in the body without being used up or changed in the process. Animals and plants contain enzymes which help break down fats, carbohydrates and proteins into smaller molecules the cells can use to get energy and carry out the processes that allow the plant or animal to survive. Without enzymes, most physiological processes would not take place. Hundreds of different types of enzymes are present in plant and animal cells and each is very specific in its function.
After the substrate solution was added, five drops of the enzyme were quickly placed in tubes 3, 4 and 5. There were no drops of enzyme added in tubes 1 and 2 and in tube 6 ten drops were added. Once the enzyme solution has been added the tubes were then left to incubate for ten minutes and after five drops of DNSA solution were added to tubes 1 to 6. The tubes were then placed in a hot block at 80-90oC for five minutes. They were then taken out after the five minute period and using a 5 ml pipette, 5 ml of distilled water were added to the 6 tubes and mixed by inversion. Once everything was complete the 6 tubes were then taken to the Milton Roy Company Spectronic 21 and the absorbance of each tube was tested.
From the table provided in the lab manual, nine solutions are prepared in a separate test tube. Each solution varied from a combination of distilled water, guaiacol, hydrogen peroxide (3%), turnip extract, and hydroxylamine. When preparing the solution, it is crucial that each tube is prepared and test one at a time before preparing the next test tube. Once a test tube is prepared, it is immediately placed in the spectrophotometer to measure light absorption. After the lid is closed, the light absorbed is recorded every thirty seconds for a total of five
Organisms cannot depend solely on spontaneous reactions for the production of materials because they occur slowly and are not responsive to the organism's needs (Martineau, Dean, et al, Laboratory Manual, 43). In order to speed up the reaction process, cells use enzymes as biological catalysts. Enzymes are able to speed up the reaction through lowering activation energy. Additionally, enzymes facilitate reactions without being consumed (manual,43). Each enzyme acts on a specific molecule or set of molecules referred to as the enzyme's substrate and the results of this reaction are called products (manual 43). As a result, enzymes promote a reaction so that substrates are converted into products on a faster pace (manual 43). Most enzymes are proteins whose structure is determined by its sequence of its amino acids. Enzymes are designed to function the best under physiological conditions of PH and temperature. Any change of these variables that change the conformation of the enzyme will destroy or enhance enzyme activity(manual, 43).
The products produced by the first reaction were used as a substrate for the second. In this case the enzyme used NADH, which resulted in the decreased absorbance due to the NADH oxidation to NAD+. In addition, the spectrophotometer was used as a measuring device to follow the change in absorbance of the NADH molecules at 340nm.
Enzymes are an important part of all metabolic reactions in the body. They are catalytic proteins, able to increase the rate of a reaction, without being consumed in the process of doing so (Campbell 96). This allows the enzyme to be used again in another reaction. Enzymes speed up reactions by lowering the activation energy, the energy needed to break the chemical bonds between reactants allowing them to combine with other substances and form products (Campbell 100). In this experiment the enzyme used was acid phosphates (ACP), and the substrate was p-nitrophenyl phosphate.
In our experiment we used a blender to separate peroxidase from a potato. We prepared four solutions, 0.3ml of guaicol, 0.3 ml of hydrogen peroxide, 1.0 ml of the enzyme solution. Together, 8.5 ml was used although there was a different ph of all four solutions: tap water, ph of four, ph of eight, and ph of seven. Nest the absorbency (nm) was measured using a colorimeter after the enzyme was placed into the solution.
Enzymes are proteins that act as catalysts and help reactions take place. In short, enzymes reduce the energy needed for a reaction to take place, permitting a reaction to take place more easily. Some enzymes are shape specific and reduce the energy for certain reactions. Enzymes have unique folds of the amino acid chain which result in specifically shaped active sites (Frankova Fry 2013). When substrates fit in the active site of an enzyme, then it is able to catalyze the reaction. Enzyme activity is affected by the concentrations of the enzymes and substrate present (Worthington 2010). As the incidence of enzyme increases, the rate of reaction increases. Additionally, as the incidence of substrate increases so does the rate of reaction.
The Industrial Application of Enzymes Enzymes are naturally occurring biological molecules found in all living organisms, plant, animal and microorganisms such as bacteria. All enzymes are proteins and, as with all proteins, are made up of a chain or polymer of amino acids held together by peptide bonds. This chain coils to form a specific three-dimensional globular shape, which, typically, means an enzyme will only work with one specific substrate. The purpose of an enzyme is to lower the activation energy required for biochemical reactions to take place.