BIOL 3380 Name:_____________________________________
Circle Session: T-PM W-AM W-PM R-AM R-PM F-AM F-PM
Experiment 9 – Pre-lab Homework
Enzyme Kinetics of LDH
This pre-lab homework assignment is due at the beginning of your lab session.
You are provided with the following portion of a protocol: • Determine concentration of enzyme stock solution, if unknown, by taking an A280 nm reading of a 1:100 dilution (in water). Use a total volume of 1 ml in the cuvette.
• Dilute some of the enzyme stock with buffer A to make a 4 mg/ml solution.
• Serially dilute the 4 mg/ml solution with buffer A to make working solutions of 400 µg/ml and 40 µg/ml.
• Prepare 30 µl of each working solution for
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B. Catalysis occurs on a specific site on the enzyme (the active site). The active site is usually less than 5% of the surface area of the protein, and is always in a cleft. The rest of the molecule serves to present the active site in a three dimensional structure that is capable of binding substrate and catalyzing the reaction. Binding to a substrate is very specific, and involves ionic interactions, H bonds and van der Waals forces. C. Only amino acids with polar side chains participate directly in catalysis (Cys, His, Ser, Asp, Glu and Lys). Some reactions require electron acceptors, and since no side chains are good electron acceptors coenzymes and cofactors are required for catalysis of some reactions. D. Enzymes catalyze reactions by several means: ▪ by orienting the substrate molecules. ▪ by increasing the effective concentration of the substrate. ▪ by acid-base catalysis ▪ by covalent catalysis ▪ by using the energy of substrate binding to promote the enzyme catalyzed reaction.
III. Criteria For Establishing The Presence Of Enzymes
Since most of the known biological catalysts are proteins two criteria are generally used for establishing the existence of enzymes. The first is that the rate of a reaction in the presence of an enzyme is greater than the rate in its absence. Because the uncatalyzed rates of most biologically important reactions are effectively zero, the mere
For enzymes to be useful in a reaction, the substrate needs to bind with the enzymes active site. The active site is specific for a
In the experiment we used Turnip, Hydrogen Peroxide, Distilled Water, and Guaiacol as my substances. On the first activity, Effect of Enzyme concentration of Reaction Rate for low enzyme concentration, we tested three concentrations of the turnip extract, and hydrogen peroxide. For the Turnip Extract I used 0.5 ml, 1.0 ml, and 2.0 ml. For hydrogen peroxide we used 0.1 ml, 0.2 ml, and 0.4 ml. We used a control to see the standard, and used a control for each enzyme concentration used. The control contains turnip extract and the color reagent, Guaiacol. We prepared my substrate tubes separately from the enzyme tubes. My substrate tube
H. How would you prepare 10 mL of a 0.25M HCl solution if 1M HCl was available? How much
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.
Note that the enzyme remains unchanged so that more of the some substrates can react.
Background and Introduction: Enzymes are proteins that process substrates, which is the chemical molecule that enzymes work on to make products. Enzyme purpose is to increase the rate of activity and speed up chemical reaction in a form of biological catalysts. The enzymes specialize in lowering the activation energy to start the process. Enzymes are very specific in their process, each substrate is designed to fit with a specific substrate and the enzyme and substrate link at the active site. The binding of a substrate to the active site of an enzyme is a very specific interaction. Active sites are clefts or grooves on the surface of an enzyme, usually composed of amino acids from different parts of the polypeptide chain that are brought together in the tertiary structure of the folded protein. Substrates initially bind to the active site by noncovalent interactions, including hydrogen bonds, ionic bonds, and hydrophobic interactions. Once a substrate is bound to the active site of an enzyme, multiple mechanisms can accelerate its conversion to the product of the reaction. But sometimes, these enzymes fail or succeed to increase the rate of action because of various factors that limit the action. These factors can be known as temperature, acidity levels (pH), enzyme and/or substrate concentration, etc. In this experiment, it will be tested how much of an effect
* By using the dropper and measuring cylinder, 3ml of 1% lipase was added into the test tube
Enzymes are very large globular proteins with three dimensional shapes which is vital for enzyme activity as natural catalyst in chemical reactions within the living organisms (7).
To prevent fluctuation in the pH, a solution known as a “buffer solution” was used in the experiment. Buffer solutions are mixtures of at least two chemicals which counteract the effect of acids and alkalis. Therefore, when a small quantity of alkali or acid solution is added the pH of the enzyme doesn’t change.
Enzymes have an active site which has a complimentary base to a specific substrate, when these bind an enzyme-substrate complex is
chemical reactions. Enzymes have a set of conditions at which they work perfectly; this is known
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.
However, the rate of reaction only increases for a certain period of time until there is lesser substrate molecules than the enzyme molecules. The increase of enzyme concentration does not have effect if there are lesser substrate molecules than enzyme molecules initially.
“Enzymes are proteins that have catalytic functions” [1], “that speed up or slow down reactions”[2], “indispensable to maintenance and activity of life”[1]. They are each very specific, and will only work when a particular substrate fits in their active site. An active site is “a region on the surface of an enzyme where the substrate binds, and where the reaction occurs”[2].
Also, the enzyme was treated with different urea concentrations before it was added to the assay mixture. The amount of the second and third enzymes was essential. Likewise, this would have had an effect on the rate of the measured aldolase activity. Hence, the concentrations for both enzymes was measured and calculated prior to the experiment whilst preparing the assay mixture. Furthermore, in order to measure thiol group reactivity the base line was adjusted to zero for each cuvette. This was done to make sure that the results were