COMPARING PROTEIN CONCENTRATIONS & PROFILES FROM …



Enzymatic Activity of CellobiaseCellulose is the structural polysaccharide in the cell walls of plants, and is the most abundant biological molecule on earth. Cellulose is a polymer of glucose monomers connected together by beta-linkages (Figure 1) In general, many animals, including humans, are incapable of digesting cellulose because they do not produce the enzyme to break these beta-linkages. Figure 1 The enzyme, cellobiase, catalyzes the hydrolysis of β1-4 glycosidic bonds found in cellulose, which is necessary for digestion. This enzyme can be found in organisms, such as fungus and some bacteria, which can breakdown cellulose. Enzymes are usually made up of linked chained amino acids, i.e. proteins, which speed up the reaction it catalyzes. For this reason, enzymes are also known as catalyst. Enzymes are specific to particular substrates, which are used to speed up most biological chemical reactions that would take too long for them to occur naturally. Below is another structural polysaccharide found in most foods, Starch, which is also a polymer of glucose monomers but connected together by alpha-linkages instead of beta-linkages (Figure 2) Animals, including humans, have the specific enzyme, amylase, to break down starch. Therefore, these organisms have the ability to break down the alpha-linkages between glucose molecules but cannot break the beta-linkages. This subtle structural difference is the reason why amylase cannot digest cellulose.Figure 2In this experiment, we will observe the reaction catalyzed by cellobiase. In order to study this enzyme’s activity, it is best to be able to either detect the substrate being used in the reaction, or the products being produced. For this reaction, the substrate cellulose, and the product, glucose, is clear and colorless making it difficult to directly quantify. We will utilize an artificial substrate that has the same structural competency of beta-linkages, but will allow us to directly observe and quantify the reaction of β1-4 glycosidic bond hydrolysis by using absorption spectroscopy.Measuring Enzymatic Activity of CellobiaseTo make this reaction easier to observe, an artificial substrate: p-nitrophenyl glucopyranoside will be used. This artificial substrate will bind to the enzyme and is broken down in a similar fashion to cellulose. When the artificial substrate, p-nitrophenyl glucopyranoside, is broken down by cellobiase, it produces glucose and p-nitrophenol (Figure 3)During the course of the experiment, you will be adding the reaction between enzyme (cellobiase) and artificial substrate into Stop Buffer Solution at various time points. The stop solution is a basic solution with a pH >7, which effectively disables the cellobiase enzyme ceasing the production of glucose and p-nitrophenol. You will also notice that the reaction solution will turn a yellow color, which is due to the p-nitrophenol that is produced during the reaction. The amount of yellow color observed in the reaction solution is proportional to the amount of p-nitrophenol present. For every one molecule of p-nitrophenol present in the solution, one molecule of p-nitrophenyl glucopyranoside has reacted with the cellobiase enzyme (Figure 3). The yellow color of p-Nitrophenol can be measured using absorption spectroscopy. Figure 3 Substrate (colorless) Products (yellow) p-nitrophenyl-?-D glucopyranoside Glucose p-NitrophenolExtraction of Cellobiase Enzyme Gather all the necessary materials for the experiment: mortar & pestle, microcentrifuge tubes, funnel, and coffee filter. You MUST lightly wet the entire coffee filter using DI water prior to placing onto the funnel. You MUST also remove any excessive liquid from the coffee filter so that your coffee filter is not dripping wet! Cut and weigh out 2 grams from the cap of a mushroom, taking care to remove dark parts including the gills, and place that sample into your mortar & pestle.Add 4 ml of extraction buffer onto your mushroom sample and grind until you have a slurry mixture. Save the 15 ml tube for step 6.Once your mushroom sample has been ground into a slurry mixture, filter it through your pre-wet coffee filter into (2) 2ml microcentrifuge tubes. After you have filtered your sample, you may gently squeeze the remaining slurry mixture out of the coffee filter into the microcentrifuge tubes. Take care to not break the filter. Centrifuge your filtered crude mushroom extract for 2 minutes at the highest RPM to pellet the debris.After centrifugation, transfer the supernatant (the liquid layer) into the 15 ml tube that your extraction solution came in. This is your enzyme extract! Make sure that you do not transfer any pelleted debris. The entire class will combine their individual enzyme extract into a single tube or beaker so that each group in the class will begin with the same enzyme concentration. Save your 15 ml tube for the next step. Speculate on the variation you might see if you were to not pool the enzyme. Where might variation be introduced?Once your class has successfully combined all of their enzyme extract, the teacher will distribute 2 ml of the Enzyme Extract to each of the groups into their 15 ml tube.Determining Cellobiase Enzymatic ActivityGather all the necessary materials for this portion of the experiment: Timer, Cuvettes, Cuvette racks, 15 ml tube with mushroom extract, and spec200. Next, label 8 cuvettes #1-8 and pipet 500 μl of Stop Buffer into each cuvette. These will be your different time points. What is the purpose of the Stop Buffer? *******Your teacher will make a “zero” cuvette by adding 500 μl of stop buffer, then add 165 μl of the class enzyme extract, and then 335 μl of reaction substrate. This will be the zero for the spec readings of your 8 cuvettes of reactions. More than one group can share a zero.*******You reaction will be conducted in the 15ml tube labeled ‘reaction’ which contains 4 ml 1.5 mM p-nitrophenyl-?-D glucopyranoside substrate. IMPORTANT: You will be adding your enzyme extract to the 15 ml tube labeled Reaction and immediately start a timer. Quickly add and mix the 2 ml of enzyme extract to your substrate solution. Immediately start the timer as the Enzyme Extract is going into the Substrate. This is the time from when everything needs to be measured! After 1 min, remove 0.5 ml of your reaction and add it to cuvette labeled #1 (which contains 0.5 ml of STOP solution). Mix briefly after each step!2 min from the start, remove 0.5 ml of your reaction and add it to cuvette labeled #2.4 min from the start, remove 0.5 ml of your reaction and add it to cuvette labeled #3.6 min from the start, remove 0.5 ml of your reaction and add it to cuvette labeled #4.8 min from the start, remove 0.5 ml of your reaction and add it to cuvette labeled #5.10 min from the start, remove 0.5 ml of your reaction and add it to cuvette labeled #6.15 min from the start, remove 0.5 ml of your reaction and add it to cuvette labeled #7.30 min from the start, remove 0.5 ml of your reaction and add it to cuvette labeled #8.CuvetteTimeAbsorptionp-Nitrophenol Concentration11 min22 min34 min46 min58 min610 min715 min830 minDetermining Standard Curve of p-Nitrophenol The concentration of p-nitrophenol produced in your reaction can be determined by comparing to a standard curve of known concentrations of p-nitrophenol. You will make this standard curve using p-nitrophenol ranging from 12.5 ?M to 100 ?M p-nitrophenol. When a graph of absorbance vs. concentration is plotted for the standard solutions, a linear relationship should result. The linear relationship between absorbance and concentration for a solution is known as Beer’s law. You can determine the p-nitrophenol concentration produced in your reaction by measuring its absorbance and then finding the corresponding concentration on the standard curve. Because the relationship is linear, you could also calculate the p-nitrophenol concentration using the formula for the standard curve best fit line (y=mx+b).For the p-Nitrophenol standards, label 5 cuvettes 1-5.Add 1.0 ml of 0 (blank) to cuvette 1. Add 1.0 ml of 12.5 ?M to cuvette 2. Add 1.0 ml of 25 ?M to cuvette 3. Add 1.0 ml of 50 ?M to cuvette 4. Add 1.0 ml of 100 ?M to cuvette 5. Set the wavelength to 410 nm.Blank the spectrophotometer with the cuvette labeled #1.Read the absorbance of your standards and record data below:Cuvettep-Nitrophenol concentrationAbsorption @ 410 nm10 (blank)0212.5 ?M325.0 ?M450.0 ?M5100 ?MUse Excel to find out the equation for the linear best fit line and write it below (these instructions are for Excel in Windows 7, yours may differ):Open an Excel spread sheet containing a column for p-Nitrophenol standard concentrations, and an additional column with their corresponding absorbances (as shown above). Highlight the values, click on insert, and choose the scatter plot with no line function. Go to chart layouts, choose trendline. In order to see the equation, go into trendline options and click “display equation on chart”.Using this equation, determine the concentration of p-Nitrophenol in your Enzyme reaction tubes. Did your enzyme extraction allow the breaking of the β1-4 glycosidic bond between glucose and p-Nitrophenol (p-Nitrophenyl-?-D glucopyranoside)? Explain.Using excel plot your product concentration (p-Nitrophenol) versus time. Is the plot linear or exponential, does the concentration of product reach a plateau, and not change with any more time? Come up with the chemical reaction for this reaction, using E for the enzyme (cellobiase) and S for the substrate (of p-Nitrophenyl-?-D glucopyranoside) and products (p-nitrophenol and glucose:Thinking in terms of the enzyme acting as a catalyst, what do you think would happen to your above reaction if you added more enzyme? If you added more substrate? If you boiled the reaction? If you changed the pH? Design an experiment asking one or these questions or one of your own questions, and predict your outcome, if time permits, conduct the experiment. Cellulose is an untapped source of glucose. Glucose is not only extremely important for animal metabolism, but it can be used to make ethanol, which can be used as fuel. Currently, glucose from corn is utilized for ethanol production for fuel. Instead of using corn to make biofuel, if we could access the glucose in cellulose, ethanol could be synthesized without impacting animal food sources. Name several sources of cellulose: Do you think it is feasible to use cellobiase to generate biofuel? Why or why not? SolutionsExtraction buffer and reaction buffer50 mM MOPS Buffer pH 6 (MW 209.26 g/ mole)-Store at room temp indefinitelyWeigh out 2.6 gDissolve in 150 mL of distilled waterAdjust pH to 6Bring to final volume of 250 mL with distilled waterSubstrate1.5 mM p-nitrophenyl-B-D glucopyranoside (pNPG) (MW 301.25 g/mole) in 50 mM MOPs Buffer pH 6.0-Store at 4°C, shelf life 1 week. Weigh out 0.023 gDissolve in 40 mL of 50 mM MOPS BufferBring to final volume of 50 mLs with 50 mM MOPS BufferStop Solution500 mM Na2CO3.H2O (MW 124.00 g/mole)-Store at room temp indefinitelyWeigh 6.2 gDissolve in 200 ml of dH2OBring to final volume of 250 ml with dH2Op-Nitrophenol Standard Stock Solution2.0 mM p-nitrophenol (MW 139.11 g/mole)-Store in at room temp indefinitelyWeigh 0.0278 g of p-nitrophenolDissolve in 75 mL of waterBring to a final volume of 100 mL0.2 mM p-nitrophenol Standard solution-Store at room temp indefinitelyDilute 2.0 mM p-nitrophenol 1:10 using distilled water by adding 1 mL of 2 mM p-nitrophenol and 9 mL of distilled water in 15 mL conical tube. ................
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