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Amylase breaks down starch into the simple sugar glucose and is one of the first steps in digestion. Starch can be detected with a simple chemical stain. In the presence of starch, a solution of potassium iodide/iodine (KI/iodine) will turn blue/black in color. This is a quantitative reaction – the darker the blue/black color, the more starch is present. As amylase digests the starch, the intensity of the blue/black solution of KI/iodine decreases. We will quantify the reaction using a spectrophotometer. Amylase can also be studied by looking at the simple sugars generated as starch breaks down. Reducing sugars such as glucose can be detected using Benedict's reagent, a solution of copper (II) sulfate, sodium carbonate, and sodium citrate. Benedict's reagent turns from a soluble blue color into a brick red precipitate. It is also a quantitative reaction, and we will use a desktop scanner and a free imaging program (ImageJ) developed by the National Institutes of Health to measure the intensity of the signal in the red and blue channels. We will then use a standard curve (similar to the curve for starch) to calculate the amount of reducing sugar present as amylase digests starch. This lab has four parts: Part I: Making a standard curve using known amounts of starch and KI/iodine solution. Part II: Determining the activity of amylase present in a given sample of saliva using KI/iodine reagent. Part III: Using Benedict’s reagent to detect the amount of glucose in a given sample. Part IV: Determining the activity of amylase present in a given sample of saliva using Benedict’s reagent Materials and Reagents The iodine reagent is made by dissolving 6.0 g of KI in 100 mL of 2% tincture of iodine. Benedict's reagent is made by dissolving 30.0 g of Cu(II) sulfate in 60 mL of hot water (may require extensive heating to fully dissolve), and in a second flask dissolving 20.0 g of sodium carbonate and 34.6 g of sodium citrate in 120 mL of hot water. While still warm, these two solutions are mixed slowly as some bubbling will occur, and then diluted to 200 mL with distilled water. Starch solution is made by dissolving 0.5g soluble starch in 100 mL of cold water (0.5% starch solution) followed by gentle boiling. A 0.1% starch solution should be made by diluting the 0.5% stock into distilled water. A 0.5% dextrose (ie. glucose) solution is made by dissolving 0.5g dextrose in 100 mL distilled water. The concentrations of these reagents are important, as volumes are chosen to optimize spectrophotometry readings and to avoid saturating conditions. Hazards Iodine is often obtained as a solution in alcohol, which is flammable. Iodine also stains clothing and skin, so special care should be taken with this reagent. The Benedict's reagent assay requires that samples be boiled for at least 5 minutes. Tubes should be boiled in a slightly
and starch solution. 7) Calculations: 1) Plot on a graph Absorbance for each tube versus the Concentration of starch in each tube.Starch Standard Curve Materials Needed: Spectronic 20 spectrophotometer 5 spectrophotometer test tubes Dropper KI/iodine reagent Regular test tube Tissue paper 0. 2 .9 mL 2.7 mL 4) 0.” Fill each subsequent tube according to the following chart: Starch water Starch Concentration Absorbance 1) 0 mL 3. PBS.1 mL 2. (making sure to clean the outside of each tube) into the spectrophotometer.0 mL 2) 0.4 mL 5) 0. (Absorbance versus Concentration) This is the Standard Curve. Set the wavelength on the spectrophotometer to 620 nm. Insert into spectrophotometer and adjust the transmittance control knob (right knob) to 100 percent. Insert each Tube 2 – 5.9 mL 3) 0. Record Tube 1’s Absorbance as zero. read and record the Absorbance for each on the chart under Step 4. Do not heat test tubes directly over a flame.1 mL 5) Add one drop of KI/iodine reagent to each tube and mix completely. 6) Adjust the left knob on the Spectrophotometer to zero. Part I.bubbling water bath and appropriate precautions against burns from the boiling water must be taken.6 mL 2.5 mg/mL (0. Label each test tube 1-5.3 mL 2. Fill Tube 1 with 3 mL distilled water – it will be the “blank. The iodine solution will tend to settle at the bottom of the tubes and must be evenly distributed. Benedict's reagent should NOT be mixed with acids. Clean the outside of Tube 1 (the “blank”) with tissue paper to remove finger prints. Each test tube will contain 3 mL total volume to enable them to be read by the spectrophotometer.05%)starch solution Deionized water Pipettes Test tube rack PBS Procedure: 1) 2) 3) 4) Turn on the spectrophotometer and allow to warm up for at least 15 minutes. then insert into test tube rack Obtain KI/iodine reagent (pipette into regular test tube). Make sure all spectrophotometer test tubes are clean and dry.
Ensure that all test tubes are clean and dry. Note that the first time point is CRITICAL.2) Using excel. KI/iodine solution should be made fresh! Materials Needed: Spectronic 20 spectrophotometer 4 large test tubes 0. collect at least 1 mL of saliva in a large test tube. It will be necessary to take time points of the reaction. Insert into spectrophotometer and adjust the transmittance control knob (right knob) to 100 percent. Prepare to time your experiment – you will be taking time points every 2 minutes. Pipette 1 mL of the saliva into a second large test tube. Clean the outside of a “blank” test tube (filled with deionized water) with tissue paper to remove finger prints. deionized water. and starch solution. Adjust the left knob on the spectrophotometer to zero. Remove 1 mL of the 1:100 saliva dilution and pipette into the 4th large test tube.5 mg/mL (0. Part II – Amylase in Saliva This part of the experiment will determine the activity of amylase present in a given sample of saliva. This is the 1:10 saliva dilution.you should make sure 8 spectrophotometer test tubes Dropper fresh KI/iodine reagent (6g. This will speed up sample processing. and recording your results. 2g I2 per 100ml) Deionized water Pipettes Tissue paper Stop watch 2) 3) 4) 5) 6) 5) 6) 7) 3 . plot a trendline using the obtained data points and calculate the linear regression. Set the wavelength on the spectrophotometer to 620 nm. Remove blank and set aside. Add 9 mL distilled water to the 1 mL saliva sample. From a volunteer. Label the spectrophotometer test tubes 1-7 and insert them into test tube rack Obtain KI/iodine reagent (pipette into regular test tube).05%) starch solution Test tube rack(s) Procedure: 1) Turn on the spectrophotometer and allow to warm up for at least 15 minutes. Absorbance will need to be measured and recorded at each given time point. Make sure to label your test tubes! Take 1 mL of the 1:10 saliva dilution and pipette into a fresh tube and add 9 mL of distilled water to make a 1:100 saliva dilution. Fill each of the 7 spectrophotometer tubes with 2 mL deionized water and add 1 drop of KI/Iodine reagent to the tubes. mix well.
time. How does the reaction rate change over time? 4) Plot the reaction rate vs [starch] (Michaelis-Menten plot). 2) Graph the concentration of starch vs. 12) Calculations: 1) Using the standard curve determined in part I. 6. and read and record the Absorbance on the chart under Step 8. mix well. 3) 4 min. What is the shape of this graph? 3) Plot the reaction rate ([starch]/∆ t) vs. Record the time on the following chart: Target time 1) 0 min. As soon as you begin adding the starch to the dilution. and 12 minutes. 8. 4) 6 min. At 2 minutes. Add 1 drop of KI/iodine reagent. Repeat these steps at every 4. 9) 10) 11) Actual time Absorbance starch concentration Immediately pipette 1 mL of the starch/saliva dilution mixture into the first spectrophotometer test tube (test tube 1) with KI/iodine reagent. 10. quickly read and record the Absorbance. 6) 10 min. (This is time = 0). calculate the concentration of starch using the determined absorbance. pipette another 1 mL of the starch/saliva dilution mixture into the second tube. 7) 12 min. What does this graph look like? 5) Make the double reciprocal plot (1/reaction rate) vs 1/[starch] (Lineweaver-Burke plot). start timing with the stop watch.to have everything ready to go and be ready to collect the first sample at the earliest possible time point. 2) 2 min. mix quickly and well. time. 5) 8 min. What is the shape of this graph? 4 . Insert the tube into the spectrophotometer. 8) You will add 9 mL of starch solution to the 1 mL of 1:100 saliva dilution in the 4th test tube and mix well.
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