EXERCISE 6 Lab Procedures I Determine the effect of substrate concentration on enzyme activity Be sure you do not confuse the enzyme glucose oxidase with the substrate glucose 1 Turn on the Spec 20 and set the wavelength to 510 nm The Spec 20 must warm up for at least 15 minutes before it will give accurate measurements 2 Examine the solutions you have been given One should be labeled ENZYME Glucose Oxidase This is the ENZYME that will catalyze the reaction you will observe Another solution should be labeled GLUCOSE Stock Solution 60 mg dL This is one of the substrates for the reaction you will observe Be very careful to use the correct solution for each part of the lab exercise 3 Using labeling tape and a Sharpie label one 5 mL pipette dH2O and another 5 mL pipette Glucose Be very careful to use the dH2O pipette only with distilled water and the Glucose pipette only with the glucose stock solution Do not cross contaminate any of your solutions It is critical that the enzyme solution not be contaminated with glucose and that the glucose solution not be contaminated with enzyme Contamination usually occurs because the same pipette has been used to measure both solutions or the tip of the pipettor has touched the other solution while dispensing If you have accidentally cross contaminated your solutions or if either solution appears pink while in its original container give the contaminated solution to your instructor and obtain uncontaminated solutions before you continue the activity To avoid contaminating your enzyme solution with glucose use only a micropipettor and clean tips to dispense the enzyme solution 4 Using labeling tape and a Sharpie label 8 clean Spec 20 cuvettes as follows label one cuvette B for blank and label the remaining 7 cuvettes with the numbers 1 7 When labeling Spec 20 cuvettes never write on the cuvette Use a small piece of label tape affixed to the top of the cuvette so that it will not interfere with the spectrophotometer reading 5 Add 3 0 mL of dH20 using the dH2O pipet and 2 0 mL of the enzyme using a 1000 L micropipettor with a clean tip to the Spec 20 cuvette labeled B and set it aside Later this cuvette will be used to calibrate the Spec 20 If this cuvette turns pink it probably means that your blank cuvette has been contaminated with glucose and must be prepared again You must locate the source of your contamination immediately to avoid making the same mistake again before continuing with the experiment 6 Starting with the stock solution containing 60 mg dL of glucose and using dH2O as your diluting solution you need to make 3 mL of each of the following glucose solutions 30 mg dL 15 mg dL 7 5 mg dL 3 75 mg dL 1 875 mg dL and 937 5 g dL As part of the Prelab you were supposed to label the diagram below to show how you would prepare these solutions using the serial dilution technique Ask your instructor to check your diagram before you proceed A B C 1 60 mg dL B B D 2 30 mg dL F E 3 15 mg dL B 4 7 5 mg dL Exercise 6 Austin Community College BIO 1406 Laboratory Manual 12th Ed 2006 B G 5 3 75 mg dL B H 6 1 875 mg dL I 7 937 5 g dL 6 7 7 Prepare your serial dilution of glucose in the Spec 20 cuvettes numbered 1 7 as follows A B C D E F G H I Use the pipette labeled Glucose to place the correct amount v1 v2 of stock solution in tube 1 Use the pipette labeled dH2O to place the correct amount v2 of dH2O in tubes 2 7 Use the pipette labeled Glucose to transfer the correct amount v1 of solution from tube 1 to tube 2 Mix the contents of tube 2 and then use a clean pipette to transfer the correct amount v1 of solution from tube 2 to tube 3 Mix the contents of tube 3 and then use a clean pipette to transfer the correct amount v1 of solution from tube 3 to tube 4 Mix the contents of tube 4 and then use a clean pipette to transfer the correct amount v1 of solution from tube 4 to tube 5 Mix the contents of tube 5 and then use a clean pipette to transfer the correct amount v1 of solution from tube 5 to tube 6 Mix the contents of tube 6 and then use a clean pipette to transfer the correct amount v1 of solution from tube 6 to tube 7 Mix the contents of tube 7 and then use a clean pipette to discard the correct amount v1 of solution from tube 7 8 Decide which student in your group will be the timekeeper which student will write down the absorbance levels measured the note taker which student will add glucose oxidase enzyme to the Spec 20 cuvettes and which student will operate the Spec 20 As you watch the readout while each tube is in the Spec 20 it will be constantly changing so careful coordination in your group is necessary The timekeeper will call out the times the operator of the Spec 20 will call out the readings and the note taker will record the data The note taker must prepare an appropriately labeled table in his lab notebook to record the data and this data must be transferred to every lab partner s notebook before starting Part II of the procedures 9 Zero the Spec 20 following the instructions for the Spec 20 or the Spec 20D Use the cuvette labeled B which you prepared in step 5 as your blank Note In the next step the timekeeper should note the exact time that the glucose oxidase enzyme is added to the glucose This is time zero The first reading is at 15 seconds so you have 15 seconds to mix the solutions wipe off the cuvette with a Kimwipe and place the cuvette in the Spec 20 You will be adding enzyme only to the diluted glucose solutions in cuvettes 2 7 not to the stock solution in cuvette 1 10 Add 2 0 mL of glucose oxidase enzyme to cuvette 2 ONLY and note the time DO NOT add enzyme to more than one cuvette at a time Quickly mix the glucose and enzyme solutions by sealing the end of the cuvette with Parafilm and gently inverting it 2 3 times 11 After cleaning the cuvette with a Kimwipe to remove any fingerprints or spills immediately place the cuvette into the Spec 20 Record the A510 values in your lab notebook every 15 seconds for 2 minutes using a table such as the one below A510 Readings after Adding 2 mL of Glucose Oxidase to Varying Concentrations of Glucose Cuvette Glucose Concentration mg dL 15 s 30s 45s 60 s 75 s 90 s 105 s 120 s 2 3 4 5 6 7 Exercise 6 Austin Community College BIO …