Lab 7 (Quiz 7) Please hand in your Lab 6 Report You have 5 minutes (from 2:10 to 2:15 pm) to complete the quiz 1. Silence your cell phone and put it away 2. Have your calculator available in case you may need it for the quiz 3. Work on your own; do not cheat 4. When done, turn your quiz upside down and raise your hand for a TA to come and collect your quiz 5. Keep quiet until all quizzes have been collected and counted Final Lab exam Wednesday December 5th (the week after Thanksgiving) Gowan Hall Rm. 126 12:40 pm to 1:30 pm (during your regular lecture time) Exam will cover the material discussed in the lab, lab manual and PowerPoint slides Lab 7: Enzymes • • • • Catalyst Specific; active site Proteins ase • Enzymes are proteins • Protein’s function is based on protein’s shape; change shape (denature). • How can denature protein? High Temp .(not low) and pH (among other factors) • Enzyme function, shape, active site • Catabolic, break down molecules and release energy • Anabolic, construct molecules • You will examine the effects of temperature and pH on the activity of an enzyme (Amylase) that catalyze the breakdown (hydrolysis) of starch into maltose molecules. Starch, a major carbohydrate of the human diet. The amylase enzyme is found in the human saliva and also secreted by the pancreas. Starch Amylase maltose • You will now use a quantitative assay to precisely measure the amount of maltose (reflect Amylase activity) in an unknown sample. Quantitative assay need ??????? The effects of temperature and pH • Prepare standard curve. • Use Exercise 6 information to design the standard curve assay. • What substance you should use for the standard curve? starch OR maltose • Your instructor or TA should approve your protocol before conducting the experiment • Stock solution maltose ________ mg/ml • C1V1 =C2V2 calculate STD. final concentration Tube # 1 2 3 4 5 6 Stock C1 (mg/ml) V1 H2O volume (μl) 0 1000 Maltose Std. concentratio n (C2) 0 Abs. at 540 0.000 Standard Curve 1 Stock (μl) 2 3 4 5 1000 1000 1000 1000 8 8 6 0 H20 (μl) 1000 DNS (μl) 1000 1000 Boil for 3 min H2O (ml) 8 8 8 Measure Abs. at 540 nm Record Abs. (table in previous slide) Draw STD. Curve (next slide/worksheet) • DNS; Stops enzyme reaction, maltose chemical indicator 8 Dependent and Independent variable Temperature Effects Temperature 2% starch 4 (Ice) 23 37 37 Crl 65 100 1 2 3 4 5 6 450 μl 450 μl 450 μl 450 μl 450 μl 450 μl Place each tube in its respective temperature for 5 minutes 100 l Amylase Amylase Amylase H2O Amylase Amylase Incubate tubes in respective temperature for 5 minutes DNS (μl) 1000 1000 1000 1000 1000 1000 Boil for 3 min H2O (ml) 8 8 8 8 Measure Abs. at 540 nm 8 8 • Record your results in table 7.1 pg. 57 • Graph your results and find optimum temperature (slide 8/worksheet). Table 7.1: Results Temperature 4C 23 C 37 C 37 C-Crl. 65 C 100 C Absorbance pH Effect 2 100 l 450 l 4 7 Amylase amylase pH 2 pH 4 amylase pH 7 7 Crl 8 10 H2O amylase amylase pH 8 pH 10 pH 7 Mix Incubate all tubes at room temperature for 5 minutes Add 450 l 2% starch to all tubes Mix Incubate all tubes at room temperature for 5 minutes Add 1 ml DNS to all tube. Boil for 3 minutes Add 8 ml of deionized H2O to all tubes Measure Abs. at 540 nm • Record your results in table 7.1 pg. 58. • Graph your results (slide 8/worksheet) and find optimum pH. 200 145 140 Absorbance Absorbance Temp. effect on Enzyme activity pH effect on Enzyme activity 160 120 100 91 80 81 60 49 40 39 150 145 100 50 99 44 20 0 3 5 pH 0 11 25 Temp. (C) 37 0 100 250 145 230 120 98 85 80 60 40 32 0.5 230 1.0 2.0 Competing Sub Concentration (ug) 185 150 145 100 50 20 0 230 200 115 100 0 4 Effect of Sub concentration Absorbance Absorbance 9 Effect of competing Sub 160 140 7 3.0 0 0.5 1.0 1.5 2.0 Sub Concentration (ug) 2.5 Biology 115 Lab Fall 2018 Lab 7 Worksheet Instructors: ___________________________________________________________________________________ I. Student Name: Section: (Mon, Tue, Wed or Thur): Group Members: _____________________________________________________________________________________ II. Personalized Title [0.5 pts] _____________________________________________________________________________________ III. Hypothesis [1.0 pts] 1. _____________________________________________________________________________________ IV. Introduction IVA. General Theory/Background [1.0 pts] IVB. Summary of Overall Approach [1.5 pts] Page 1 of 5 Biology 115 Lab Fall 2018 Lab 7 Worksheet Instructors: _____________________________________________________________________________________ V. Observations & Results VA. Data [1.5 pts] Page 2 of 5 Biology 115 Lab Fall 2018 Lab 7 Worksheet Instructors: Page 3 of 5 Biology 115 Lab Fall 2018 Lab 7 Worksheet VB. Calculations Instructors: [0.5 pts] _____________________________________________________________________________________ V. Discussion Questions 1. True or false. Continuous increase in temperature will result in continuous increase in enzyme activity. Explain you answer. [0.5 pt] 2. Why is is important to have enzymes catalyze biological reactions? [0.5 pts] 3. List two advantages of using DNS in detecting maltose [0.5 pt] _____________________________________________________________________________________ Page 4 of 5 Biology 115 Lab Fall 2018 VI. Conclusion(s)/Inference(s): Lab 7 Worksheet Instructors: [0.5 pt] _____________________________________________________________________________________ Total Score: _____ / 8.00 (Includes _____ Bonus Points for ________________________________________________) Comments: ___________________________________________________________________________ _____________________________________________________________________________________ Page 5 of 5 
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