BIO 181 LAB: ENZYMES II - Lab Write Up, To Go with Online Enzyme Labs Be sure to complete the online enzyme labs before following these directions. The regular in- class Lab Manual chapter 'Enzymes I' is included with this document for your reference. It is worth reading for its details on enzymes introduction), beta-galactosidase & ONPG, and how to use the spectrophotometer (similar to the one used in the online simulation). The experiments described here are more detailed than the online versions, thus the manual is worth reading for examples of general experimental protocol. Here are the basic results of these two experiments. Experiment 1: Enzyme Specificity B2 Tube Enzyme Present Substrate Present Color of Solution Reaction (+/-) AI Beta-galactosidase ONPG Yellow A2 Diastase ONPG clear A3 Buffer (no enzyme ONPG clear 1 Beta-galactosidase Starch Blue/black Diastase Starch Yellowish B3 Buffer (no enzyme Starch Blue/black Note: B tubes had IKI iodine) added. The blue/black response indicates presence of starch; the reaction to hydrolyze starch did not occur. Conclusion: Enzymes are highly specific. Beta-galactosidase can hydrolyze ONPG, a molecule very similar to the disaccharide lactose. However, it cannot hydrolyze the bonds between two glucose molecules, as found in starch. Similarly, diastase can hydrolyze starch, a long chain of glucose molecules, but it cannot hydrolyze the bond in ONPG, an analog of a different disaccharide. Try to answer the questions in the lab manual associated with this experiment. Experiment 2: Enzyme Concentration You performed a similar experiment online. As expected, Tube A in this experiment, with the highest concentration of the enzyme (8% beta-gal), had the brightest yellow, the highest absorbance (from the spectrophotometer) and the highest rate of reaction. Try to answer the questions in the lab manual. Note that the substrate used in these experiments, ONPG, is the same used in the BioIndustrial Production online enzyme lab. However, the in-class lab uses a different enzyme, beta- galactosidase (b-gal) which is synthesized by the bacterium Eschericia coli (E. coli). Enzyme Lab II - Your Assignment: A lab report in two parts During in-class labs, the second enzyme lab would provide an opportunity for students to create their own experiment and test their own hypotheses regarding the effect of temperature, pH or a competitive inhibitor on the rate of this reaction, the hydrolysis of ONPG by beta-galactosidase. Your assignment is to write a lab report in two parts. First, you will write a Title and the Introduction to your lab report, including background facts, an explanatory hypothesis and specific predictions. Second, you will be given a table of results and you will write the Results, Discussion and Literature Cited sections of the lab report. Report on The Effect of Temperature on the rate of an Enzyme-Catalyzed Ron Your report should follow the standard format of a scientific journal. Part I will be limited to the Title and Introduction. Following that submission, you will receive a data set. You will then write Part II of the report with these sections: Results, Discussion and Literature Cited. Use these terms as headings at the start of each section (except Title): they provide an outline for your paper. Title: A supported hypothesis makes an ideal title - it is concise and informative. You may assume your hypothesis will be supported (if your hypothesis is well grounded in the facts presented in your text, lab manual and lectures) Introduction: Why did you perform this study? What observation or question stimulated you to perform this experiment? What background information relates to the question? For this paper, begin with a definition of enzymes, referenced from your text (Urry et al 2020). Describe the specific enzyme-catalyzed reaction and how the factor you are studying affects enzymes (proteins). Who made the enzyme? Where is it normally found? Be sure to use all of the information above to make an educated hypothesis: a statement that explains how temperature affects the rate of this enzyme-catalyzed reaction what is the optimum (why?), what happens at other temperatures (why?). Finally, provide clear predictions of your hypothesis, in terms of reaction rates. "If my hypothesis is supported, I predict... Simply restate the reaction will be greatest at the optimum temperature (what is it?) and slower at higher and lower temperatures, as explained by your hypothesis Rubric: Your background should contain at least five facts relevant to your hypothesis. Your hypothesis should identify and explain the optimum temperature (why this temp?) and what happens in cooler and warmer conditions (and why?). Directions for Part 2 to come. The following two pages provide information given to students in preparation for their second enzyme lab. You may want to consider these details when writing your lab report. General information possibly useful for lab report The enzyme beta-galactosidase is made by certain bacteria (e.g. Escherichia coli) when lactose (the sugar in milk) is present as a food source. Escherichia coli are some of the common bacteria found in your intestines. ONPG (CHNO.) "an analog of lactose" (p. 78) means that ONPG has a structural formula very similar in shape to that of lactose. ONPG fits in the active site of the enzyme beta- galactosidase. ONPG was created in the lab for the purpose of studying the activity of this enzyme. GLUCOSE HCON H OHH OH HCOH OH OH HO 0 OH HCOM H OH H OHH C C HO LACTOSE OH OHH GALACTOSE NO2 ONP pellav NO2 HO-C CE HO H HCON H OHH HO OM ONPG H OH GH H . H OH GALACTOSE Tips for ALL experiments: Each test tube must contain a total of at least 3 mL in order to be read in the Spec 20. However, ONPG is expensive, so don't use excessive amounts (ie. making up test tubes with a total of 6 mL is wasteful and therefore unacceptable). More than one control tube may be helpful or necessary for some experiments. As indicated on your graded experimental design, you must have a tube that contains everything except enzyme; use buffer to substitute for the enzyme volume used in the other tubes (= control tube). The control tube is required to calibrate the spectrophotometer. If you decided to use something else as your control sample, please be sure to have this calibration tube also. See "tips" below for other suggestions For your controlled variables, use the same conditions as in last week's experiment and be sure to specify these conditions in your lab report Consider the possibility of using replicates of your tubes and/or doing the experiment more than once if there is time (especially if something goes wrong the first time). Your lab report will be a lot more interesting if you have reasonably good data to interpret. Tips for pH experiments: Since pH alone can destabilize molecules, you might want to consider having a control tube for each pH, without enzyme, so you can tell what else the pH may be doing other than affecting the cnzyme, Be sure you measure the pH after adding the appropriate buffer, since the final pH may differ from the pll of the buffer alone. The actual pH in the tube is what you need to use in the lab report. Be sure not to add anything to your tubes that is already buffered Unbuffered ONPG and enzyme will be available. Tips for temperature experiments: Be sure to bring both enzyme and substrate to the desired temperature BEFORE mixing them together. Tips for inhibitor experiment: Be sure that all tubes contain the same total volume of sample. While varying volume of inhibitor, adjust total volume added to cach tube with pH 7 buffer.