Analysis of Polyphenoloxidase Enzyme Activity from Potato Extract Biochemistry Lab I (CHEM 4401) Student Supplement Background (Bring

your Practical Buffer Preparation Supplement to lab) Enzymes are protein molecules (primarily) that serve as biological catalysts. They are responsible for the synthesis and degradation of lipids, amino acids, carbohydrates, proteins, vitamins, steroids, hormones, neurotransmitters, nucleic acids, polysaccharides and all their metabolic intermediates. Enzymes are able to perform their functions by binding to reactants in a very specific manner, straining them to increase their reactivity and providing the chemical environment necessary to allow the reaction to proceed quickly and efficiently. The rate at which enzymes can catalyze particular reactions can be truly astonishing. For example, a single catalase enzyme, which catalyzes the conversion of H2O2 (hydrogen peroxide) to H2O and O2, can perform this reaction on 40,000,000 molecules of H2O2 per second! In order to understand these biological transformations and how they are catalyzed we need to purify enzymes and study their activity. Enzyme activity concerns factors such as how fast the reaction is catalyzed, how strongly it binds its substrate, sensitivity of the catalysis to changes in pH, substrate or cofactor concentration, temperature or other variables. Today we will be performing a very crude isolation of the enzyme polyphenoloxidase from potato. This enzyme catalyzes the hydroxylation of phenolic compounds such as intermediates in amino acid synthesis or degradation pathways. It also catalyzes the oxidation of diphenol compounds, such as those that lead to the production of various melanin pigments. We will use 3,4-dihydroxyphenyalanine (DOPA) as the substrate for our reaction. Polyphenoloxidase will oxidize DOPA to dopachrome (figure 1), an orange -colored Oquinone that absorbs light at 475 nm (max). We will follow the production of dopachrome spectrophotometrically using several different initial concentrations of DOPA. Data from these reactions will be used to produce graphs (Michaelis-Menten, Lineweaver-Burke) in our next laboratory section. These graphs, in turn, will be used to estimate kinetic data on our enzyme, such as the maximum velocity at which it can catalyze the reaction (Vmax) and the affinity it has for the DOPA substrate (Km). We will also examine the effect of pH and temperature on enzymatic activity.
HO COOO COONH3+ OH O N H

DOPA Figure 1. Conversion of DOPA to dopachrome

dopachrome

Prep Sheet Materials and Reagents Potatoes Mortars & Pestles Cheesecloth 15 ml centrifuge tubes 15 ml snap-cap tubes 13 x 100 mm test tubes razor blades DOPA (L--3,4-dihydroxyphenylalanine)(20 mM) Phosphate buffer (0.1M, pH 6.8) Acetate buffer (0.1M, pH 4.0) Borate buffer (0.1M, pH 10) Potato peelers aluminum foil

per Group 10 g 1 4 in2 2 1 7 1 10 ml 40 ml 5 ml 5 ml 2 per section 6 in2

Modifications to Experimental Protocol in Laboratory Manual Preparation of Potato extract (Crude Polyphenoloxidase) 1. After weighing potato sample, mince with a razor blade on a paper towel. 2. Grind without sand 3. Line a small funnel with 4 in piece of cheesecloth. Scoop ground potato mash onto cheesecloth and strain into a 15 ml, screw-top centrifuge tube. Give to your instructor 4. Centrifuge only after all groups have prepared their extract. Use the S4180 rotor in the Beckman Allegra centrifuge. Be sure to indicate proper rotor in centrifuge controls. Spin at 3500 rpm 5. Pour supernatant (liquid phase) into a fresh 15 ml centrifuge tube. 6. Do not make this dilution. Use concentrated extract (supernatant from step 5) for assays in tubes 1-7
2

Determination of Specific Activity 1. As in manual 2. As in manual. 13 x 100 mm test tubes for cuvettes. Make up only 1 tube at a time for taking spec. readings. DO NOT MAKE UP ALL TUBES AT ONCE. 3. As in manual 4. As in manual 5. Each student group should keep 10 ml of DOPA in a foil-covered snap-cap tube at their bench. Wipe down test tube with a kimwipe after mixing. 6. As in manual. Have students record an absorbance reading of 0 for their zero time point. One student keeps time and records absorbance values. Lab partner calls out absorbance readings at 20 second intervals. 7. As in manual

Determination of pH and Temperature Inhibition on Specific Activity 1. As in manual 2. As in manual. Again, make up only 1 tube at a time. 3. Pipet 3.2 ml of buffer in tubes 5 and 6, and 3.0 ml buffer in tube 7. Ignore phosphate adjective, as we will be using three different buffers for tubes 5-7 4. As in manual 5. Use 0.6 ml DOPA (20 mM) in tubes 5 and 6, and 0.8 ml DOPA in tube 7. 6. As in manual 7. Repeat steps for tubes 6 & 7, we will not perform the assay on tube 8. Take 0o C phosphate buffer (pH 6.8) from stock on ice when needed. This will keep the buffer cold until just before the assay is to be run. Etc. 1. 2. 3. 4. 5. 6. Collect excess DOPA and enzyme assay waste in specified waste bottle Excess buffers, enzyme prep can go down the sink (Put potato waste in trash) Glass test tubes to glass waste. Plastic test tubes to trash. Be sure to also clean mortars & pestles. Check out with your TA before leaving. We will go over data prep in lab next week (supplement to be posted to web site). Be sure to read next weeks posted supplement and the Lab Report Expectations section in the lab manual carefully beforehand. You will be better prepared and able to perform your tasks that much more quickly if you do.