Polyphenol Oxidase from Grapes
One enzyme that can be purified in a short time and is easily available is the polyphenol oxidase from fruit. This enzyme catalyzes the oxidation of diphenols to quinones in the presence of oxygen. The standard assay method relies on observing the colored quinone. However, since the phenol is oxidized, O2 is reduced, and we could also measure the rate of production of superoxide or hydrogen peroxide. It might be interesting to compare them.
Protocol. Preparation of 25% Triton X-114: Add 10 g of Triton and 16 mg butylated hydroxytoluene (this will probably not all dissolve) to 490 mL 0.1 M NaPi, pH 7.3. Dissolve at 0o. Incubate at 30o (this is most efficiently done in some kind of water bath) until the detergent aggregates and the mixture separates into two phases. The larger, aqueous phase is depleted of detergent, and the smaller phase is detergent rich. Discard the aqueous phase and replace with the same amount of phosphate buffer, mix at 0o, and allow to separate again at 30o. Repeat once more. The third condensed phase is 25% Triton. You should have about 40 mL of this solution (why?). If you don't you may have done something wrong.
Unless otherwise indicated, all steps are carried out in the cold and the solutions are maintained at 4o or below. Add 50 g grapes (fresh or frozen) to 25 mL grinding buffer (0.1 M NaPi, 10 mM ascorbate, pH 7.3) and homogenize for 15 s. Filter through 8 layers of cheesecloth and centrifuge the filtrate at 4000 g for 15 min. The pellet is resuspended in 20 mL 0.1 M NaPi, pH 7.3, containing 1.5% Triton X-114, and incubated on ice for 30 min. Centrifuge at 30,000 g for 30 min. Carefully pour off the supernatant and bring it to a final concentration of 4% Triton X-114 by the addition of 25% Triton X-114. Incubate on ice for 15 min, and then warm to 35o for 10 min (the solution must be at 35o for 10 min - a water bath of some kind will speed up this step), or until the solution becomes cloudy, due to aggregation of the detergent. Triton X-114 at this concentration is not stable in aqueous solution and forms two phases at 35o. The upper phase has very little detergent in it. The lower phase is about 25% detergent. The enzyme should be in the upper phase. As you carry out this procedure, save small samples of every fraction (as in Cell & Molec, remember to record the volume of each fraction, as well). It will be important to look for enzyme activity in each of them.
Enzyme assay. PPO catalyzes the oxidation of o-diphenols to quinones. Of course, the activity of the enzyme differs for different substrates. We have both 4-methylcatechol and t-butylcatechol. The quinones absorb at 400 nm, so activity is measured by following the absorbance increase at that wavelength. The extinction coefficient will be determined by allowing a sample of the catechol of known (low) concentration to oxidize completely (overnight in the dark, for instance, with the enzyme present; or you can add a strong oxidant, like periodate). The assay mixture consists of 50 mM acetate buffer, pH 5.0, about 2-20 mM catechol (you will need to make sure the enzyme is saturated), and a sample of enzyme. The volume of the assay mixture should remain fixed for all assays. Prior to assay, you must adjust the pH of the enzyme sample to 5.0 by the addition of 0.1 M acetic acid. (It is possible to adjust the pH by adding the enzyme to the assay buffer and allowing it to sit for a predetermined length of time before adding the substrate to start the reaction.) Specific activity is usually reported per mg of protein or per mg of chlorophyll.
Kinetic parameters. This refers to Km and Vmax. The velocity should be given in units of mol (or mmol or mmol ...) per unit time per unit enzyme (this could be a volume unit, it would preferably be a mass unit). Inhibitor studies are also typically done. Cinnamic acid is a typical inhibitor of PPO. Other inhibitors of this enzyme include ascorbic acid and metabisulfite, both of which are pretty good reducing agents. Inhibitors of this enzyme are of some importance in the wine industry, to prevent the production of unpleasant flavors and odors. Of course, the toxicity of anything added to a food product is of concern. With these reducing agents (cysteine could also be tried), it might be interesting to see if there is any difference when the agent is added to the enzyme before the substrate (preincubation), as compared to having it present only during reaction.
Enzyme activation. Many enzymes exhibit latent activity. This means that some chemical/physical change is required for their activity. PPO is normal activated by lowering the pH to 5. What is seen when the enzyme is added without having lowered the pH initially? Other treatments known to affect this enzyme’s activity are trypsin or detergent treatment. When these alternate treatments are used for activation, the pH is left at 7.3.