This experiment revolves around isolation of Polyphenol Oxidase (PPO) and the determination of this enzyme’s optimal pH and temperature. Polyphenol Oxidase (PPO) is an enzyme that catalyses the oxidation of phenols to quinones upon exposure to oxygen. The quinones are converted to dark pigments (black, brown or red) which results in enzymatic discolouration thereby leading to browning of plant materials such as in apples and pears. This results in negative effects on appeal, nutritional value, taste, colour and flavour. An example of a browning apple is shown below:
PPO is a tetramer containing four Copper atoms per molecule, which is the binding site for oxygen and two aromatic compounds. PPO is an oxygen transferring enzyme which is inactivated by increasing the concentration of Copper (II) ions. In addition, the Worthington Enzyme manual states that the Molecular weight of PPO is 128000 Da.
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The aim of this experiment is to isolate Polyphenol Oxidase (PPO) from a frozen, cut pear sample and determine its protein content using the Bradford method and then eventually, determining the optimum pH and temperature of this enzyme using 4-methylcatechol substrate exposed to differing pH and temperatures respectively. The enzyme is present within the pear cells. To access the enzyme, blending and centrifuging in the presence of an extraction buffer (Trizma with Triton and Polyvinyl Pyrrolidone) was carried out (at low temperatures so enzymes are inactive) to ensure the disruption of cell membranes gaining access to the enzyme. Furthermore, protein concentration determination was carried out using Bradford reagent, Bovine Serum Albumin (BSA) and Comassie Blue dye allowing the visualisation of the proteins. Bradford standards were calculated using the equation C1V1=C2V2, where C=concentration and V=volume. Finally, to determine the optimum pH and optimum temperature, 4-methylcatechol substrate was used and exposed to different pH and temperatures. The amount of product formed was determined by using the absorbance coefficient, 4-methylquinone. Hydrochloric acid was used to stop the reaction since this is a strong acid and would therefore denature the enzyme. All steps were incubated in a water bath for at least 5 minutes to allow all reagents to reach the required temperature uniformly since this is a temperature driven reaction. The overall experiment was carried out over 5 weeks.
Based on Graph 1, the equation of the linear plot is y = 0.0003x+0.049, where y = Absorbance and x=Protein concentration of BSA standards (ug/ml). When the experiment was carried out, the enzyme reaction mixture had absorbance values of 0.907 and 0.725, thereby giving an average absorbance value of 0.8160, from which the blank of 0.6645 is deducted giving 0.1515. If this value is substituted into the equation above, the protein concentration of PPO may be obtained:
y = 0.0003x+0.049
0.1515= 0.0003x+0.049
x = (0.1515-0.049)/0.0003
x = protein concentration= 341.7 ug/ml
If extrapolated from Graph 1 above, a protein concentration value of about 310 ug/ml is obtained, therefore it can be suggested that the protein concentration of Polyphenol Oxidase in the pear sample lies between 310 – 341.7 ug/ml.
Secondly, from Graph 2, it can be seen that the PPO activity remained fairly constant and high until 25°C, when there was a sudden drop until 35°C and then activity started to rapidly increase again. The optimum temperature of Polyphenol Oxidase is about 30°C (CP, et al., 2000), however from the graph above, the optimum temperature seems to be about 25°C because this is the point where maximum activity was observed and then after his point, activity started to decrease. It can be seen that activity is fairly high from 0-25°C. Even though there may be no trend between these values, the high activity could be due to the fact that as temperature increases, the reactant molecules are supplied with more kinetic energy, thus collide with each other more often and therefore increase the chances of a reaction taking place. However, above this point, the activity decreases steeply until 35°C, this is probably due to this temperature being too high resulting in the breakdown of hydrogen bonds between molecules thereby denaturing the enzyme which cannot catalyse the reaction anymore. An odd situation occurs after 35°C where the activity starts to rapidly increase again and reaches a steady level which shows that maybe for this enzyme, the denaturing may be reversed at higher temperatures or it could be due to experimental errors. The slight difference in expected versus actual outcome of optimum temperature may be due to errors made during the experiment such as the individual reaction mixtures may not have been kept in respective water baths for as long as 5 minutes leading to lower temperatures achieved than required.
Thirdly, as observed from Graph 3, PPO activity increased steadily from pH 3.0 to about 5.5, after which, activity decreased slightly until pH 6.0 and began to rise again until pH 7.0. The optimum pH for Polyphenol Oxidase is 6.0 to 7.0. However, from the graph above, the peak performance occurs at pH 5.5 which is close to the range given but slightly lower, this may be due to experimental errors such as not cleaning the well plate sufficiently thereby obscuring the measurement of absorbance. In the experiment done, a faulty pipette was used which gave differing volumes of liquids in each test tube of the reaction mixture. pH is a measure of hydrogen ion concentration, changes in the concentration above and below optimum pH results in disruption of ionic bonds causing the enzyme to denature thereby hindering its activity in catalysing reactions. The sudden rise in the graph from pH 6.0-7.0 could be as a result of the enzyme showcasing its actual optimum pH rather than the experimental peak at pH 5.5.
In conclusion, the aims of the experiment were achieved, the isolation of Polyphenol Oxidase was successfully done, after which the optimum pH and optimum temperature were determined as 5.5 and 25°C respectively. The protein concentration of Polyphenol Oxidase in the pear sample lies between 310 – 341.7 ug/ml. Therefore, we could successfully say that Polyphenol Oxidase is present within pears and are the main cause of the browning of fruits.
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