Understanding Browning of Fruits and Vegetables by an Enzyme 2019

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Understanding Browning of Fruits and Vegetables by an Enzyme: Polyphenoloxidase in Potatoes Recall the following about enzymes: Enzymes act as biological catalysts. Almost all enzymes are proteins. The enzymatic reaction is catalyzed in a region of the enzyme called the active site. Enzyme activity is influenced by a number of factors:  enzyme concentration,  substrate concentration,  pH of the reaction,  temperature,  possibly cofactors, inhibitors and/or activators. Some enzymes are absolutely specific for their substrate: this means they will catalyze a reaction only on one substrate structure, whereas other enzymes may show “group specificity”, meaning that they can catalyze similar reactions with two or more substrates of related chemical structure (e.g. peptides, alcohols, etc). POLYPHENOLOXIDASE A number of fruits and vegetables contain an enzyme called polyphenoloxidase, which is responsible for the browning we observe when these fruits and vegetables are cut and exposed to the air, or bruised (e.g. raw potatoes, apples). This enzyme contains a metal cofactor, copper, and it can catalyze an oxidation reaction of dihydroxyphenols and trihydroxyphenols to give quinones. For example, if the starting substrate is catechol, the reaction equation would be as follows: You will work with an extract of raw potatoes which contains polyphenoloxidase, and by carrying out several experiments, you will test the effect of different variables on the observed extent of reaction, or rate of reaction. IMPORTANT: In all work with enzymes, it is very important to use very clean glassware, rinsed with distilled water. Enzymes can be easily denatured by leftover detergent on glassware, or inhibited by metal ions that are present in tap water. So keep this in mind as you prepare for and do your work.

POLYPHENOLOXIDASE EXPERIMENTS: For all the following experiments you are comparing reactions in 3 or 4 tubes. It is important to cooperate within your group: since you are basically comparing enzymatic reaction rates, you want all reactions to start pretty much at the same time, so add enzyme to all tubes quickly and keep track of what is being done (one experimentor, one scribe). Materials: 13x100 glass tubes, rack P1000 pipettors, tips Solutions: enzyme extract (on ice), 0.01M catechol, water, 0.01M phenol, 0.01M hydroquinone, 0.4M HCl, 0.1M lactic acid, 0.5M sodium carbonate I. THE BASIC REACTION OF POLYPHENOLOXIDASE Assemble the following reactions: Tube A Tube B Tube C 10 drops distilled water 10 drops 0.01M catechol 10 drops 0.01M catechol 10 drops enzyme extract 10 drops enzyme extract 10 drops distilled water  Gently shake tubes to mix well and place them in the 37 degree waterbath  Every 5 minutes, shake the tubes to aerate the contents. This adds oxygen  Examine the contents of the tubes every 5 minutes, by holding up to the light or against a sheet of white paper. Continue for a total of 15 minutes, or until tube B has a dark, deep red color. Record the intensity of the red color observed in all tubes, by using the notation +, ++, +++, -. Table 1: Time (min) Tube A Tube B Tube C 0 - ++ - 5 - ++ - 10 - ++ - 15 - ++ - Questions: 1) What do tubes A and C control for? Tube A was control for Enzyme extract, C was for 0.01M catechol. 2) Since the intensity of color is proportional to the concentration of product benzoquinone, what do you conclude about how the amount of benzoquinone changed over time for tube B? For our experiment over time the color did not change much which means the concentration stayed the same. 3) Did the reaction occur in tube C without enzyme? The reaction did not occur. 4) Did you observe some color change in tube A? Since A did not contain any catechol, what is your explanation for what you observed? Minimal color change in tube A not clear but no red color enzyme had minimal reaction with water.

5) In one sentence state your overall conclusion for what Part I has shown you. Catechol is reacting with enzyme extract to give us a red color.

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II. SUBSTRATE SPECIFICITY In this part of the experiment, you will be comparing the ability of polyphenoloxidase to catalyze the oxidation of three different phenol compounds: phenol, catechol, and hydroquinone. Each of these compounds can become oxidized to form colored compounds (red or brown) but you do not know whether polyphenoloxidase can act as a catalyst for all three reactions. This experiment should give you that information. The structures of the three phenol compounds are as follows: Assemble the following reactions, making sure that enzyme is added to the tubes at as closely the same time as possible: Tube A Tube B Tube C Tube D 15 drops phenol 15 drops catechol 15 drops hydroquinone 15 drops dist. Water 5 drops enzyme extract 5 drops enzyme extract 5 drops enzyme extract 5 drops enzyme extract  Shake each tube gently and place in the water bath at 37 degrees  Observe colors at the start (zero time) and then at 5 minutes and at 10 minutes. At each 5 minute interval, shake the tube briefly to aerate. Record your results in the table below, always comparing color development to tube D = no substrate control. Record the color intensity as above, using -, +, ++, +++ etc., for each time. Table 2: Time (min) Tube A Tube B Tube C Tube D 0 - ++ + - 5 + ++ + - 10 + +++ ++ - Questions: 5) Based on your results, were all substrates equivalent in the speed at which reaction was catalyzed? No because it did not change at same times. 6) Which substrate shows the fastest (“best”) reaction? B reacted the fastest. 7) Did any substrates appear NOT to react with polyphenoloxidase as the enzyme? Tube D did not react. Tube A did not untill after 5 mins. 8) Based on your results, would you consider that polyphenoloxidase shows group specificity or absolute specificity ? If your answer is “group”, what type of structure seems to be needed? Group, An alcohol group needs to be present.

III. EFFECT OF ENZYME CONCENTRATION Enzyme reactions such as the present one are carried out with the enzyme concentration much lower than the concentration of the substrate. Thus the rate of the reaction should be directly dependent on the amount of enzyme used (ie enzyme concentration). Assemble the following reactions, making sure that enzyme is added LAST to the tubes and at as closely the same time as possible: addition Tube A Tube B Tube C Tube D Distilled water 6 drops 3 drops 0 drops 8 drops 0.01M catechol 20 drops 20 drops 20 drops 20 drops Enzyme extract 2 drops 5 drops 8 drops 0 drops Incubate the tubes at 37 o C. Shake the tubes to mix them every 2-3 minutes. Examine color development at 5 minute intervals over 15 minutes. Record your results in the following table, using -, +, ++, +++ etc as before: Table 3 : Time (min) Tube A Tube B Tube C Tube D 0 5 + + ++ - 10 + + +++ - 15 ++ + ++ - Questions: 9) Does the rate of reaction show an increase with increasing enzyme concentration? Yes because tube C had more of enzyme extract and it developed the darkest color AND tube D had no enzyme extract. 10) In one sentence, state your overall conclusion for this part of the experiment. Higher amount of enzyme extract will have more color and a greater reaction. IV. EFFECT OF SUBSTRATE CONCENTRATION The rate of enzyme catalyzed reactions depends on the concentration of the substrate, but (as we discussed in lectures) this follows a hyperbolic shape. At low concentrations of the substrate, increasing the substrate concentration should result in an increased rate, but above a certain level, the rate no longer changes when substrate concentration is increased because the enzyme is “saturated” with substrate. We call this maximum rate for a given amount of enzyme the “ Vmax” . The purpose of this experiment is to see whether you can actually observe this effect. Notice that you are not actually observing the rate, but instead you are observing something related to the rate – at each time, you are observing the total amount of reaction that has occurred up to that time, by the intensity of the red color of the product of the reaction.

addition Tube A Tube B Tube C Tube D Distilled water 35 drops 14 drops 0 drops 7 drops 0.01M catechol 7 drops 28 drops 42 drops 42 drops Enzyme extract 7 drops 7 drops 7 drops 0 drops Assemble the following reactions: Place tubes in 37 degree waterbath Shake each tube every 2-3 minutes Examine tubes at 5 minute intervals, starting at “zero” time, for 20 minutes and record your results in the table below: use +, ++, +++ etc to denote increases in red color. Table 4: Time (min) Tube A Tube B Tube C Tube D 0 + + ++ - 5 + ++ ++ - 10 + ++ ++ - 15 + +++ ++ - 20 + +++ ++ - Questions: 11) Based on your results, do you see evidence that a Vmax was reached? Tube B reached the Vmax. 12) In one sentence, indicate your overall conclusion for this part of the experiment. Water and Catechol are the determinates the color change V. EFECT OF TEMPERATURE For most chemical reactions, an increase in temperature leads to an increase in the rate of the reaction: for every 10 degree increase in temperature, the rate approximately doubles. For many enzymes, however, this is only true at lower temperatures: higher temperatures can lead to denaturation of the enzyme and loss of activity. The temperature which shows the fastest rate is sometimes considered the “optimum temperature” but really, at this temperature some denaturation may already take place, so it is best to work with an enzyme below this temperature. Two water baths are supplied – at 37 degrees and 70 degrees C. You will prepare your own cold water bath with crushed ice and a little water mixed in a 150 mL beaker. This will be called 4degrees. 25 degrees will be the room temperature (ie no waterbath, just leave the tube on the benchtop). Assemble the following reactions: addition 2 degrees 25 degrees 37 degrees 70 degrees Enzyme extract 15 drops 15 drops 15 drops 15 drops Preincubate tubes at their respective temperatures for 5 minutes catechol 15 drops 15 drops 15 drops 15 drops 14 drops 0.01M catechol 0.01M catechol 14 drops 14 drops

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After adding the catechol, shake the tubes to mix and quickly return them to their respective waterbath; After 10 minutes examine the tubes for red color changes; don’t allow the 2 degree tube to warm up while examining it! Record results in table, using +, ++, +++ etc as above Table 5: Temperature, degrees C Extent of reaction or color change 2 - 25 ++ 37 +++ 70 ++ Questions: 13) Of the four temperatures examined, which one was optimal? C 14) Was this the result you expected? yes 15) Explain carefully what you think accounts for the differences you observed at the four temperatures. The temperature differences had an effect on the color. Materials per group:  tubes, glass, 13x100  Dist water: 10 ml  Enzyme extract: 10ml  Catechol: 20 ml  Phenol, hydroquinone: 1ml each  8 Plastic droppers

Understanding Browning of Fruits and Vegetables by an Enzyme 2019

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