Some factors affecting polyphenol oxidase activity Lab 2 Some factors affecting polyphenol oxidase activity ARWA AL- KHYYAT .KSU.BIOCHEMESTRY

ARWA AL- KHYYAT .KSU.BIOCHEMESTRY Enzyme kinetics Enzyme kinetics is the study of the chemical reactions that are catalyzed by enzymes. In enzyme kinetics the reaction rate is measured and the effects of varying the conditions of the reaction investigated. ARWA AL- KHYYAT .KSU.BIOCHEMESTRY

Factors affecting enzyme action There are many factors which effects the rate of enzyme reaction: 1- Temperature 2- pH 3- Activators' and Inhibitors 4- Time 5- Substrate concentration 6- Enzyme concentration ARWA AL- KHYYAT .KSU.BIOCHEMESTRY

ARWA AL- KHYYAT .KSU.BIOCHEMESTRY 1- Temperature Low activity Optimum Tm: is that Tm at which the activity of the enzyme is maximum ARWA AL- KHYYAT .KSU.BIOCHEMESTRY

ARWA AL- KHYYAT .KSU.BIOCHEMESTRY 2- pH The rate of enzymatic reaction also depends on pH of the medium (rang 4-9) The enzyme activity is maximum at a particular pH which called its optimum pH ARWA AL- KHYYAT .KSU.BIOCHEMESTRY

ARWA AL- KHYYAT .KSU.BIOCHEMESTRY 3- Time 4- activators' and Inhibitors Enzyme inhibitors are molecules that bind to enzymes and decrease their activity Enzyme activators bind to enzymes and increase their enzymatic activity. ARWA AL- KHYYAT .KSU.BIOCHEMESTRY

ARWA AL- KHYYAT .KSU.BIOCHEMESTRY 5- Enzyme Concentration 6- Substrate Concentration ARWA AL- KHYYAT .KSU.BIOCHEMESTRY

ARWA AL- KHYYAT .KSU.BIOCHEMESTRY Enzyme activity: Amount of reaction that a certain amount of enzyme will produce in a specified period of time Activity determined by measuring the amount of product formed or substrate that disappeared IU of enzyme activity is – The amount of enzyme necessary to produce 1 μmole of product (or the loss of 1 μmol of substrate) per minute under specified conditions of substrate concentration, pH and Temperature ARWA AL- KHYYAT .KSU.BIOCHEMESTRY

polyphenol oxidase activity Polyphenol oxidase (PPO) enzymes catalyzes the oxidation of di or tri-hydroxy phenols to produce o-quinones. One example of a substrate for these enzymes is catechol that consider as di- hydroxy phenols, hence the alternative name ‘catechol oxidases’ for these enzymes. It is copper containing enzyme. pH is 6.7 This oxidation reduction is accompanied by a color change that absorb by visible region of the spectrophotometer at 540nm This reaction commonly occurs in nature and accounts for the browning of potatoes ,apples, banana……. ARWA AL- KHYYAT .KSU.BIOCHEMESTRY

Medical Reports and Nutrition Information's Perhaps the greatest green tea benefit is the presence of polyphenol oxidase – an abundant supply of antioxidants. Antioxidants can be termed as the “true elixir” of life. The activity of antioxidants in tea is more than that in over 21 fruits and vegetables. One of the most encouraging recent studies, published in the September 13, 2006 Journal of the American Medical Association, found that among 40,530 Japanese adults followed for 11 years, there was a 16 percent lower risk of death from all causes among those who drank more than 5 cups of green tea a day compared to those who drank less than one cup daily. ARWA AL- KHYYAT .KSU.BIOCHEMESTRY

ARWA AL- KHYYAT .KSU.BIOCHEMESTRY Objectives: 1- To demonstrate activity of the enzyme polyphenol oxidase in crude extract prepared from potato. 2- To demonstrate the chemical nature of the enzyme. 3- To investigate the substrate specificity of the enzyme. 4- To investigate the effects of various temperatures on the activity of the enzyme. ARWA AL- KHYYAT .KSU.BIOCHEMESTRY

ARWA AL- KHYYAT .KSU.BIOCHEMESTRY Experiments TEST TUBE ENZYMIC ACTIVITY: Label three clean test tubes A, B and C. Prepare each tube as follows: TUBE A: 15 drops of enzyme extract. 15 drops of 0.01M catechol solution. TUBE B: 15 drops of enzyme extract. 15 drops of distilled water. TUBE C: 15 drops of 0.0M catechol solution. c. Place all three tubes in a water bath at 37 ºC. d. Shake each tube every 5 minutes to aerate, thereby adding oxygen to the solution. e. Every 5 minutes, after shaking, hold the tubes up to the light and examine. Record the color in each tube, according to the scheme described in this page, in the table in the "Results" section. Continue for 25 minutes. ARWA AL- KHYYAT .KSU.BIOCHEMESTRY

Incubation time (minutes) Results: 1-TEST TUBE ENZYMIC ACTIVITY Incubation time (minutes) Degree of color intensity (Symbol: −, +, ++ or +++) TUBE A TUBE B TUBE C 5 10 15 20 25 ARWA AL- KHYYAT .KSU.BIOCHEMESTRY

ARWA AL- KHYYAT .KSU.BIOCHEMESTRY CHEMICAL NATURE OF POLYPHENOL OXIDASE: a. Label four clean test tubes A, B, C and D. b. Prepare, and teat, each tube as follows: TUBE A: Add 15 drops of enzyme extract. Add 15 drops of 0.01M catechol solution. Shake tube and place in water bath at 37 ºC for 10 minutes. Set tube aside as control with which to compare results of tubes B, C and D. TUBE B: Add 10 drops of enzyme extract. Add 10 drops of 5% trypsin solution. Shake tube thoroughly. Place tube in a water bath at 37 ºC for 10 minutes. Add 10 drops of 0.01M catechol solution. Replace in the same water bath for 10 minutes. Examine and compare with tube A. Record your observations in the table in the "Results" section. ARWA AL- KHYYAT .KSU.BIOCHEMESTRY

ARWA AL- KHYYAT .KSU.BIOCHEMESTRY TUBE C: Add 10 drops of enzyme extract. Add 10 drops of 5% trichloroacetic acid. Shake tube thoroughly and wait 5 minutes. Add 10 drops of 0.01M catechol solution. Place tube in water bath at 37 ºC for 10 minutes. Examine and compare with tube A. Record your observations in the table in the "Results" section. TUBE D: Add 15 drops of enzyme extract. Add a few crystals of phenylthiourea. Shake tube thoroughly and continue shaking it frequently during a period of 5 minutes. Then add 15 drops of 0.01M catechol solution. ARWA AL- KHYYAT .KSU.BIOCHEMESTRY

Degree of color intensity (Symbol: −, +, ++ or +++) 2-CHEMICAL NATURE OF POLYPHENOL OXIDASE Tube Treatment Degree of color intensity (Symbol: −, +, ++ or +++) A Control B Trypsin C TCA D Phenylthiourea ARWA AL- KHYYAT .KSU.BIOCHEMESTRY

ARWA AL- KHYYAT .KSU.BIOCHEMESTRY SUBSTRATE SPECIFICITY: a. Label three clean test tubes A, B and C. b. Add 15 drops of enzyme extract t oeach tube. c. Prepare each tube as follows: TUBE A: Add 15 drops of 0.01M catechol solution. TUBE B: Add 15 drops of 0.01M phenol solution. TUBE C: Add 15 drops of 0.01M hydroquinone solution. d. Shake the tubes gently and place them in a water bath at 37 ºC. e. Examine the tubes after 5 minutes and after 10 minutes. Record the color in each tube, according to the scheme described on the page 3, in the table in the "Results" section. The three compounds used as substrates in this part of the experiment are structurally related, as shown in the figure below. Each is capable of reacting with oxygen to form various colored products. ARWA AL- KHYYAT .KSU.BIOCHEMESTRY

3-SUBSTRATE SPECIFICITY Degree of color intensity (Symbol: −, +, ++ or +++) 5 minutes 10 minutes Catechol Phenol Hydroquinone ARWA AL- KHYYAT .KSU.BIOCHEMESTRY

ARWA AL- KHYYAT .KSU.BIOCHEMESTRY TEMPERATURE AND ENZYMIC ACTIVTIY: Label three clean test tubes A, B and C. Add 15 drops of enzyme extract to each tube. Place each tube containing the enzyme extract, in a water bath for 10 minutes at the following temperatures: TUBE A: 0 ºC (in a container of crushed ice). TUBE B: 37 ºC. TUBE C: 70 ºC. Add 15 drops of 0.01M catechol solution to each tube. Shake each tube gently and quickly return it to its proper temperature condition. Wait for 15 minutes. After this time, examine each tube, without removing it from its temperature condition, and record the color in each tube, according to the scheme described on page 3, in the table in the "Results" section. ARWA AL- KHYYAT .KSU.BIOCHEMESTRY

4-TEMPERATURE AND ENZYMIC ACTIVTIY Temperature (ºC) Degree of color intensity (Symbol: −, +, ++ or +++) 37 70 ARWA AL- KHYYAT .KSU.BIOCHEMESTRY

ARWA AL- KHYYAT .KSU.BIOCHEMESTRY References: Paum, S.J. and Bowen, W.R. (1372), Exercises in organic and biological chemistry, The Macmillan Company, 566 Third Avenue, New York 10022. Carl A,B and Edward R,A.(1996),Titaz Fundamentals of Clinicak Chemistry, W.B.Saunders Company,4th Edition,USA. Chatterjea, M, N and Shinde, R.(2007), Medical Biochemistry, Jaypee Brothers Medical Publishers(P) Ltd, 7th Edition,New Delhi110 002. ARWA AL- KHYYAT .KSU.BIOCHEMESTRY