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Catechol Oxidase Reaction in Apples

By Ashalee Joseph and Sean Suppo

Due: Thursday 3/13/2014

Lab Instructor: Professor Dunn

“This experiment was developed as a collaboration between Ashalee and Sean”

Conclusion

Hypothesis

The rate of the reaction will increase as temperature increases. Based on the experimental plan we expect the rate of reaction to increase as temperature increases. We expect our hypothesis to be true and be proven by the experiment. We expect that as the temperature increases the rate of reaction will increase up to a point where the reaction cannot go any faster.Temperature plays a role in why the rate of reaction increases. Molecules are colliding faster so the product is being produced faster.

From this experiment we learned different ways to deal with catechol oxidase. Research helped and it helped us make a decision on which way we wanted to go. We decided to work with temperatures. The 40 degree water bath offered the smallest standard deviation and 20 degree had the highest.

Experimental Design

1. Obtain Apple solution by mixing apples, water and the phosphate buffer in a blender

2. Measure out 30g of apples

3. Add phosphate buffer ( 2.5ml per gram of apples)

4. Place in blender, mix well but do not overgrind

5. Pour ending mixture into a funnel with cheesecloth lined around it

6. The liquid obtained will be used as the catechol oxidase

7. Keep extract cold on ice and also covered

8. Heat the solutions in the tube in a waterbath, increase A at 20 degrees, B at 40 degrees, and C at 60 degrees

9. Pipette the extract into cuvettes labeled A B C

Tube A: 2ml phosphate buffer, 0.20ml water, .22ml extract, .28ml 10mM Catechol

Tube B: 2ml phosphate buffer, 0.20ml water, .22ml extract, .28ml 10mM Catechol

Tube C: 2ml phosphate buffer, 0.20ml water, .22ml extract, .28ml 10mM Catechol

10. Shake the cuvettes well to mix

11. Leave the solution out and record observations

12. Use a spectrometer to record the rate of absorption

13. Record rates, mean, and standard deviation

Heating our solutions is

what help us address our

hypothesis. Our data was

collected by using a

spectrometer and we

recorded our results

after a minute, 10 times

each

References

Biology 1: Principles and Themes - Lab 2. (n.d.). Retrieved from http://umanitoba.ca/Biology/BIOL1020/lab2/biolab2_4.html

Catechol Catalysis. (n.d.). Retrieved from http://science.csumb.edu/~hkibak/241L_web/03_Catalysis/Catalysis_01.html

Catechol oxidase - Wikipedia, the free encyclopedia. (n.d.). Retrieved February 28, 2014, from http://en.wikipedia.org/wiki/Catechol_oxidase

Results

Introduction

  • Catechol is present in small quantities in the vacuoles of cells of many plant tissues. Catechol oxidase is present in the cell cytoplasm. If the plant tissues are damaged, the catechol is released and the enzyme converts the catechol to ortho-quinone, which is a natural antiseptic. Catechol oxidase, therefore, has a role in plant defence mechanisms, helping to protect damaged plants against both bacterial and fungal disease.It has been suggested that the quantity of catechol oxidase produced by a plant may be related to its susceptibility to fungal infection. Benzoquinone inhibits the growth of microorganisms and prevents damaged fruit from rotting. Catechol Oxidase is a plant enzyme that oxidizes catechol and converts it to benzoquinone (Wiki). In plants catechol is compartmentalized in the vacuole while the enzyme is in the cytoplasm. When a plant cell is damaged enough to rupture the vacuole, catechol and catechol oxidase come into contact and the reaction proceeds. Benzoquinone is toxic to bacteria and therefore prevents decay in wounded plant tissues(http://science.csumb.edu/). In experiment 2 we observed catechol oxidase in the presence of phosphate buffer and we observed that in the presence of the phosphate buffer the solution turned brown. We seen slight discoloration and the solution seemed to be murky. We recorded the absorbance over the time period by using spectrophotometer. Spectrophotometer is the quantitative instrument used to measure the amount of light the sample itself absorbs.

20 degrees water bath

A .591 .700 .755 .804 .837 .883 .916 .963 .972 .1.003

B .330 .331 .338 .355 .350 .351 .357 .372 .377 .383

C .807 .967 1.044 1.089 1.115 1.140 1.150 1.156 1.194 1.206

Mean .576 .666 .706 .743 .767 .791 .823 .830 .847 .864

S.D. .195 .261 .289 .311 .316 .329 .333 .334 .345 .350

40 degrees water bath

A .719 .851 .937 1.030 1.050 1.100 1.137 1.152 1.184 1.189

B .677 .781 .878 .947 .989 1.018 1.052 1.075 1.102 1.136

C .741 .865 .949 1.006 1.044 1.100 1.104 1.140 1.174 1.195

Mean .712 .832 .921 .994 1.03 1.07 1.097 1.12 1.16 1.21

S.D. .027 .037 .031 .035 .027 .039 .035 .034 .037 .032

60 degrees water bath

A .696 .803 .904 .924 .967 .987 1.034 1.072 1.089 1.120

B .730 .847 .934 .973 1.036 1.048 1.081 1.111 1.141 1.161

C .955 1.102 1.174 1.221 1.297 1.304 1.329 1.371 1.391 1.400

Mean .794 .917 1.004 1.039 1.100 1.113 1.148 1.185 1.205 1.227

S.D. .115 .132 .121 .130 .142 .137 .129 .133 .132 .137

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