Bio report

The effect of temperature on the benzoquinone production of catechol oxidase

Fanny Falcon, Ollie Burrowing Owl, Pete Prairie

BIOL1106.015

21 December 2012

Introduction:

Enzymes are biological catalysts that speed up chemical reactions. The reaction studied in this report is

catechol + oxygen - >> - benzoquinone + water. The enzyme involved is catechol oxidase. This is a

common reaction in fruit and vegetables that darken when cut. The product benzoquinone is dark. In

general, temperature increases enzyme activity because the molecules are moving more. The question

studied was ‘What effect does temperature have on catechol oxidase activity?’. The hypothesis tested

was ‘Catechol oxidase activity will increase with an increase in temperature’.

Materials and Methods:

Ten 13x100 test tubes were prepared following the mixing table in the instructions. 1mL of catechol

oxidase from prepared potato extract was added to each test tube. All tubes were placed in their

respective temperature baths for 5 minutes: 1&2 – 0C (ice), 3&4 – 27C (room temp), 5&6 – 40C water

bath, 7&8 – 70C water bath, 9&10 – 100C boiling water bath. All tubes were left in their temperatures

while 1mL of water was added to the odd tubes (1, 3, 5, 7, 9) and 1mL of catechol was added to the even

tubes (2, 4, 6, 8, 10). A square of Parafilm was used to cover and seal each tube. All tubes were inverted

to mix contents. All tubes remained in their temperatures for 10 more minutes. After time was finished,

each odd number was blanked on a spectrophotometer set at 540nm. The corresponding even number

was read. Absorbance readings for the even tubes were recorded. Tubes were inverted and wiped

down before inserting into the spectrophotometer. Tube 1 was blanked and tube 2 was read. Tube 3

was blanked and tube 4 was read. Tube 5 was blanked and tube 6 was read. Tube 7 was blanked and

tube 8 was read. Tube 9 was blanked and tube 10 was read.

Results

The absorbance numbers ranged from -0.16A at 100C and 2.38A at 40C. The lowest reading are negative

in the highest temperatures 70C and 100C. On a spectrophotometer, any time the sample is more clear

or lighter than the blank, the sample is read as negative absorbance. The darkest tube with the highest

reading was on the 40C tube. The absorbance readings changed with the different temperatures.

Raw data:

Tube Temperature Tube Contents Absorbance at 540 nm

2 0oC Ice Catechol oxidase & catechol Enzyme & Substrate 1.54

4 27oC Room Catechol oxidase & catechol Enzyme & Substrate 1.04

6 40oC Warm Catechol oxidase & catechol Enzyme & Substrate 2.39

8 70 oC Hot Catechol oxidase & catechol Enzyme & Substrate -0.02

10 100 oC Boiling Catechol oxidase & catechol Enzyme & Substrate -0.16

Chart:

-0.5

0

0.5

1

1.5

2

2.5

3

0 27 40 70 100

A b

so rb

an ce

a t

5 4

0 n

m

The effect of temperature on catechol oxidase activity

Temperature C

Discussion

The hypothesis tested was ‘Catechol oxidase activity will increase with an increase in temperature’. The

data only supports the hypothesis in two readings 1.04A at 27C and 2.39A at 40C. None of the other

data supports the hypothesis. In general the data does not support the hypothesis. In repeating this

experiment, I would more quickly blank and read the tubes at the cold temperatures. I think the ice

tubes warmed up in my hands while waiting to use the spectrophotometer. I would do more repetitions

at each temperature and more temperatures. The change in absorbance between 40C with 2.39A and

70C with -0.02A is dramatic. I wonder what it would be like at every 5 or 10 degrees in between. I would

change the hypothesis. The higher temperatures denatured the enzymes and caused it to fall out of

solution. Each tube has a greyish precipitate at the bottom of the tube. The tube overall was a light

color showing none of the dark benzoquinone product seen in the cooler tubes. I would change the

hypothesis to ‘Catechol oxidase activity increases with temperature until extreme temperature

denatures the enzyme’. In conclusion, catechol oxidase activity varies with temperature.