Maranan, Yvonne R.

October 2, 2018
EXPERIMENT No. 4
ENZYME KINETICS AND INHIBITION

A. Isolation of Polyphenol Oxidase

Cool 0.200 M
phosphate
buffer in an
Filter
icebath.
homogenate Collect extract
using on a beaker.
cheesecloth.
Wear your gloves
then peel and finely
chop the potato. Homogenize
Weigh a 15.0 g using a blender Cover with
sample on a clean at a high speed aluminum foil.
dry watch glass. for 30 seconds.

Add 40.0 mL of ice-cold Cool the sample

Transfer to a phosphate buffer and in an icebath
blender. 10.0 mL of 0.100 M NaF thtoughout the
on your sample. experiment.

B. Enzyme Kinetics of Uninhibited Reaction

Prepare the following solutions for analysis of the uninhibited reaction.

Test Tube 0.100 M Catechol 0.200 M Phosphate buffer

1 0.20 mL 2.6 mL
2 0.40 mL 2.4 mL
3 0.80 mL 2.0 mL
4 1.60 mL 1.20 mL
5 2.00 mL 0.80 mL
 Shake vigoruously fo 5
Add 0.20 mL of the seconds.
extracted enzyme to DO NOT ADD THE
tube No. 1 ENZYME YET TO OTHER
TEST TUBES!

Read absorbance
Repeat the above
immediately at 420 nm
procedure one at a
at 5 seconds interval
time for the remaining
against phosphate
tubes.
buffer until 1 minute.

C. Enzyme Inhibition

0.100 M Ascorbic 0.200 M

Test Tube 0.100 M Catechol Acid Phosphatee Buffer
1 0.20 mL 0.20 mL 2.4 mL
2 0.40 mL 0.20 mL 2.2 mL
3 0.80 mL 0.20 mL 1.80 mL
4 1.60 mL 0.20 mL 1.00 mL
5 2.00 mL 0.20 mL 0.60 mL

Repeat procedures
No. 2 to 4 as in B using
your samples in this
section