ENZYME KINETICS

I. Objectives
 Study the kinetic activity of the o-diphenol oxidase on catechol substrate obtained from banana
 Assess the type of inhibition done by p-hydroxybenzoic acid on the enzyme

II. Figure and set ups

Lakatan Blender Uv vis

III. MSDS
Reagent Physical Properties Hazards and safety
catechol Molecular Weight: 110.11 Corrosive, causes burns.
Boiling Point: 245 °C Harmful by inhalation,
Solubility in Water: Soluble ingestion and through skin
Appearance and Odor: Clear, absorption. Readily
colorless crystals with odor of absorbed through skin. Very
phenolph destructive of mucous
membranes and upper
respiratory tract, eyes and
skin.

p-hydroxybenzoic Physical state and appearance: Solid. Hazardous in case of eye

acid Molecular Weight: 138.12 g/mole contact (irritant). Slightly
Color: White. Off-white. hazardous in case of skin
contact (irritant), of
ingestion, of inhalation.

IV. Schematic diagram of Procedure

Sample Preparation
 Peel the fruits and cut into small pieces
 Weigh 50g + 5mL 0.1M phosphate buffer pH 7
 Homogenize sample
 Filter the crude extract through cheesecloth into E flask in ice bath
 Collect filtrate. Centrifuge for 10 mins or until clear
 Collect supernatant and maintain in ice bath

Determination of Km and V max

 Prepare 50ml 4.8mM stock catechol solution in 0.1Mphosphate buffer pH7
 Set up 4 10mL solutions of catechol diluted with phosphate buffer with the following
concentrations: (do twice for part III)
Solution A = 4.8 mM

Enzyme Kinetics
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 B = 1.2 mM
C = 0.6 mM
D = 0.3 mM
 Use mixture of 1 ml of the sample extract and 2ml of the phosphate buffer as blank
 Mix 1 ml of the sample extract to 2 ml of solution a and immediately read the absorbance at 540
nm. Continue to record the absorbance at 20-sec intervals for 5 mins
 Repeat previous step using sol’n BCD

Inhibition Studies
 Prepare 0.3mM of p-hydrobenzoic acid and another set of solutions A-D
 Dispense 1 ml of the 0.3mM p-hydrobenzoic acid into each solution
 Repeat steps 3-5 of part II

V. Waste Disposal
 Collect all solid wastes –bin
 Mercaptoethanol – organic waste
 All other solution – sink with running water

VI. Calculations

Enzyme Kinetics
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