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LAB 2 Enzyme Catalysis. H 2 O 2 HYDROGEN PEROXIDE. 2 H 2 O 2 → 2 H 2 O + O 2. In aerobic organisms Toxic byproduct of metabolism Strong oxidizer (grabs electrons from other molecules). Substrate Products.

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Lab 2 enzyme catalysis

LAB 2Enzyme Catalysis


H 2 o 2 hydrogen peroxide
H2O2 HYDROGEN PEROXIDE

2 H2O2 → 2 H2O + O2

  • In aerobic organisms

  • Toxic byproduct of metabolism

  • Strong oxidizer

  • (grabs electrons from other molecules)

Substrate Products

Reaction happens spontaneously (slowly)


Catalase
Catalase

ENZYME

Speeds up reaction to break down hydrogen peroxide

Tetramer (4 polypeptide subunits)

Contains Porphyrin groups with iron cofactors = HEME

ANTI-OXIDANT

http://en.wikipedia.org/wiki/File:Catalase_Structure.png

http://semicrystalline.wordpress.com/2007/05/


Other molecules with porphyrin rings
Other molecules with porphyrin rings

Hemoglobin

Chlorophyll

http://drvsrs.com/spimgs/HEMOGLOBIN.jpg

http://www.fkp.jku.at/fkp/forschung/PorSil.aspx


2A

Watch for bubbles

  • Repeat with boiled enzyme

  • Repeat with diced potato

http://www.nealbrownstudio.com/adm/photo/163_nb_fried_egg.jpg


2B

DETERMINE BASELINE AMOUNT of H2O2

+10 mL H2O2

+1 mL H2O

+10 mL H2SO4

REMOVE A 5 mL SAMPLE

Assay for Hydrogen Peroxide

http://www.nealbrownstudio.com/adm/photo/163_nb_fried_egg.jpg


ASSAY for presence of H2O2

5 H2O2 + 2 KMnO4 + 3 H2SO4 → K2SO4 + 2 MnSO4 + 8 H2O + 5 O2

Potassium permanganate (KMnO4 ) = Purple

Add KMnO4 until solution stays a faint pink or brown

Syringe

Numbers runbackwards

BURET


2C

DETERMINE UNCATALYZED RATE of H2O2 DECOMPOSTION

15 mL H2O2LET SIT OVERNIGHT

Use 10 mL (overnight) H2O2

ADD 1 mL waterADD 10 mL sulfuric acid (H2SO4)

ASSAY for H2O2

REMOVE A 5 mL SAMPLE


2D

Enzyme Catalyzed Rate of H2O2 Decomposition

360 sec

180 sec

60 sec

90 sec

120 sec

30 sec

RedoBaseline

10 sec

+ 10 mL H2O2 to each beaker

For each time point+ 1 mL Catalase (yeast) Swirl gentlyAt appropriate time: add sulfuric acid( H2SO4 )

Assay for H2O2


µmolesH2O2

TIME (seconds)


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