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LAB 2 Enzyme Catalysis

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

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  1. LAB 2Enzyme Catalysis

  2. 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)

  3. 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/

  4. Other molecules with porphyrin rings Hemoglobin Chlorophyll http://drvsrs.com/spimgs/HEMOGLOBIN.jpg http://www.fkp.jku.at/fkp/forschung/PorSil.aspx

  5. 2A Watch for bubbles • Repeat with boiled enzyme • Repeat with diced potato http://www.nealbrownstudio.com/adm/photo/163_nb_fried_egg.jpg

  6. 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

  7. 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

  8. 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

  9. 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

  10. µmolesH2O2 TIME (seconds)

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