BIO131 Lab - Enzyme Action PDF

BIO131 LAB ENZYME ACTION Deeba S. Jairajpuri, PhD Department of Medical Biochemistry Metabolism ■ Metabolism: Chemical reactions in cells ■ Metabolism: Either Anabolic or Catabolic – Anabolic (Synthesis): substrates joined together to form products. Reactants (Substrates) Product – Catabolic (...

BIO131 LAB ENZYME ACTION Deeba S. Jairajpuri, PhD Department of Medical Biochemistry Metabolism ■ Metabolism: Chemical reactions in cells ■ Metabolism: Either Anabolic or Catabolic – Anabolic (Synthesis): substrates joined together to form products. Reactants (Substrates) Product – Catabolic (degradation/breakdown): substrates broken down to form products Reactant (Substrate) Products How does an enzyme work? ■ Enzymes are biological catalyst that speed up the chemical reactions in our body ■ Substrate: the substance which the enzyme acts upon ■ Active site: pocket-like structure on the enzyme surface where the substrate can bind enzyme enzyme enzyme enzyme Substrate-Enzyme complex substrate products Substrate-Enzyme complex Active sites Enzyme Enzyme Action ■ Enzymes activity known to be: 1. Specific. 2. Affected by temperature (less active in cold temp. & denatured in hot temp.) 3. Affected by change in concentration (Substrate & Enzyme) 4. Optimum pH. Enzyme action: is specific Catalase + H2O2 Catalase + HCL Catalase + NaOH Enzyme action: is affected by temperature Enzyme action: is affected by changes in enzyme and substrate concentration Enzyme action: is affected by changes in pH Lab experiments ■ You will carry out 5 experiments to assess the four important features of enzyme action (i.e. specificity, and effects of temperature, pH and substrate/enzyme concentration). ■ In your experiments you will use catalase as the enzyme, and H2O2 as the substrate. ■ Catalase is a common antioxidant enzyme produced naturally in almost all living organisms. ■ It helps the body to break down H2O2(harmful oxidizing agent) into oxygen and water ■ Catalase uses H2O2 to oxidize potentially harmful toxins in the body including formaldehyde, formic acid, alcohol, and phenol. 1. Enzyme specificity experiment ■ Mark three tubes at 0.5 & 1.0 cm level. ■ Fill them with H2O, sucrose and H2O2 to the 0.5 cm Mark. ■ Fill the tubes with catalase till 1.0 cm Mark. ■ measure the height of bubble column. 1.0 cm 0.5 cm H2O or Sucrose or H2O2 2. Temperature & enzyme activity experiment ■ Mark three tubes at 0.5 & 1.0 cm level. ■ Fill them with catalase to the 0.5 cm Mark. ■ Place the tubes in three temps: ice, 37ºC and 100 ºC. Wait for 10 mins. ■ Add H2O2 till 1.0 cm Mark. ■ measure the height of bubble column. 1.0 cm 0.5 cm 3. Enzyme concentration experiment ■ Mark one tube at 0.5 & 1.0 cm level. ■ Mark the second tube at 1.0 & 1.5 cm level ■ Mark the third tube at 1.5 & 2.0 cm level ■ Fill them with catalase to the first Mark. ■ Add H2O2 till the second Mark. ■ measure the height of bubble column. catalase catalase catalase 4. Substrate concentration experiment ■ Mark one tube at 0.5 & 1.0 cm level. ■ Mark the second tube at 1.0 & 1.5 cm level ■ Mark the third tube at 1.5 & 2.0 cm level ■ Fill them with H2O2 to the first Mark. ■ Add catalase till the second Mark. ■ measure the height of bubble column. H2O2 H2O2 H2O2 5. pH and enzyme activity experiment ■ Mark three tubes at 0.5 & 1.5 cm and 2.0 cm level. ■ Fill them with catalase to the 0.5 cm Mark. ■ Fill the tubes till the second mark with solutions of a pH: 3, 7 and 11. ■ Add H2O2 till the third Mark. ■ measure the height of bubble column. catalase catalase acidic catalase neutral Basic