2.14B: Practical: Investigate How Enzyme Activity can be Affected by Changes in pH

INVESTIGATING EFFECT OF pH ON ENZYME ACTIVITY

Diagram Showing the Spots of Starch and Amylase in Wells

INDEPENDENT VARIABLE: pH of Buffer Solution (pH 2, 4, 6 , 8, 10, 12) - buffer solution maintains reaction mixture at a constant pH

METHOD: Test tube of Starch solution is placed in Water bath of set temperature (30 - 40°C as optimum temperature of Amylase is approximately 37°C) Solution of Amylase enzyme is added into test tube of Starch solution Immediately after, buffer solution of set pH is added which will maintain reaction mixture at set pH Pipette is used to add droplets of mixture into wells on tile A few drops of Iodine solution is added Process is repeated in increments of fixed time (seconds) until Iodine turns orange, indicating that Starch has completely broken down into Glucose Time taken for Starch to be completely broken down is compared to that of each respective pH (time taken for Iodine solution to turn orange with each respective pH of buffer solution)

RESULT: As pH increases towards optimum (pH 6.7 - 7.0), the rate of amylase activity increases as shown with the decrease in time taken for Iodine solution to turn orange (as pH increases towards optimum) This is because as pH increases, the shape and structure of the active site, and the charge of substrate molecule will both be optimal to allow Starch to bind to the active site of Amylase This allows more frequent successful enzyme-substrate collisions to occur, increasing the rate in which Starch is broken down into Glucose (which causes Iodine solution to turn orange quicker as Starch is not present), hence increasing enzyme activity However, as pH exceeds optimum, Amylase will denature as shown with the increase in time taken for Iodine solution to turn orange (as pH increases away from optimum) This is because the increase/decrease from optimum pH causes the shape and structure of active site to change, additionally changing the charge fo substrates, therefore diminishing the ability of Starch to bind to active site of Amylase Therefore, Amylase denatures and Starch cannot be broken down into Glucose (which causes Iodine solution to turn orange slower as Starch is present), hence decreasing enzyme activity