Enzyme Lab

Enzyme: Phenolase Reaction catalyzedReaction catalyzed Catechol + ½ O > 0-benzoquinone Catechol + ½ O > 0-benzoquinone 0-benzoquinone + H 2 O---> 0-benzoquinone + H 2 O---> pink/gold melanin ---> ---> brown melanin pink/gold melanin ---> ---> brown melanin Reaction detected by change in Absorbance at 475 nm due to production of the pink/gold melaninReaction detected by change in Absorbance at 475 nm due to production of the pink/gold melanin phenolase

Standard Reaction Mixture 1 ml Catechol (substrate) Catechol (substrate) Potato Extract (enzyme) 1 ml Phosphate Buffer 3 mls For Part A: begin timing when extract is added Read A 475 at 30 seconds and 5 minutes For Part B: After pre- treating the enzyme, begin timing when substrate is added

Experimental Design Choose independent variable to studyChoose independent variable to study Choose a range of values for the independent variableChoose a range of values for the independent variable Hold all other factors constant, including reaction volumeHold all other factors constant, including reaction volume Plan the composition of experimental samples, using the standard reaction mixture as a modelPlan the composition of experimental samples, using the standard reaction mixture as a model Make sure to have a control sampleMake sure to have a control sample

Adjusting the Standard Reaction Mixture to Produce Experimental Samples 1 ml Catechol (substrate) Catechol (substrate) Varying the Enzyme Concentration Potato Extract (enzyme)+ Phosphate Buffer 4 mls Choose different volumes of enzyme, add buffer to reach 4 ml Varying the Substrate Concentration Phosphate Buffer + Catechol (substrate) 4 mls Choose different volumes of substrate, add buffer to reach 4 ml Potato Extract (enzyme) 1 ml Must have at least 2 ml of buffered solution, including extract volume

Experimental Design Use a 5 ml reaction volumeUse a 5 ml reaction volume At least 2 ml of this volume must contain solutions that have phosphate buffer at pH 6.0 (except for pH experiment)At least 2 ml of this volume must contain solutions that have phosphate buffer at pH 6.0 (except for pH experiment) –You can satisfy this requirement with 1 ml of potato extract (already buffered) and 1 ml of buffer You have a choice of potato extract in phosphate buffer at pH 6 or potato extract in H 2 OYou have a choice of potato extract in phosphate buffer at pH 6 or potato extract in H 2 O Determine the order in which you will mix the reactants and decide whether some components should be preincubated together (under certain environmental conditions) before the reaction startsDetermine the order in which you will mix the reactants and decide whether some components should be preincubated together (under certain environmental conditions) before the reaction starts

Planning Next Week’s Experiment Propose two possible hypotheses to studyPropose two possible hypotheses to study One of these two will be assigned to your groupOne of these two will be assigned to your group Prepare a detailed experimental plan for testing the assigned hypothesisPrepare a detailed experimental plan for testing the assigned hypothesis Obtain your instructor’s approval for your plan BEFORE you begin the experimentObtain your instructor’s approval for your plan BEFORE you begin the experiment