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Enzyme Lab

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

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Enzyme Lab

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  1. Enzyme Lab

  2. Enzyme: Phenolase phenolase • Reaction catalyzed Catechol + ½ O2-----------> 0-benzoquinone 0-benzoquinone + H2O---> pink/gold melanin ---> ---> brown melanin • Reaction detected by change in Absorbance at 475 nm due to production of the pink/gold melanin

  3. PotatoExtract (enzyme) 1 ml PhosphateBuffer 3 mls Catechol(substrate) 1 ml Standard Reaction Mixture For Part A: begin timing when extract is added Read A475 at 30 seconds and 5 minutes For Part B: After pre-treating the enzyme, begin timing when substrate is added

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

  5. PotatoExtract (enzyme) + PhosphateBuffer PotatoExtract (enzyme) 1 ml 4 mls PhosphateBuffer + Catechol(substrate) Catechol(substrate) 4 mls 1 ml Choose different volumes of substrate, add buffer to reach 4 ml Adjusting the StandardReaction Mixture to Produce Experimental Samples Choose different volumes of enzyme, add buffer to reach 4 ml Must have at least 2 ml of buffered solution, including extract volume Varying the Enzyme Concentration Varying the Substrate Concentration

  6. Experimental Design • Use 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) • 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 H2O • 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 starts

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

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