Enzyme LabEnzyme Lab

Enzyme: PhenolaseEnzyme: Phenolase• Reaction catalyzedReaction catalyzed Catechol + ½ OCatechol + ½ O22-----------> 0-benzoquinone -----------> 0-benzoquinone

0-benzoquinone + H0-benzoquinone + H22O---> O--->

pink/gold melanin ---> ---> brown melanin pink/gold melanin ---> ---> brown melanin • Reaction detected by change in Absorbance Reaction detected by change in Absorbance

at 475 nm due to production of the pink/gold at 475 nm due to production of the pink/gold melaninmelanin

phenolasephenolase

Standard Reaction MixtureStandard Reaction Mixture

1 ml CatecholCatechol

(substrate)(substrate)

PotatoPotatoExtractExtract

(enzyme)(enzyme)1 ml

PhosphatePhosphateBufferBuffer

3 mls

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

Experimental DesignExperimental Design

• Choose independent variable to studyChoose independent variable to study• Choose a range of values for the independent Choose a range of values for the independent

variablevariable• Hold all other factors constant, including Hold all other factors constant, including

reaction volumereaction volume• Plan the composition of experimental Plan the composition of experimental

samples, using the standard reaction mixture samples, using the standard reaction mixture as a modelas a model

• Make sure to have a control sampleMake sure to have a control sample

Adjusting the StandardAdjusting the StandardReaction Mixture to Reaction Mixture to

Produce Experimental SamplesProduce Experimental Samples

1 ml CatecholCatechol

(substrate)(substrate)

Varying the Varying the Enzyme ConcentrationEnzyme Concentration

PotatoPotatoExtractExtract

(enzyme)(enzyme)++

PhosphatePhosphateBufferBuffer

4 mls

Choose different

volumes of enzyme, add

buffer to reach 4 ml

Varying the Varying the Substrate ConcentrationSubstrate Concentration

PhosphatePhosphateBufferBuffer

+ + CatecholCatechol

(substrate)(substrate)

4 mls

Choose different

volumes of substrate,

add buffer to reach 4 ml

PotatoPotatoExtractExtract

(enzyme)(enzyme)1 ml

Must have at least 2 ml of

buffered solution, including extract volume

Experimental DesignExperimental Design

• Use a 5 ml reaction volumeUse a 5 ml reaction volume• At least 2 ml of this volume must contain solutions At least 2 ml of this volume must contain solutions

that have phosphate buffer at pH 6.0 (except for that have phosphate buffer at pH 6.0 (except for pH experiment)pH experiment)– You can satisfy this requirement with 1 ml of potato You can satisfy this requirement with 1 ml of potato

extract (already buffered) and 1 ml of bufferextract (already buffered) and 1 ml of buffer• You have a choice of potato extract in phosphate You have a choice of potato extract in phosphate

buffer at pH 6 or potato extract in Hbuffer at pH 6 or potato extract in H22OO• Determine the order in which you will mix the Determine the order in which you will mix the

reactants and decide whether some components reactants and decide whether some components should be preincubated together (under certain should be preincubated together (under certain environmental conditions) before the reaction environmental conditions) before the reaction startsstarts

Planning Next Week’s ExperimentPlanning Next Week’s Experiment

• Propose two possible hypotheses to Propose two possible hypotheses to studystudy

• One of these two will be assigned to One of these two will be assigned to your groupyour group

• Prepare a detailed experimental plan for Prepare a detailed experimental plan for testing the assigned hypothesistesting the assigned hypothesis

• Obtain your instructor’s approval for your Obtain your instructor’s approval for your plan BEFORE you begin the experimentplan BEFORE you begin the experiment