Development of an enzyme assay EZA

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Development of an enzyme assay (EZA)

• Hydrolysis of PNPG by ?-galactosidase

Rachel Nelson Rbnelson2_at_wisc.edu Biotech 2330
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Goals

• Improve techniques
• Learn how to set up an activity assay
• Get data you can work with
• Think like a scientist - designing your own
  experiment

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Lecture overview

• Background
• Lab Protocols
• Lab advice
• Lab write-up guidelines

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E. coli ?-Galactosidase

• Hydrolyses lactose
• 1021 residues per protein
• Quaternary structure is a symmetrical tetrad
• reporter constructs - i.e. blue/white screening

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Reactions Catalyzed
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Spectrophotometry
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Enzyme assays

• Enzyme assay method to detect and quantitate
  the presence of an enzyme
• Often used to determine the purity of an enzyme
• Used to determine mechanism and kinetic
  parameters of a reaction
• Features of a good assay
• Fast, convenient, and cost effective
• Quantitative, specific, and sensitive

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Factors affecting an assay

• pH
• Temperature
• Buffer
• Cofactors
• Inhibitors
• Activators
• Other substrates
• Allosteric effects
• Stabilizing agents (detergent, salt, reducing
  agent, etc)

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Types of assays

• Time resolved
• continuous
• Single point (fixed time) assay
• Incubate each sample with substrate for a fixed
  time
• Quench rxn and detect product formation

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Designing a fixed time assay

• Choose a high substrate concentration so the S
  does not change appreciably over the course of
  the reaction
• Pick a random (relatively high) enzyme
  concentration
• Monitor product formation as a function of time
• Want a point such that DP/Dt V0

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Creating a progress curve
1. Mix E S 2. Remove at various time points
and quench 3. Measure product formation
(A400) 4. Plot A400 (y-axis) vs. time (x-axis)
12 mL PNPG solution
4 mL b-gal solution
1 M Na2CO3
Blank
1 min
2 min
3 min
4 min
6 min
8 min
10 min
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Quenching the reaction

• Na2CO3 is basic
• Addition of Na2CO3 raises the pH to gt10
• High pH
• Stops the enzyme reaction
• Coverts p-nitrophenol to p-nitrophenolate
  (absorbs light at 400 nm)
• pKa 7.15

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Progress Curves

• Determine initial slope of reaction curve
• Ratio of reaction slope to ideal slope will give
  a dilution factor for using the maximum range of
  the spec

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Calculating the dilution factor

• Ideal slope 0.09
• real slope 0.369
• DF real slope 0.369/0.09 4.1 4
  ideal slope
• Dilute 1 part enzyme in 3 parts buffer

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Underestimating initial activity using a
fixed-time assay
measured rate, not the initial rate!
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Single measurement to check your dilution factor
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found
A400
ideal
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Time (minutes)
May have to run a couple dilutions to find the
right one. Anything between 0.75 to 1.0 is
okay Please check with me before diluting
the rest of your enzyme!!!
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Designing a fixed time assay - Part 2

• Demonstrate the valid range of the assay by
  varying the E and showing that product
  formation ? to E over the entire range -
  Validation Run

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Validation runs

• Choose 3 (or more) different volumes of diluted
  enzyme
• Mix substrate and diluted enzyme at 1 minute
  intervals
• 2. Quench each after 10 minutes
• 3. Observe A400

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A400
You will need to convert from A400 to
international units for your lab write-up.
.1
.2
.3
.4
0.5
mL of enzyme
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Enzyme Kinetics

• How can I determine kinetic parameters for my
  enzyme?
• Michaelis-Menten plots - vary S with E
  constant and visually determine Km and Vmax

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Michaelis-Menten Theory

• Assumption 1 S gtgt E
• Assumption 2 ES state is in equilibrium with ES
  state
• Velocity of reaction is measured at varying S
  until Vmax is reached
• Drawbacks assymptotic relationship only allows
  for visual estimation of Vmax and Km
• What is Km?

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Eadie-Hofstee Plot
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Determining Kinetics

• Design an experiment to create a Michaelis-Menten
  plot for your enzyme
• Choose appropriate volume of diluted enzyme
• Choose 5 (or more) appropriate substrate
  concentrations
• Perform all reactions in triplicate

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Determining Kinetics

• Be careful when choosing your substrate
  concentrations!

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Practical Tips

• Label clearly all test tubes, pipets, and beakers
  at the beginning
• Mix all reactions thoroughly but gently
• Timely quench the reactions
• Do not hold tube in your hand
• Pipet precisely
• Beware of contamination
• Warm up spectrophotometer for 10 minutes.
• For parts 4 and 5 stagger the start times of each
  reaction by 15s to 1min according to your comfort

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Use appropriate blanks

• What should you add in a blank?
• Order matters!
• add enzyme last

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Lab write-up

• Beers law Aebc
• A absorbance (400 nm)
• e extinction coefficient
• b path length (typically 1cm)
• c concentration
• International units (IU, or U)
• U mmol/min (conc.)(vol)/(time)
• U/mL slope of your validation plot
• Volume activity U/mL
• Specific activity U/mg
• Q-test see statistics textbook
• Eadie-Hofstee plot see Enzymology textbook

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Calculate absorbance values for continuous assay

• Henderson Hasselbach
• pH pKa log A-
• HA
• A- phenolate
• HA phenol
• Remember to correct for dilution

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Things to bring

• Calculator
• Lab book