Enzyme Assay

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Enzyme Assay

• Enzyme assays are done to study the kinetics of
  the particular enzyme from any source and the
  factors that affect its activity like substrate
  concentration, pH and temperature

• Related LOs Enzyme kinetics
• gt Prior Viewing IDD-32. Buffer preparation
  for western analysis, IDD-50 basic
  instrumentation
• gt Future Viewing IDD-17. SDS-PAGE, IDD-33.
  Western blot assay
• Course Name Enzyme Assay
  
• Level(UG/PG) UG
• Author(s) Dinesh Raghu,Vinayak Pachapur
• Mentor Dr. Sanjeeva Srivastava

The contents in this ppt are licensed under
Creative Commons Attribution-NonCommercial-ShareAl
ike 2.5 India license
2
Learning objectives
1

• After interacting with this Learning Object, the
  learner will be able to
• Define the enzyme extraction from the sample.
• Determine the importance of enzyme kinetics
• Operate to work with UV-Visible spectrophotometer
• Infer the mechanism behind the spectrophotometer

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Master Layout
1
Reagent preparations (Slide 5-11)
Enzyme extraction (Slide 12-14)
2
UV-Visible spectrophotometer (Slide 15-16)
3
Beer-Lamberts Law (Slide 17-18)
Enzyme assay (Slide 19-20)
4
Spectrophotometric analysis (Slide 21-24)
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4
Definitions and Keywords
1
1. Beer Lamberts law the law relates the
absorbance and the concentration of the solution
In the equation L signifies the path
length, C concentration, E(epsilon) absorption
coefficient and Io and I corresponds to the
intensity of light before entering the solution
and the intensity after coming out of the
solution. The intensity of the light coming out
of the cuvette decreases when the concentration
of the substances in the cuvette increases. This
is the law that governs the spectrophotometric
analysis 2. Peroxidase the enzyme that acts on
hydrogen peroxide and convert it to water and
oxygen 3.TMB 3,3,5,5 Tetramethyl benzidine ,
an organic substrate which remains colorless in
reduced form and once oxidized it turns blue 4.
Rate of the reaction Velocity at which the
reaction occurs, substrate getting converted into
the product
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Step 1
1
T1Reagents for enzyme assay
2
3
Audio Narration (if any)?
Description of the action
Show a measuring balance, with display, ON, OFF
and TARE/0 buttons on it. let user ON it, display
reading as 0.000g, let user picks up the paper
from the rack, makes 1/10 of folding on the sides
and places it on the balance. Now the display
reading changes to 0.003g. Instruct user to TARE
the reading. And animate to click the tare
button. Once user clicks it, reading must show
0
When measuing with paper, the weight of the paper
need to be tared from actual reading.
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5
6
Step 1
1
T1Reagents for enzyme assay
2
Beaker
Magnetic bead
3
Description of the action
Audio Narration (if any)?
Show magnetic stirrer instrument. Let user place
the beaker on it. Display the beaker containing
powder at bottom, liquid layer on top and a
magnetic bead at the bottom. Instruct user to ON
the instrument, let user cotrol the speed nob and
regulate it accordingly to control the mixing
speed in the beaker. Animate powder getting into
the solution. Show a turbid solution turning
colorless
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5
7
Step 2
1
T1Reagents for enzyme assay
TMB
water
2
Audio Narration
Description of the action
3
Let user pick up tetramethyl benzidine
bottle,(TMB) spatula, measuring cylinder from
the rack and keeps it on the table next to
balance. Instruct user to weigh 0.5g of TMB, let
user tare the balance, user should click on the
TMBbottle, uncap it, with help of spatula weigh
the required amount on a paper over the balance.
Display a gradual increase in reading with
quantity addition. if the gram exceeds user
should remove some quantity or if it less add the
quantity to get the exact required amount. After
weighing transfer the quantity to beaker. Now
instruct the user to measuring cylinder and water
pour the water in the cylinder till the volume
shows 90ml and show like mixing as in slide 6
and add 10ml to make the volume to 100ml.
Prepare 0.5 of 3,3,5,5 tetramethylbenzidine
4
5
8
Step 1
1
T1Reagents for enzyme assay
Citric acid monohydrate
water
2
Audio Narration
Description of the action
3
Prepare 1.0M citrate buffer of pH 6
Let user pick up citric acid monohydrate,
spatula, measuring cylinder from the rack and
keeps it on the table next to balance. Instruct
user to weigh 210g of citric acid, let user tare
the balance, user should click on the citric acid
bottle, uncap it, with help of spatula weigh the
required amount on a paper over the balance.
Display a gradual increase in reading with
quantity addition. if the gram exceeds user
should remove some quantity or if it less add the
quantity to get the exact required amount. After
weighing transfer the quantity to beaker. Now
instruct the user to measuring cylinder and water
pour the water in the cylinder till the volume
shows 900ml and show like mixing as in slide 6.
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5
9
Step 2
1
T1Reagents for enzyme assay
2
STD 1
STD 2
3
Audio Narration
Description of the action
Before the pH reading, pH instrument need to be
calibrated with standards. Once with STD 1 at pH
4 and with STD 2 at pH 9.
Display standard pH bottles and pH instrument and
deionized water, discard placed on a table.
Instruct user to caliberate the instrument. Let
user ON the instrument. Initially for the pH rod
is dipped in water, when user clicks on read
button, display must show a reading 7. Now show
like taking out the rod and washing it with
deionized-water, let user cleans the rod with
tissue. Now pick the STD 1, uncap it, dip the
cleaned rod into the solution, user must click
read button with display showing 4. now clean
the rod and repeat the step to note down the
reading for STD 2 and now the display should show
9.

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5
10
Step 3
1
T1Reagents for enzyme assay
2
NaOH
HCl
3
Audio Narration
Description of the action
Instruct user to set the pH for TBST pH at 6. Now
take the TBST bottle, uncap it, dip the cleaned
pH rod into the solution. User need to click on
read button. Initially display must show a
reading 4. now instruct user to add NaOH to
adjust the pH. Now allow the user to click on
NaOH bottle so that drops of NaOH should be added
with filler, user need to mix the solution with
glass rod, click on read button and the reading
should anywhere near 4.1- 4.3. let user keeps
adding the NaOH drop till the pH display shows
6 and later transfer the beaker solution to
1000ml measuring cylinder to makeup the volume to
1000ml by clicking on water and adding it to
that. All action should happen when the user
clicks the hand image.
Prepare 1.0M citrate buffer of pH 6
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5
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Step 4
1
T1Reagents for enzyme assay
Mortar/pestle
2
ice
Audio Narration
Description of the action
3
Animator should show a radish on the table, and a
knife let the user click on knife to remove the
coat of the radish, now let the user cut a small
piece of radish and weigh it to 0.05g , Now show
a ice bucket and the user should place a mortar
and pestle on it , let the user place the radish
pieces in the mortar and click on the bottle
labeled as citrate buffer and set the pipette
1000ul and take the buffer and add to pestle, now
instruct the user to click on pestle for grinding
the tissue after it was ground well, instruct the
user to add the buffer 3 times as shown earlier.
Weigh the required amount of radish and place it
in the prechilled mortar and pestle, grind it
properly for a uniform phaste.
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Step 5
1
T2 Enzyme extraction
2
Audio Narration
Description of the action
3
Add 4ml of Citrate buffer and grind well, Filter
the ground paste using the filter.
Now show a tea filter and a beaker, instruct the
user to filter the ground radish solution paste.
Let user place place the tea filter over the
beaker and lift the motor, to tilt it to pour the
liquid solution through the filter into the
beaker. Let user collect the solution in the
beaker for further processing.
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Step 6
1
T2 Enzyme extraction
Make up the volume of the extract to 100ml using
the buffer such that the extract contains
100units of enzyme/ml of the buffer
Now instruct the user to take the measuring
cylinder and citrate buffer, animate like the
user pouring the citrate buffer to the cylinder
till the volume reaches 96 ml and add to the
beaker with radish extract.
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Step 7
1
T2 Enzyme extraction
Hydrogen peroxide
water
2
TMB
Citrate buffer
Extract
Audio Narration
Description of the action
1 2
3
Let user pick up water bottle and the pipette to
take 2.77ml in the pipette and add to the tube-1
in stand again repeat the same for other to
tubes, Now instruct the user to take the pipette
set to 30ul and take the citrate buffer pipette
out and add to the tube-1 follow the same for
tube-2.
Prepare the tubes for the assay, do not add the
enzyme or the TMB or Hydrogen peroxide before
everything is ready.
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5
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Step 8
1
T3UV-Visible spectrophotometer
Display
Options like number 0-9, set wavelength,
autozero, absorbance
2
Lid that can be opened
3
cuvette
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Step 8
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T3 UV-Visible spectrophotometer
Animate the instrument as in figure and redraw
the instruments with the specification mentioned
in the figure and zoom the instrument and show a
schematic as shown in the figure with the
labelings but redraw completely
UV-Visible spectrophotometer has a monochromator,
light source and sample holder and detector,
Light from the source are converted to a
monochromatic light of particular wavelength and
allow it pass through the sample and amount of
light that emerges is detected by a detector.
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Step 9
1
T4 Beer-Lamberts Law
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3
L
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Step
1
T4 Beer-Lamberts Law
Now show the figure as in slide above (redraw)
followed by the formula which is given in the
slide , audio narration should take place
simultaneously
UV-Visible spectrophotometer works on the basis
of Beer-Lamberts law, the law relates the
absorbance and the concentration of the solution,
. In the equation L signifies the path length, C
concentration, E(epsilon) absorption coefficient
and Io and I corresponds to the intensity of
light before entering the solution and the
intensity after coming out of the solution. The
intensity of the light coming out of the cuvette
decreases when the concentration of the
substances in the cuvette increases.
2
3
4
5
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Step 10
1
T5 Enzyme assay
Hydrogen peroxide
water
2
TMB
Citrate buffer
Extract
Audio Narration
Description of the action
1 2
3

• Instruct the user to set the spectrophotometer by
  switching on click set wavelength and type
  655nm and press enter. Let user pick up TMB and
  instruct the user to take the pipette set to 50ul
  and take the TMB, pipette out and add to the
  tube-1 and follow same for tube-2.
• Let user pick up extract and instruct the user to
  take the pipette set to 50ul and take the
  extract, pipette out and add to the tube-1 follow
  the same to add to tube-2. Show like the user
  taking the tubes and mixing by shaking it.
• .

Set the wavelength of 655 nm in UV visible
spectrophotometer Prepare the control and assay
solution.
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Step 10
1
T5 Enzyme assay
Let the user take the tube-1 and take 2 cuvettes
as in figure . Instruct the user to press the
open lid show two opening inside it one after the
other in longitudinal way. Show like pouring the
contents from tube-1 to both the cuvettes, show
like taking the tissue and wiping on the sides
and placing it in the openings, now animate like
closing the lid and press absorbance . (before
keeping the cuvette the reading should be 0.000,
once the cuvette is kept and absorbance is
pressed it should be 0.123) Now instruct the user
to press auto zero and the reading should be
0.000 and remove the cuvette by opening the lid
and taking out the cuvette from opening 2 discard
the solution out from the cuvette.
Auto zero and callibrate the instrument using the
control solution without hydrogen peroxide
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Step 11
1
T6 spectrophotometeric analysis

• Instruct the user to click on hydrogen peroxide
  and a pipette set to 100ul and take the peroxide
  solution and add to the tube-2 and show like
  mixing and show like the user switching on stop
  clock. Once it has reached 15sec.
• Let the user take the tube-2 and take cuvette as
  in figure. Show like pouring the contents from
  tube-2 to one of the cuvettes , show like taking
  the tissue and wiping on the sides and placing it
  in the openings, now animate like closing the lid
  and press absorbance .
• (before keeping the cuvette the reading
  should be 0.000, once the cuvette is kept and
  absorbance is pressed it should be 0.223 and
  animate like user making a note of the reading.
  Once this has done show like removing cuvette 2
  and discarding the solution and wait till the
  stop clock shows 30 sec again start step-2 as in
  slide and repeat it for every 15 seconds until
  2minutes. Animate the steps as per the
  instructions. Show the solution in blue color and
  as the time goes on show the color getting darker.

Add the hydrogen peroxide to the assay tube and
take readings for every 15seconds for a total of
2mins. The peroxidase in the extract reacts with
the hydrogen peroxide and releases water and
oxygen molecule, the released oxygen molecule
oxidize the tetramethyl benzidine thereby giving
the blue color as the time increases, oxygen
release increases and color intensity also
increases
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Step 15
1
T6 spectrophotometeric analysis
Time (seconds) OD at 655nm
15 0.223
30 0.334
45 0.45
60 0.56
75 0.666
90 0.79
105 0.89
120 0.99

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Step 16
1
T6 spectrophotometeric analysis
2
OD at 655nm
3
Time in seconds
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5
24
Step 15 and 16
1
T6 spectrophotometeric analysis
Instruct the user to plot the graph as OD in y
axis and the time in x axis and show like a
straight line is drawn (red line) connecting time
and OD. Instruct the user to determine the rate
of the reaction.
Plot the graph between OD at 655nm and the time
in seconds of the sample and draw the line joing
the points and determine the rate of the reaction
by calculating the ration of change in absorbance
at 655nm and the time elapsed since the start of
the reaction.
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Slide 17-18
Slide 19-20
Slide 21-24
Slide 5-11
Slide 12-14
Slide 15-16
Tab 02
Tab 03
Tab 04
Tab 05
Tab 06
Tab 07
Tab 01
Name of the section/stage
Interactivity area

• Animation area

Interactions Slide 19-22 Show like the user
set the wrong wavelength say 400nm and started
taking the readings, show like the readings are
in the negative region Instruction Instruct
the user to reset the wavelength to 655nm and
start taking the reading. If the exact
wavelength range has not set , the readings will
be deviating from the exact value to be obtained
Instructions/ Working area
Credits
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Questionnaire
APPENDIX 1

• Question 1
• Absorbance can be taken using
• Calorimetry
• Spectroscopy
• spectrophotometry
• Refractometry
• Question 2
• UV-Visible spectrophotometer works based on
• Beers law
• Lamberts Law
• Beer-Lamberts Law
• Raman spectrum

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Questionnaire
APPENDIX 1

• Question 4
• Rate of the reaction means
• a)Mechanism at which the reaction occurs
• b) Speed at which the reaction occurs
• c) Velocity at which the reaction occurs
• d) Reaction time
• Question 5
• TMB on oxidation gives
• Orange color
• Red color
• Purple color
• Blue color

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APPENDIX 2
Links for further reading

• Reference websites
• 1.http//www.indiana.edu/l113/independent_project
  s/addlprocedures/AP-VEnzymes.pdf
• 2.http//www.fondriest.com/pdf/thermo_colorimeter_
  theory.pdf
• Book
• Biochemistry by Voet Voet, 3rd edition

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APPENDIX 3
Summary
The method mostly involves the study of kinetics
of the enzyme reactionn. Steps involved are
preparation of control and assay tube,
preparation of citrate buffer, TMB, enzyme
extract and hydrogen peroxide addition,
spectrophotometric analysis.