Announcements • Chapter 2 post‐lab write‐ups due on Chapter 3 week 2 lab • B1‐B3: Wednesday, October 2 nd • B4: Thursday, October 3 rd • B8: Friday, October 4 th • B9: Monday, October 7 th • BA: Tuesday, October 8 th • You are getting your Chapter 1 post‐lab back this week • You have exactly 7 days to return them to your TFs or Jose for a regrade • Blackboard announcement to follow on the official regrade policy and guidelines for the BI421 lab
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Announcements• Chapter 2 post‐lab write‐ups due on Chapter 3 week 2 lab
• B1‐B3: Wednesday, October 2nd• B4: Thursday, October 3rd• B8: Friday, October 4th• B9: Monday, October 7th• BA: Tuesday, October 8th
• You are getting your Chapter 1 post‐lab back this week• You have exactly 7 days to return them to your TFs or Jose for a regrade• Blackboard announcement to follow on the official regrade policy and guidelines for the BI421 lab
Chapter 3: Purification of an Enzyme
Biochemical Isolation of Lactate Dehydrogenase (LDH)
from bovine tissue
Overall goals of this chapter:
Learn the step‐wise purification techniques for isolating an enzyme
Analysis of enzyme activity & yield
Lactate Dehydrogenase (LDH)● LDH catalyzes the last step of anaerobic glycolysis
● Multiple forms of LDH found in different tissues – Isozymes
● Each isozyme has slightly different kinetic and structural properties, but same function and overall structure
Ch. 3 Protein Purification Process• Homogenization achieved by blendingminced tissue + buffer
The TWO MOST IMPORTANT FACTORS for Chapter 3 success:
1) Maintaining HIGH ENZYME ACTIVITY
*Keep your enzyme COLD& AVOID BUBBLES!!!
2) Maximum recovery of protein (HIGH YIELD)*Don’t be sloppy… Do the procedures correctly.*PAY ATTENTION to what needs to be saved eachweek!!!
Comportment grade penalties for throwing out critical fractions!
Why is Chapter 3 such a big deal??
Top 5 reasons:
5) Notebook write‐up is worth A LOT… 3x CH1 pts
4) The notebook write‐up will take FOREVER.
3) Enzyme purification is DIFFICULT.[effort + attention = insanely critical]
2) Will need your purified LDH for Ch 4 & 5 too.
1) Increasing SPECIFIC ACTIVITY is your main focus. *Should go up with each purification step.Low/no activity = sad/useless enzyme
Objectives for Chapter 3 Week 1:
A) Prepare a crude enzyme extract from bovine heart or skeletal muscle*Note: You will be assigned heart ormuscle by your TFs!
B) Obtain spectra of NAD+ and NADH to determine εapp of NADH
C) Assay for LDH activity of crude extract*Mastery of enzyme activity assay is critical for Chapters #3‐5!!!
Part A: Preparation of Crude ExtractSteps:1) Homogenize beef sample2) Centrifuge to clear lysate3) Collect crude extract
+ +K/PO4 buffer
1) Homogenize beef sample
Part A: Preparation of Crude ExtractSteps:1) Homogenize beef sample2) Centrifuge to clear lysate3) Collect crude extract
Purpose of Centrifugation Uses centrifugal force to separate sample components
Separates lysate from cell debris
Cell debris, organelles
Carbohydrates and other small molecules
LDH
Extraneous proteins and other macromolecules
High speed
centrifugation
(“Meat smoothie”)
Collect cell lysate
Discard pellet
1S crude extractCentrifuged sampleHomogenate
Part A: Preparation of Crude ExtractSteps:1) Homogenize beef sample2) Centrifuge to clear lysate3) Collect crude extract (contains fat deposits)
1P Pellet (discarded)
1S supernatant (collected)
Centrifugetubes:
After Step 2 centrifugation…
‐ Pour centrifuged 1S sample through cheesecloth‐ Collect filtered 1S sample
Your TFs may advise you to skip the cheesecloth step if your centrifuged 1S sample is clean and free of fat
Tips: Preparation of Crude Extract• Before blending, weigh & recordmassof initial beef sample*TFs will have already pre‐weighed & mincedapproximate amounts for you
• After blending, record total volume of homogenate
• After cheesecloth, record volume of filtered crude 1S sample
= ? g
= ? mL
= ? mL
Tips: Preparation of Crude Extract (cont’d)• Before centrifugation, balance tubes & record total volume that went into the tubes
**DO NOT OVERFILL centrifuge tubes! (*only allowed two)You will need to discard some of the volume. Always note
any discarded volumes for correct back calculations.
• Record volume of 1S crude extract (supernatant) fraction• Collect in 50 mL conical tubes
*Leave some room for freezing!• Take 1 mL 1S aliquot to use for activity & dye‐binding assays
Label all tubes before turning in to TFs!Always keep samples & aliquots on ice!
Part B: NAD+ & NADH spectraSteps:1) Obtain spectra from 250 to 400 nm for NAD+ &
solutions on Cary‐60 UV/Vis stations*Use UV plastic cuvettes
2) Record NAD+ and absorbance readings on benchtop specs at 340 nm*Use glass test tubes and the same samples you
used from the Cary‐60
Use the dilution scheme in Chapter 3 part B
Cofactors of LDH● LDH uses pyridine nucleotide coenzymes as cofactors to transfer
reducing equivalents
● H+ and a pair of electrons
● NAD+ – Nicotinamide adenine dinucleotide Oxidized form
Reduced form
● Pyridine nucleotides have characteristic absorbance spectra
● NAD+: No absorbance at 340 nm
: λmax = 340 nm, ε = 6210 M‐1cm‐1
● To calculate εapp for benchtop spectrophotometer since using these specs to measure enzyme activity
– Given ε340 for NADH (6210 M‐1 cm‐1) is for rectangular cuvettes with 1 cm path length
– When test tube is used, light path length (l) may differ Recall Beer’s Law! A = ε l c
• Measure A340 of NADH on both benchtop spec & UV‐Vis spec, then calculate:
ε340(apparent) = [ε340(UV)] x [A340(test tube)/A340(UV cuvette)]
Purpose of NAD+ & NADH spectra
How to Measure Activity of LDH● Enzymes are catalysts – speed up the rate of the reactions
● Activity assay measures:● Rate of consumption of reactant (NADH)
● NADH has a visible absorbance at 340 nm – can follow its rate of oxidation as reaction progresses
ΔA340/min
Part C: The LDH activity assaySteps:1) Make cocktail (*enough for 10 assays to start)
2) Aliquot into test tubes3) Add diluted enzyme to one test tube at a time
& run assay
Reagent Volume 1 M KPO4, pH 7.4 200 μl
6 mM Sodium pyruvate 300 μl1 mM NADH 300 μlDI Water 2.15 ml
Diluted Enzyme Solution* 50 μlTotal Volume 3.00 ml
“The Cocktail”(page 78)
(*Enzyme is added to test tube just before running the assay)
Activity Assay Procedure (Week 1)● To run activity assay:
– Use LDH Kinetics program– Blank spectrophotometer – Add cocktail to test tube– Adjust volume with water– Add diluted enzyme, mix, and start run
● Obtain rate from linear portion of graph● ΔA340/min readout rate will be a negative value‐Why??
•Need at least 4 volumes in range ΔA340/min = ‐0.05 to ‐0.25•Need to record table of time vs. A340 for ONE run only
A340
time
0.65
0.33
0.01150 s
What is wrong with this activity assay graph?!
The absorbance hasn’t changed from its initial value - Enzyme was too diluted; go back to a previous dilution- Enzyme is dead; did you dilute with water and not EDB?
A340
time
0.65
0.33
0.01150 s
What is wrong with this activity assay graph?!
Absorbance started off and stayed at a low value - Your NADH is dead, check with cocktail + water – enzyme tube- Your enzyme is too concentrated and you caught the end of rxn
A340
time
0.65
0.33
0.01150 s
What is wrong with this activity assay graph?!
Visible nose-dive with a sharp plateau - Enzyme is too concentrated but you are close; try an additional
1:10 dilution
A340
time
0.65
0.33
0.01150 s
What is wrong with this activity assay graph?!
Jagged yet steadily decreasing trend - Tube has active enzyme, but tube was not vortexed properly
before reading; heterogeneous pockets of activity are diffused through solution
A340
time
0.65
0.33
0.01150 s
What is wrong with this activity assay graph?!
Nothing, perfect trendline- This has a change in abs of -0.32 over 2.5 minutes, therefore the
ΔA/min = -0.128
Tips: LDH activity assay• DO NOT put *cold* cocktail in the spec! Condensation will skew reading!*Cocktail can be kept at RT for ~1 hour (after too long, NADH oxidizes)
• Will need to dilute enzyme substantially before starting assays*Typically 1:200 or 1:400 dilutions of enzyme are required;TFs will give you a starting point
• Dilute enzyme with ENZYME DILUTION BUFFER (EDB)• Blank the spec with EDB or water (Why not cocktail??)
• WORK FAST! Addition of enzyme starts the reaction, so get tube in spec & start run ASAP to obtain most accurate rate!
• Ask TFs how to adjust plot on spec to get slope from linear portion only (if needed)
Tips: LDH activity assayMaster performing dilutions and serial dilutions
Know exactly how much volume you need from a dilution and always make a little more than what you need.(e.g., if you need 50 μL of a 1:10 dilution, then make two additional volumes just in case = 150 μL of diluted enzyme [15 μL of enzyme + 135 μL of EDB])
Common dilutions Serial dilution strategies (enzyme + EDB)
1:100 1:10 x 1:10 = (20 + 180) x (100 + 900)1:200 1:20 x 1:10 = (10 + 190) x (10 + 90)1:300 1:30 x 1:10 = (10 + 290) x (10 + 90)1:400 1:20 x 1:20 = (10 + 190) x (10 + 190)1:500 1:100 x 1:5 = 1:100 x (30 + 120)1:600 1:300 x 1:2 = 1:300 x (75 + 75)
Activity = Units = μmol of Substrate Consumed or Product Formed / minActivity concentration (ΔC) = [Activity] = μmol/min*mL = Units/mL
From Beer’s Law: A = (ε)(l)(C) ‐> We are looking at Kinetic Rates: ΔA = (ε)(l)(ΔC)Apparent extinction coefficient, εapp, in mM‐1 combines ε and l
IF ΔA340/min = (εapp in mM‐1) (ΔC)THEN [Activity] = ΔC = ΔA340/min / εapp in mM‐1
● Total Protein = ([Protein])(Total Volume) = mg/ml* ml = mg
● Specific Activity = Total Activity/Total Protein = Units/mg
● % Yield = Total Activity in Given Step
Remember to account for the dilutions of your protein!
Do not procrastinate! Start on calculations ASAP! See purification table on pg. 99
Calculation of Specific Activity
Total Activity in Crude Extractx 100
Week 1: Protein Determination● Protein Concentration – Bradford (Dye‐binding) Assay
● Assay protein concentration for 1S fraction
• Dilute your protein with dH2O, NOT EDB!!!(Do you know why?)
• What will you use to blank spec?● Do Bradford only if time allows, otherwise save for Week 2
● Always prioritize enzyme activity assays first & do same week(Why is this important?)
● CANNOT reuse old standard curves!● Doesn’t matter how “good” curve was; NEW standard curve must be made for each week dye‐binding assay is performed
Standard curves for the semester1 mg/mL BSA di‐water Dye‐binding reagent
0 μL 0.5 mL 4.5 mL10 μL 0.49 mL 4.5 mL20 μL 0.48 mL 4.5 mL30 μL 0.47 mL 4.5 mL40 μL 0.46 mL 4.5 mL50 μL 0.45 mL 4.5 mL60 μL 0.44 mL 4.5 mL
• Set-up your other experimental readings at the same time (protein sample volume, adjust to 500 μL with di-water, add 4.5 mL of dye-binding reagent)
• Let tubes sit for 5 minutes at room temperature then read at 595 nm• Redo any experiments that are outside of standard curve interpolation
region (10 μg – 60 μg of BSA)• DO NOT PLOT the 0 μg of BSA on standard curve, this tube was used
to blank the spec and is not a real data measurement
Chapter 3, Week 1 Pre‐lab Checklist:For your part 2 data collection sheets:
Purification flowchart for all of Chapter 3 – don’t need to do it again for subsequent weeks
Tissue assignment and original mass of tissue sample
All volume recordings at different steps – you will discard sample at the centrifugation step
A table for your activity assays – trial number, amount of enzyme added, dilution factor, resulting ΔA/min
A table for a tabulation of one of your activity assays (time and corresponding absorbance at 340 nm).
A table for your dye‐binding standard curve and assay (if time permits, but have it ready just in case, never hurts).
Chapter 3, Week 1 Checklist:
At the end of lab, you should have:Obtained UV‐vis spectra printout for NAD+ & NADH (micro‐UV plastic cuvettes)
Recorded A340 for NADH and NAD+ on benchtop spec (glass test tube)
Performed enzyme activity assays using 1S crude extract fractiono Recorded rates for 4 different dilutions within ΔA340/min = ‐0.05‐0.25 rangeo Recorded tabular data (time vs. A340) for ONE of the dilutions
(If time permitted) made a new standard curve & performed Bradford (dye‐binding) assay for 1S crude extract fraction
Labeled & turned in 1S crude extract fractions & 1S aliquot to your TFs