C2-3. Page 144Table C.2-3. Lactate Dehydrogenase Reaction Time Courses

Reading number

time(seconds)

A340 readings

50 µlsample

100 µlsample

200 µl sample

300 µl sample

400 µl sample

1 0

2 15

3 30

4 45

5 60

6 75

7 90

8 105

9 120

Protein Purification Lab C2Pages 115 to 168

Lab C.2

Four Periods

Protocol Page 135-160

Benchtop Protocols begin on page 398

Be sure to read theory starting page 120

Exam

• Exam October 19,20

• Includes Carbohydrates, Enzyme kinetics, and all protein labs and material related there to.

• Pay attention to the powerpoints– Read theory sections in the lab manual

• Will be about one hour in length

• Example of exam with answers is posted on web

This Lab

• 4 lab periods

• Prelab= 12 points

• Lab Report= 55 points

• First exam in period 4

You Have:

• Become skilled at using micro pipetters• Have learned to use the spectrophotometer

– To determine concentration of an unknown• Beers Law

– To measure activity of an enzyme

• Have learned how to organize experimental protocols

• Have learned how to prepare a report.

In the next days

• You will use all of these skills to perform a fundamental exercise in Biochemistry/Molecular Biology

• Will learn basic protocols in protein purification and analysis

Protein Purification

• A black art (proteins have personality)• Requires knowledge of protein

– What kind of cell is it coming from– What part of cell– What does it do

• Particularly helpful– Size– Composition

Strategy

• Move from organism to pure protein in as few steps as possible with as little loss of activity (assayable quality) as possible– Time and temperature are factors

Next 4 sessions

• Day one: Protein fractionation by centrifugation, salt precipitation and dialysis

• Day 2: Purification by affinity Chromatography

• Day 3: Determination of concentration by BCA assay

• Day 4: Determine purity by PAGE

Will fill out this critical table as we proceed page 162 (day 4)

Table C.2-4. Enzyme Purification Table

Net volume(ml)

V0 units per

ml

V0 units

Total(an “amount”)

Protein content

(% of total)

Proteinconcentration

(mg/ml)

Net amount

of protein(mg)

Specific Activity(V0/mg

protein)

Step A B C D E F G

1. Cleared

2. (NH4)2SO4

Supernatant

3. diluted dialyzed sample/ solution placed on column

4. pooled peak tubes from column

Column C = (Column A)(Column B)Column F = (Column A)(Column E)Column G = Column C/Column F = Column B / Column EColumn D = Column C/first value in Column C

Protocols for Protein Purification

• Highly individualized

• Use a common approach– Fractionate crude extract in a way that protein

of interest always goes into the pellet or the supernatant.

– Follow progress with functional assay

Lactate Dehydrogenase

• NADH + H+ + Pyruvate =NAD+ + Lactate• Enzyme clears lactic acid from working muscles• The obvious source of enzyme is muscle tissue

(heart & skeletal muscle, H&M, isomers)• We will assay for the enzymes ability to convert

Pyruvate to Lactate

Begin with intact tissue• Disrupt (step4&5)

– Blender, homoginizer

• Remove debris (step7)– Centrifugation

• Precipitate/concentrate (step 14-16)– Ammonium sulfate

• Remove salt (step 22)– dialysis

• Purify (next Lab)– Chromatography

• Analyze (Part B and week 3 & 4)– Activity, molecular weight

Ammonium Sulfate pptpage 124

• Has a wide range of application• Relies on fact that proteins loose solubility as

concentration of salt is increased– Is characteristic of particular protein– Results in a partial purification of all proteins with

similar solubility characteristics– Must determine [amm sulf] to precipitate your protein

empirically.

• Produces “salt cuts”

Salting in / Salting out

• Salting IN• At low concentrations,

added salt usually increases the solubility of charged macromolecules because the salt screens out charge-charge interactions.

• So low [salt] prevents aggregation and therefore precipitation or “crashing.”

• Salting OUT• At high concentrations

added salt lowers the solubility of macromolecules because it competes for the solvent (H2O) needed to solvate the macromolecules.

• So high [salt] removes the solvation sphere from the protein molecules and they come out of solution.

Kosmotrope vs. ChaotropePage 125

• Ammonium Sulfate• Increasing conc

causes proteins to precipitate stably.

• Kosmotropic ion = stabilizing ion.

• Urea• Increasing conc

denatures proteins; when they finally do precipitate, it is random and aggregated.

• Chaotropic ion = denaturing ion.

Dialysis Page 180

• Passage of solutes through a semi-permeable membrane.

• Pores in the dialysis membrane are of a certain size.

• Protein stays in; water, salts, protein fragments, and other molecules smaller than the pore size pass through.

Column Chromatography2nd Day Page 126

Available in any volume

Gel Filtration

Ion Exchange

Affinity Chromatography

We will use bound Adenosine-5’-monophosphate (page 128 & 152). This is part Of NAD+. LDH will Bind. Release LDH by adding NADH

NAD+

AMP

Affinity chromatography

• Remember: NADH is a co-substrate for lactate dehydrogenase.

• We use AMP-Sepharose: AMP is covalently bound to the affinity gel, which will not pass through the filter.

• LDH binds to the AMP b/c it looks like half an NADH.

• Thus LDH remains immobilized in the column until we add NADH which binds tighter to the LDH.

Protein Purificationpage 130

A280

Activity

NADH

Protein Concentration

• Lowry ( most cited reference in biology)– Color assay

• A280

– Intrinsic absorbance Page 132– Relies on aromatic amino acids

• BCA page 133– Modification of Lowry: increased sensitivity and

consistency

• Bradford– Shifts Amax of dye from 465nm to 595nm

A280 Page 132

• Uses intrinsic absorbance

• Detects aromatic residues – Resonating bonds

• Depends on protein structure, native state and AA composition

• Retains protein function

Protein separation using SDS-PAGE(Laemmli system)

Stackinggel

Resolvinggel

1. Apply protein/dye samples into polyacrylamide gel wells

2. Run the electrophoresis until dye reaches the end of the gel

3. Remove the gel from the apparatus and stain for proteins

Page 158

SDS PAGE of Purification Process

1. Complete mix of proteins2. High Salt3. Ion exchange4. Gel-filtratio5. Affinity

10micrograms loaded in each lane

IMPORTANT

• Do not throw away anything until you are certain you no longer need it– Biggest source of problem in this lab

• Label everything clearly copy labels into lab book

• Throwing out wrong fraction results in starting over– 3 days into experiment huge problem

Will follow Flow sheet: Page 138

We will do only one NH4SO4 cutAmmonium sulfate precipitation, Centrifuge

Precipitate

Supernate

Step 2b

Remove dialysate,Store at -20oC

Ground sirloin(or alternative LDH source)

Place in blender, add buffer,homogenize

Initial meat suspension

Centrifuge

Cleared meat extract

(save 1 ml)

Discard precipitate

(save 1 ml)

Step 1

Discard remainder

Resuspend in buffer

Add PMSF,Dialyze

Step 2a(save 1 ml)

Save 3 samplesWill determine protein concentrationactivity and purity

Will fill out this critical table as we proceed page 162

Table C.2-4. Enzyme Purification Table

Net volume(ml)

V0 units per

ml

V0 units

Total(an “amount”)

Protein content

(% of total)

Proteinconcentration

(mg/ml)

Net amount

of protein(mg)

Specific Activity(V0/mg

protein)

Step A B C D E F G

1. Cleared

2. (NH4)2SO4

Supernatant

3. diluted dialyzed sample/ solution placed on column

4. pooled peak tubes from column

Column C = (Column A)(Column B)Column F = (Column A)(Column E)Column G = Column C/Column F = Column B / Column EColumn D = Column C/first value in Column C

Today. Page 138 (part of group)

• Steps 1-5: Weigh muscle sample place in blender with 50ml ice cold buffer homogenize for 2 minutes.

• Steps 6&7: remove large debris by centrifugation Save Supernatant (remove 1ml (Microfuge tube) for later analysis).

• Steps 9-13: Measure the volume of the supernatant determine amount of ammonium sulfate required for precipitation, weigh out 0.4 grams per/ml (NH4)2SO4

Today group 1 continued

• Step14-16: Slowly add salt to gently stirred supernatant . Keep Cold!!See step 12

• Step 17: Centrifuge precipitate to a pellet• Step 18-21: Save supernatant (1ml in microfuge

tube). Suspend pellets in 5ml cold buffer• Step 22, 23: Add PMSF and place suspended

pellet in dialysis tubing and give to TA

Today group 2

• Set up standard assay as on page 142– Measure loss of absorbance as NADH is converted to

NAD+• Step 4 is similar to Kinetic curve you did for ADH

(page 124) only reversed as measure loss of absorbance

• Steps 8-12: You will determine the velocity of LDH catalyzed reaction by varying the concentration of LDH with constant substrate and cofactor. Be sure to adjust the amount of reaction buffer to give 3.2 ml final volume in each assay

Very Important: Page 145

0

0.2

0.4

time (sec)60 120 1800

0

0.2

0.4

time (sec)60 120 1800

A B

observed

observed

extrapolated timecourse

Blank without NADH Blank with NADH

Today group 2 continued

• You are establishing the assay conditions you will use next week to follow the purification of LDH. You must become proficient at this assay.

Flow chart 1B (page 142)Prepare the reaction mixtures

Each reaction will contain 3.200 ml:

3.00 ml 50 mM buffer, pH 7.5

50 l NADH

100 l Enzyme solution, column fraction or diluted Step 1, 2, 3 or 4

Zero the spectrophotometer:Add buffer and pyruvate to the cuvette then set the zero.

Add NADH and check the A340 value.

Determine A340 at 15 sec and 45 sec after adding the enzyme sample.

Note: You may have to adjust the time frame of the rate measurement or the amount of added enzyme to achieve a non-spurious V0 value.

50 l pyruvate

Calculate V0.

Divide the raw answer by the product of 340 (for NADH) times the cuvette path length to convert the units to mole/liter per sec units.

Spurious Vo MeasurementsSame as with ADH

(this is similar to your [ADH] exp)

0

0.2

0.4

0.6

0 15 30 45 60

more enzyme

time (sec)

A340

0

0.2

0.4

0.6

0 15 30 45 60

time (sec)

A340

A) Small [E] B) Increasing [E]

75

Procedure (Page 143)

• 1 Step 1-6. Will create a kinetic curve for LDH (adjust volume of buffer to make 3.2ml)– Similar to ADH

• 2. Repeat kinetic curve with different concentrations of enzyme– This is protocol you will use as you purify LDH

• Do this assay on the unknown samples from step one and 2a from group 1.

C2-3. Page 144Table C.2-3. Lactate Dehydrogenase Reaction Time Courses

Reading number

time(seconds)

A340 readings

50 µlsample

100 µlsample

200 µl sample

300 µl sample

400 µl sample

1 0

2 15

3 30

4 45

5 60

6 75

7 90

8 105

9 120

Today 200 microliter

Next Week Column Chromatography

• Due next time: Prelab assignment for period 2 of ‘LDH Purification’

• You really should write up or otherwise arrange what you did today as soon as possible. Do Not Trust Your Memory

Next lab

• Need member of group to be here at 1:30 to begin washing column

• Will need to measure absorbance at 280 to determine that contaminating protein is lost from column. Wash and measure until A280 is constant.

Strategy

• For samples generated determine amount of protein (A280 ) and activity

• Activity per microgram of protein =s specific activity

• You strive for maximal activity per unit of protein. (table C2-4 Column G, Page 162)

Will generate this elution profilePage 153

A280 V0

fraction (tube) number (approximate only)

NADH added

0

0 10 20 30 40 50 60 70 80

contaminant protein

LDH

Will fill out this critical table as we proceed page 162 (day 4)

Table C.2-4. Enzyme Purification Table

Net volume(ml)

V0 units per

ml

V0 units

Total(an “amount”)

Protein content

(% of total)

Proteinconcentration

(mg/ml)

Net amount

of protein(mg)

Specific Activity(V0/mg

protein)

Step A B C D E F G

1. Cleared

2. (NH4)2SO4

Supernatant

3. diluted dialyzed sample/ solution placed on column

4. pooled peak tubes from column

Column C = (Column A)(Column B)Column F = (Column A)(Column E)Column G = Column C/Column F = Column B / Column EColumn D = Column C/first value in Column C