University:
Boston University
Course:
CAS BI 525 | Biology of Neurodegenerative Diseases
Academic year:

2021
Views:

270

Pages:

17
Author:

versfussjyt4

Purification of Lactate Dehydrogenase (LDH) Purpose:  Understand the effects of ionic strength on proteins and precipitate proteins via ammonium sulfate  Use dialysis to de-salt a protein sample Protein Solubility ● Depends on number of hydrophilic and hydrophobic residues on protein surface ● ● ● ● Majority of hydrophobic residues on inner globular core of protein Hydrophilic residues tend to stay on outer surface to interact with aqueous solution Charged and polar groups more likely to form salt bridges and hydrogen bonds Also depends on properties of the solution in which the protein is dissolved Blue = hydrophillic White = hydrophobic Major Factors Influencing Protein Stability ● Ionic Strength ● ● pH ● ● Extremes tend to have poor protein stability Presence of Denaturants – SDS, Urea, Guanidinium chloride, [SCN-], etc. ● ● Extremes tend to have poor protein stability Temperature ● ● Many ions effect stability of other ions in solution Necessary in some complex mixtures Dielectric Constant of the Solvent ● Relative polarity of solvent Ionic Strength Effects ● Salts have different effects on proteins depending on ionic strength ● ● ● ● Protein solubility increases with neutral salts at low ionic strength – “Salting-in” Protein solubility decreases with neutral salts at high ionic strength – “Salting-out” Salting-in stabilizes charged groups of proteins Salting-out is competition between protein and salt for waters of hydration ● As salt concentration increases protein molecules aggregate and some fall out of solution Ammonium Sulfate Precipitation ● ● ● ● Method that allows us to use the relative ionic strength of different proteins to purify individual proteins Different proteins precipitate at different levels of ionic strength due to different secondary and tertiary structure Ammonium sulfate used in protein purification and crystallography to help “salt-out” proteins ● Need to watch pH ● % Saturation is unit used to denote ionic strength “Salted-out” proteins are separated after salt addition by centrifugation ● ● ● ● Method to separate solvents from the rest of the protein Semi-permeable membrane allows salt and solvent molecules out Protein molecules remain inside membrane Water diffuses in as salt diffuses out ● ● [Salt] inside and outside diffuse to equilibrium Buffer switched 2-3 times ● [Salt] from ~4 M to 4 μM Dialysis Selectively permeable membrane Large Enzyme Molecules Small salt/solvent molecules Add (NH4)2SO4 (aq) to 40% Sat. Centrifuge 12krpm, 10 min 1S Supernatant – CRUDE EXTRACT 2P Pellet – Extraneous proteins Add (NH4)2SO4 (s) to 75% Sat. Centrifuge 12krpm, 10 min Flow Chart for LDH Purification 2S Supernatant – LDH + Other proteins 3P Pellet – LDH + Other proteins Dialysis – 2 Buffer exchanges by TF’s – 3P–Dialyzed Affinity Purification Ultrafiltration PURE LDH! 3S Supernatant – Extraneous proteins Week 2: Procedure ● Ammonium Sulfate Precipitation ● Activity Assays ● Protein Concentration via Dye Binding ● Dialysis Keep everything on ice! Especially LDH extract! Procedure ● Ammonium Sulfate Precipitation ● ● ● ● ● ● ● Thaw protein from week 1, save 1 mL aliquot Set up buret with saturated (NH4)2SO4 solution Bring to 40% Saturation  Add 33 ml (NH4)2SO4 (aq) for 67 ml of crude extract Use appropriate proportion Add dropwise in beaker, and let equilibrate 5 min after adding Centrifuge 10 min at 12,000 rpm After Spin: 2S and 2P  Where is LDH? Resuspend 2P in 15 ml of buffer SAVE ALIQUOTS OF EVERYTHING! Procedure ● Ammonium Sulfate Precipitation ● Measure volume of 2S ● Add solid (NH4)2SO4 to bring final solution to 75% – See table p. 65 (21.2 g/100 mL) – Add while stirring over ~15 min – Check pH midway through, ~7-8, adjust with NH3(aq) ● Equilibrate 5 min after addition ● Centrifuge 10 min at 12,000 rpm ● After Spin: 3S and 3P  Where is LDH? ● Resuspend 3P in 15 ml of buffer SAVE ALIQUOTS OF EVERYTHING! Procedure ● Activity Assays ● Do LDH Activity Assays on: – 1S – Compare to week 1 activity – 2S – 2P – No dilution – 3S – No dilution – 3P ● Use 1 ml aliquots for assays ● All assays need to be in range of ΔA340/min of 0.05-0.25 What do you use to blank your spectrophotometer? Procedure ● Protein Concentration – Dye Binding Assay ● ● Make new standard curve if necessary Find protein concentration for: – – – – – ● ● 1S 2S 2P 3S 3P Use 1 ml aliquots for protein concentration A595 should be within linear region of your standard curve – Dilute protein when necessary What do you use to blank your spectrophotometer? Procedure ● Dialysis ● TF’s will show to how prepare bag ● Put all but 1 ml aliquot of 3P sample in dialysis bag ● ● ● Leave some head space at top of bag for expansion during dialysis Label tape in clamp with initials and leave same labeled tube with TF’s TF’s will switch dialysis buffer at least twice before your next lab and then will freeze your protein sample – 3P Dialyzed Fraction Activity Calculation [Activity] = Units/ml = μmol of Substrate Consumed or Product Formed min * ml [Activity] = ΔC = ΔA340/min / εapp in mM-1 = (0.05/min)/(6.21 mM-1) = 0.0081 units/ml in the assay You must account for the dilutions of your protein! [ActivityUndiluted] = (ΔC)(Total Volume of Assay)(Dilution Factor) (Volume of enzyme used in assay) [ActivityUndiluted]= (0.0081 units/ml)(3.0 ml)(400) = 193 units/mL (0.05 ml) More Enzyme Calculations ● Total Activity = (Activity)(Total Volume) = Units/ml* ml = Units ● Protein = Mass Protein/Volume Extract = mg/ml ● Total Protein = (Protein)(Total Volume) = mg/ml* ml = mg ● Specific Activity = Total Activity/Total Protein = Units/mg ● % Yield = Total Activity in Given Step Total Activity in Crude Extract x 100 Remember to account for the dilutions of your protein! Purification Table Calculations Specific Activity (units/ ΔA340/ Dilution mg) Yield (%) min Factor Corrected Activity Total Total Volume Volume (units/ Protein Activity Protein Fraction (mL) (mL) mL) (mg/mL) (units) (mg) 160 160 Homogenate 1S 80 160.0 272.5 125 43594 20000 2.2 100.0 0.071 400 2S 30 60.0 456.0 30 27362 1800 15.2 62.8 0.118 400 2P 15 30.0 10.0 44 301 1320 0.2 0.7 0.208 5 3S 35 70.0 12.0 17 839 1190 0.7 1.9 0.248 5 3P 17 34.0 560.4 26 19053 884 21.6 43.7 0.145 400 3P-D 18 36.0 510.1 22 18365 792 23.2 42.1 0.132 400 Pooled 8 32.0 499.7 4 15991 128 124.9 36.7 0.129 400 Conc. 2 8.0 1959.4 15 15675 120 130.6 36.0 0.169 1200 Only used 80 ml of original homogenate, therefore everything is multiplied by 2 Only loaded 9 ml for 5000 units for affinity column, therefore volume is multiplied by 4 (8 ml*4 = 32 ml) Final Example Purification Table Fraction Corrected Volume (mL) Activity (units/mL) Protein (mg/mL) Total Activity (units) Total Protein (mg) Specific Activity (units/mg) Yield (%) Homogenate 160 1S 160.0 272.5 125 43594 20000 2.2 100.0 2S 60.0 456.0 30 27362 1800 15.2 62.8 2P 30.0 10.0 44 301 1320 0.2 0.7 3S 70.0 12.0 17 839 1190 0.7 1.9 3P 34.0 560.4 26 19053 884 21.6 43.7 3P-D 36.0 510.1 22 18365 792 23.2 42.1 Pooled 32.0 499.7 4 15991 128 124.9 36.7 Conc. 8.0 1959.4 15 15675 120 130.6 36.0

Purification of Lactate Dehydrogenase Lecture

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