Protein Purification Process

Protein Purification Process • Disruption by: • Ultrasonic vibration • French press • Blending • Separation by centrifugation • Pellet – Cell Debris • Supernatant – Cell Extract Tissues or Cells Homogenate or Cell Extract Impure Proteins Further Purified Proteins • Separation by column chromatography or ionic strength • Ammonium Sulfate Precipitation • Affinity Chromatography • Separation by column chromatography • Gel-Filtration Chromatography • Ion-Exchange Chromatography Pure Protein Centrifugation  Uses centrifugal force applied to sample to separate complex mixtures     Breaking down of cells into various components Largest units experience largest centrifugal force, moving rapidly to bottom of tube Speed is observed in RPM – revolutions per minute Can compare between different centrifuges using RCF – relative centrifugal force: RCF = [r(2πN)2]/g = (1.118x10-5)(rcm)(NRPM)2 g = gravitational acceleration = 9.8 m/s2 r = radius of rotor N = rotational speed in RPM Lactate Dehydrogenase (LDH) ● ● ● LDH catalyzes the last step of anaerobic glycolysis Known kinetic parameters and crystal structure Multiple forms of LDH found in different tissues – Isozymes ● Each isozyme has slightly different kinetic and structural properties, but same function and overall structure 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 ● NADH  Reduced form ● Pyridine nucleotides have characteristic absorbance spectra ● NAD+: No absorbance at 340 nm ● NADH: λmax = 340 nm, ε = 6210 M-1cm-1 How to Measure if LDH is Present? ● ● ● ● Enzymes are catalysts – speed up the rate of the reactions without being consumed We can measure: ● Rate of consumption of reactants (Pyruvate or NADH) ● Rate of formation of products (L-Lactate or NAD+) NADH has a visible absorbance at 340 nm – can follow its consumption during reaction Always want to monitor the initial rate because concentrations change during the reaction Mince, blend, and centrifuge 15krpm, 15 min Bovine Heart or Skeletal Muscle 1P Pellet – Cell Debris, Nuclei  DISCARD 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% Centrifuge 12krpm, 10 min Flow Chart for LDH Purification 2S Supernatant – LDH + Other proteins 3P Pellet – LDH + Other proteins Dialysis, Affinity Purification, Ultrafiltration PURE LDH! 3S Supernatant – Extraneous proteins Week 1: Procedure ● ● Purify Beef Heart or Skeletal Muscle Absorption Spectra & Calculate Extinction Coefficient ● Activity Assay ● Protein Concentration – Dye Binding Assay Keep everything on ice! Especially LDH extract! Week 1: Procedure ● Purify Beef Heart or Skeletal Muscle ● Blend with buffer, measure volume of homogenate ● Centrifuge 15,000 rpm for 15 minutes ● Discard 1P fraction, measure total volume of 1S fraction ● ● Save ~1 mL of 1S fraction in separate aliquot for activity assays and dye-binding Save rest of 1S fraction for Week 2 Record everything! Masses, volumes, concentrations etc! When in doubt, don’t throw it out! Week 1: Procedure ● Absorption Spectra & Calculate Extinction Coefficient ● ● Obtain spectra of NAD+ and NADH with UV-Vis Spectrometer Calculate the apparent extinction coefficient for your bench top spectrophotometers – Given extinction coefficients are for square cuvettes with 1 cm path length – Measure A340 of NADH with your spectrophotometer and UVVis spectrophotometer and calculate: ελ(apparent) = [ελ(UV)][Aλ(test tube)/Aλ(UV cuvette)] Week 1: Procedure ● Activity Assay ● ● Monitors A340 nm to observe of the conversion of NADH to NAD+ as the reaction proceeds Requires cocktail with all other reagents except enzyme and water – “The Cocktail” ● Make enough for 10 assays Reagent Volume 1 M KPO4, pH 7.4 200 μl 6 mM Sodium pyruvate 300 μl 1 mM NADH 300 μl DI Water 2.15 ml Diluted Enzyme Solution 50 μl Total Volume 3.00 ml Will need to dilute enzyme substantially before starting assays – Typically 1:200 or 1:400 dilutions of enzyme are required Week 1: Procedure ● Activity Assay ● To run assays: – Use LDH Kinetics program – Blank spectrometer – Add cocktail and water to cuvette – Add enzyme, mix, and start run ● Want to look at linear portion of graph ● Readout is a RATE: -ΔA340/min – Why Negative? •Need at least 4 dilutions in range ΔA340/min -0.05-0.25 •Need table of time vs. A340 for 1 run Week 1: Procedure ● Protein Concentration – Dye Binding Assay ● ● Do only if time allows, otherwise save for week 2 You can use your standard curve from Lab 1 if it goes through the origin and is a good fit • ● ● When in doubt, make a new standard curve Record protein concentration for 1S fraction • Dilute your protein with water! • Blank? Use protein concentration with activity for calculations Activity Calculation Activity = Units = μmol of Substrate Consumed or Product Formed / min Activity 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 ΔA340/min = (εapp in mM-1) (ΔC) [Activity] = ΔC = ΔA340/min / εapp in mM-1 [Activity] = (0.05/min)/(6.21 mM-1) = 0.0081 mM/min = 0.0081 µmol/min*ml = 0.0081 units/ml mM = µmol/ml mM-1 = ml/µmol Enzyme Activity in the Assay 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!