Protein Purification You are a biochemist working at pharmaceutical company. Your boss tells you that we are starting to research metabolism in cows. As it turns out, a hormone enhancing drug that was given to the cows is suspected to increase the metabolism! The company thinks that the drug might be activating the enzyme Lactate Dehydrogenase (LDH). You, the biochemist, needs to isolate this 1 enzyme to study it.... Is this possible?

Protein properties Molecular weight (size) pI, isoelectric point (charge) Solubility (hydrophobicity) pH, Temp., (stability) Contaminant properties (proteases)

KEEP IT SIMPLE !! (but be smart !!)

Begin with intact tissue Disrupt Blender, homoginizer Remove debris Centrifugation Precipitate/concentrate Ammonium sulfate Purify Chromatography Analyze Activity, molecular weight

Protein Purification!

AS precipitation (NH 4 ) 2 SO 4 Very cheap! Very soluble in H 2 O Relies on fact that proteins loose solubility as concentration of salt is increased When protein precipitates is characteristic of particular protein Results in a partial purification of all proteins with similar solubility characteristics Can rid solution of other non-proteins also! Produces “salt cuts”

Salting out At high concentrations added salt lowers the solubility of macromolecules because it competes for the solvent (H 2 O) needed to solvate the macromolecules. Salt interacts with water via electrostatic interactions So high [salt] removes the solvation sphere from the protein molecules and they come out of solution. Proteins interact with other proteins via hydrophobic interactions

Water AS protein Solution with protein Solution with protein and AS

Dialysis 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 Chromatography

Columns Common types of column chromatography: Ion-exchange chromatography - separation based upon the overall charge of molecules Gel-filtration chromatography - separation based upon molecular size Affinity chromatography - separation by specific binding interactions between column matrix and target proteins

Ion-exchange chromatography

KCl + H 2 O  K + + Cl -

cation exchange chromatography: positively charged ions bind to a negatively charged resin anion exchange chromatography: negatively charged bind to a positively charged resin Anion Exchange Chromatography Cation Exchange Chromatography

Gel filtration chromatography

Affinity Chromatography

resin have Ni ++ attached His tag on protein binds to Ni ++ Elute with imidazole Ni ++ his Ni ++ Solvent flow histidine imidazole Affinity Chromatography

High Performance Liquid Chromatography: can be applied to many different resins Most common: separation is based on the molecule’s relative solubility in H 2 O or polarity Material will be eluted with a gradient of non-polar solvent HPLC

Protein Concentration Lowry ( most cited reference in biology) Color assay A 280 Intrinsic absorbance Relies on aromatic amino acids Bradford Shifts A max of dye from 465nm to 595nm Lowry, OH, NJ Rosbrough, AL Farr, and RJ Randall. J. Biol. Chem. 193:

Beer’s Law Beer ’ s Law is stated in a way to make certain quantities easy to compare and interpret. Parameters: l – sample pathlength (usually 1cm) c – concentration (M)  – molar absorption coefficient A – light intensity (absorbance) A=cl

Beer’s Law A = abc =  cl A c x x x x x *

What: sample dilute by ½ dilute by ½ again dilute by ½ Conc: 1g/mL1/2g/mL1/4g/mL 1/8g/mL Abs: AbsorbanceConcentration

Where’s the protein?

A 280 Uses intrinsic absorbance Detects Y and W residues and little S-S Depends on protein structure, native state and AA composition Retains protein function Layne, E. Spectrophotometric and Turbidimetric Methods for Measuring Proteins. Methods in Enzymology 3:

What is SDS-PAGE? Polyacrylamide gel electrophoresis (PAGE) Separates molecules on a polyacrylamide gel matrix when an electric field is applied SDS-PAGE. Sodium dodecyl sulfate (SDS) coats proteins with negative charges. Coated polypeptide chains then separate by molecular mass (method to determine molecular weight)

Why do we need to denature the proteins?

gels Dr Caran JMU Chemistry

(a) SDS-PAGE Electrophoresis (b) Protein banding pattern after run

pH 8.3 Stacking Gel pH 6.6 Separating Gel pH 8.8 Proteins separated by molecular weight

“Ladder” Myosin – blue  -galactosidase – magenta Bovine serum albumin – green Carbonic anhydrase – violet Soybean trypsin inhibitor – orange Lysozyme – red Aprotinin - blue Kaleidoscope standard

Proteases will cleave amide bonds at specific locations Then the puzzle can be solved! Proteases (peptidases): Enzymes that catalyzed the hydrolysis of the amide bonds of peptides and proteins. trypsin: cleaves at the C-terminal side of Arg, Lys

chymotrypsin: cleaves at the C-terminal side of aromatic residues, Phe, Tyr, Trp

E-A-Y-L-V-C-G-E-R F-V-N-Q-H-L-F-S-H-L-K G-C-F-L-P-K L-G-A F-V-N-Q-H-L-F S-H-L-K-E-A-Y L-V-C-G-E-R-G-C-F L-P-K-L-G-A F-V-N-Q-H-L-F F-V-N-Q-H-L-F-S-H-L-K S-H-L-K-E-A-Y E-A-Y-L-V-C-G-E-R L-V-C-G-E-R-G-C-F G-C-F-L-P-K L-P-K-L-G-A L-G-A F-V-N-Q-H-L-F-S-H-L-K-E-A-Y-L-V-C-G-E-R-G-C-F-L-P-K-L-G-A Align the sequences of the peptide fragments from the two complementary cleavage methods.

Quantification of protein, an Enzyme: Activity versus specific activity