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Lehninger Principles of Biochemistry Fourth Edition

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Lehninger Principles of Biochemistry Fourth Edition David L. Nelson and Michael M. Cox Lehninger Principles of Biochemistry Fourth Edition Chapter 3: Working with Proteins: Protein Isolation and Characterization Reading material: p. 89 – 95, Lehninger Chapter 3 [1] Protein Source: 1. Tissue 2. Microbial cells. If we isolate a protein from a tissue or cell, we might first need to isolate a particular part of the cell i.e. organelle. We can do this by differential centrifugation, where the spin speed determines what remains in the pellet.. 1. Grind tissue (blender/homogenizer) 2. Centrifuge – the speed determines which organelles appear in the pellet and this is based on density (which ones will pellet first?) (Fig 1-8) [2] Solubilization 1.Osmotic lysis: simple and gentle - place cells in a hypotonic solution 2. Mechanical: good if cells have cell wall crushing or grinding of cells, French press (pressure), sonication (sound) 3. Detergents: used if protein is in lipid membrane • pH: use an appropriate buffer. Figure 2-16; p.13, H(3) • Temperature: 0-4 °C – can be unstable once outside of cell • Proteases: must be inhibited (Proteases (proteinases) cut amide bonds of protein) • Adsorption: keep concentrated (denatures at surfaces) minimize foaming/exposure to air (reduce unfolding) • Storage: store under inert gas, frozen at -80 °C or -196 °C [3] Stabilization Proteins are most stable under pH and ionic strength close to physiological conditions. pH: 7.4, (lysosomal enzymes, pH 5) I (ionic strength) = 0.15 M How is Ionic Strength determined? 2 I = ½ciZi where ci is the concentration (mol/L) of ionic species, and Zi is the net charge of the species. ① 0.1 M sodium acetate, CH3COONa + 2 - 2 I = ½ ([Na ]1 + [CH3COO ]1 ) = ½ (0.11 + 0.11) = 0.1 M (equal to molarity) ② 0.1 M Na2HPO4 + 2 2- 2 I = ½ ([Na ]1 + [HPO4 ]2 ) = ½ (0.21 + 0.14) = 0.3 M (larger than molarity) Solubility of carboxy-hemoglobin at its isoelectric point as a function of ionic strength and ion type. NaCl KCl S log S’ MgSO4 Na2SO4 K4SO4 Ionic Strength [4] Solubilities of Proteins Salting In Addition of salt at low ionic strength can increase solubility of a protein by neutralizing charges on the surface of the protein, reducing the ordered water around the protein and increasing entropy of the system. Salting out (Can be used for Fractionation) If the concentration of neutral salts is at a high level (>0.1M), in many instances the protein precipitates. This phenomenon apparently results because the excess ions (not bound to the protein) compete with proteins for the solvent. The decrease in solvation and neturalization of the repulsive forces allows the proteins to aggregate and precipitate. This effect is called "salting out". The effect of salt on different proteins may differ: Certain proteins precipitate from solution under conditions In which others remain quite soluble. Once the protein is precipitated (not denatured) – can separate by centrifugation pellet can be redissolved in buffer for further purification Ⓠ Which protein will ppt first? (hydrophobic or hydrophilic?) [5] Dialysis • Following a salting-out step, the solution will contain a high concentration of salt that can be disruptive to subsequent chromatographic steps. • The salt can be removed by dialysis – dialysis tubing has pores with a specific molecular weight cut-off that allows smaller molecules (salt) to pass. Buffer– large volume Dialysis tubing with protein and high salt Exchange buffer > 3 times [6] Separation - Chromatography Makes use of a mobile phase (fluid - usually buffer) & a stationary phase (usually small beads). Ion exchange chromatography pH and [salt] dependent Separates by ionic charge: cations & anions Cation exchange Anion exchange Where on the pH scale should the buffer be compared to the pI of the protein? This is an example of cation exchange. How will anion exchange work? Cation exchanger: - CM-cellulose -CH2-COO Anion exchanger: C2H5 DEAE-cellulose -CH2-CH2-NH-C2H5 + Choosing an ion exchanger will depend on: The pI value of the target protein. The pH of buffer used. An (acidic) protein of a pI of 5. At pH 7 (phosphate buffer or Tris buffer), the protein will carry a net negative charge. The protein will bind to an anion exchanger (DEAE) Will be repelled from a cation exchanger (CM). • Apply the protein mixture containing the target protein. • Remove neutral and basic proteins in flow-through. • Recover the target protein with increasing [salt]. Size exclusion chromatography Gel filtration chromatography Contains porous beads Separates according to size and shape Larger proteins excluded from the small pores Quaternary structure determination, & Mr estimation using a standard curve (log Mr vs elution volume) Ⓠ Fibrous proteins Spherical vs rod-shaped proteins Affinity chromatography Separates by specific interactions Contains a ligand: enzyme ‒ substrate receptor ‒ hormone antigen ‒ antibody 2+ (His)6 protein – Ni Electrophoretic Analysis Separates according to size and charge concentration Matrix is polyacrylamide (proteins) or agarose (nucleic acids) SDS-PAGE (polyacrylamide gel electrophoresis): uses sodium-dodecyl sulphate (SDS) to coat the proteins to give them all equivalent (negative) charge concentration – proteins can then separate by M.W. only SDS-PAGE Trx-STS Trx-CHS s i p s i p 97 66 60 K 45 31 22 14 s - soluble fraction i - insoluble fraction p - post-Ni2+ column SDS-PAGE Animation Isoelectic focusing Ampholytes are low M.W. organic acids and bases that distribute along the electric field across the gel. Each protein of a mixture distributes across the gel according to their pI. Isoelctric focusing can be described as electrophoresis in a pH gradient set up between a cathode and anode with the cathode at a higher pH than the anode. Because of the amino acids in proteins, they have amphoteric propertites and will be positively charged at pH values below their pI and negatively charged above. This means that proteins will migrate toward their pI. Most proteins have a pI in the range of 5 to 8.5. Under the influence of the electrical force the pH gradient will be established by the carrier ampholytes, and the protein species migrate and focus (concentrate) at their isoelectric points. The focusing effect of the electrical force is counteracted by diffusion which is directly proportional to the protein concentration gradient in the zone. Eventually, a steady state is established where the electrokinetic transport of protein into the zone is exactly balanced by the diffusion out of the zone. Isoelectic focusing carrier ampholite; oligoamino, ologicarboxylic acids of 600~900 Da R-NH-(CH2)n -N-(CH2)n-COOH R=H, -CH2COOH, CH2-N-R2R2 2-D electrophoresis Perhaps certain proteins have identical pIs or molecular weights 1. Separate by IEF – 1st dimension 2. Separate by SDS-PAGE – 2nd dimension this gives a second dimension to the analysis .
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