Isolation & Purification of Proteoglycans (PGs) and Glycosaminoglycans (GAGs) PEG Trainee Lecture July 23, 2012
Most Common Extraction Procedure for PGs 4 M Guanidine-HCl Detergents such as 2% CHAPS or TX-100 Protease inhibitors ph of 6.0 Guanidine HCl Urea Formamide Charged Uncharged Hascall & Kimura, PGs Isolation & characterization, Methods Enzymol. 82;769-800, 1982. 2
Purification & Separation of PGs PGs in 4 M GnHCl extraction solution can be purified by several procedures: Isopycnic CsCl Density Gradients Direct Dissociative Gradients Associative Conditions Gradients Molecular Sieve Chromatography 4 M guanidine-hcl plus detergent (0.5% TX-100 or CHAPS) Ion Exchange Chromatography Must exchange 4 M GnHCl with 7-8 M Urea or 10 M Formamide Hascall & Kimura, PGs Isolation & characterization, Methods Enzymol. 82;769-800, 1982. 3
Direct Dissociative Gradients In the presence of 4 M guanidine-hcl, low buoyant density glycoproteins can be separated from different PGs of higher buoyant densities 4
Glycerol spraying & rotary shadowing Electron Microscopy Aggrecan Monomers Aggrecan Aggregate 1 hyaluronan + ~100 aggrecan monomers Produced by Matthias Morgelin 5
Associative Conditions Gradients 4 M guanidine-hcl followed by dialysis to ~0.5 M GnHCl allows HAbinding PGs to re-form aggregate super-structures monomer aggregate 6
Chromatography Chromatography (chroma "color" & graphein "to write") is a set of laboratory techniques for the separation of mixtures. A sample is dissolved in a fluid (or gas) mobile phase, which carries it through a stationary phase structure. Separation of a sample s constituents is based on differential partitioning between the mobile and stationary phases. Differences in constituent chemistry result in differential retention on the stationary phase (partition coefficient) thereby separating constituents in the mobile phase. Chromatography may be preparative or analytical. Preparative chromatography separates the components of a mixture for subsequent analysis. Analytical chromatography uses relatively smaller amounts of material for measuring the relative proportions of analytes. 7
Types of Chromatography Adsorption Chromatography Partition Chromatography Molecular Exclusion Chromatography aka, molecular sieve chromatography, gel filtration chromatography, size exclusion chromatography Ion Exchange Chromatography Affinity Chromatography 8
Types of Chromatography Adsorption Chromatography Adsorption chromatography is probably one of the oldest types of chromatography around. It utilizes a mobile liquid or gaseous phase that is adsorbed onto the surface of a stationary solid phase. The equilibration between the mobile and stationary phase accounts for the separation of different solutes. Partition Chromatography This form of chromatography is based on a thin film formed on the surface of a solid support by a liquid stationary phase. Solute equilibrates between the mobile phase and the stationary liquid. 1997 Kevin Yip - Introduction to Biochemical Engineering 9
Types of Chromatography (continued) Molecular Exclusion Chromatography Also known as gel permeation or gel filtration, this type of chromatography lacks an attractive interaction between the stationary phase and solute. The liquid or gaseous phase passes through a porous gel which separates the molecules according to its size. The pores are normally small and exclude the larger solute molecules, but allows smaller molecules to enter the gel, causing them to flow through a larger volume. This causes the larger molecules to pass through the column at a faster rate than the smaller ones. 1997 Kevin Yip - Introduction to Biochemical Engineering 10
Gel Filtration Chromatography Simple column-based means to separate small from large sized molecules in solution Sephadex G-50 desalting columns 11
Molecular Exclusion Chromatography Column-based means to separate varying sized molecules in solution Sepharose CL-4B separation of PGs 12
Molecular Exclusion Chromatography MW Separation Ranges 13
Molecular Exclusion Chromatography Separation of Proteoglycans HU Choi et al, J Biol Chem 264:2876-2884, 1989 14
Types of Chromatography (continued) Ion Exchange Chromatography In this type of chromatography, the use of a resin (the stationary solid phase) is used to covalently attach anions or cations onto it. Solute ions of the opposite charge in the mobile liquid phase are attracted to the resin by electrostatic forces. 1997 Kevin Yip - Introduction to Biochemical Engineering 15
Ion Exchange Chromatography: Principle Analyte molecules are retained on the column based on ionic interactions allowed by solution chemistry. The stationary phase surface displays ionic functional groups that interact with analyte ions of opposite charge. Cation exchange chromatography retains positively charged cations Anion exchange chromatography retains negatively charged anions Cation exchange example 16
Ion Exchange Chromatography: Principle Bound analytes can be eluted from the column by: Increasing the concentration of a similarly charged species that will displace them from the stationary phase Shifting the elution buffer ph towards the pi of proteins thereby neutralizing their intrinsic charge Goat SA 17
Anion Exchange Chromatography: Different Approaches for PG Isolation Pre-packed commercial exchangers versus self-packed units Low Medium Pressure Limits Filter based Units Medium High Pressure Limits 18
Anion Exchange Chromatography: Different Approaches for PG Isolation 1. Introduction of samples to top of column versus; 2. Binding sample to beads first then packing them into a column 1. 2. G50 Void Sample Extract in Urea or Formamide Sample added to pre-packed column Sample concentrates at the top of the prepacked column G50 Void Sample Extract in Urea or Formamide Sample binds to beads Sample bound to beads is distributed evenly in column M Yanagishita et al, Methods Enzymol, 138:279-289, 1987 19
Anion Exchange Chromatography: Different Approaches for PG Isolation Continuous gradient elution Best used for complex sample unknowns and the need for high resolution separation Step wise elution Fast separation of several types of molecules of known chemistry differences Batch elution Fastest separation of bound versus unbound molecules (no further separations) M Yanagishita et al, Methods Enzymol, 138:279-289, 1987 20
Anion Exchange Chromatography: Different Approaches for PG Isolation Sophisticated commercial devices versus simple-crafted, custom-built units HPLC M Yanagishita et al, Methods Enzymol, 138:279-289, 1987 21
Anion Exchange Chromatography: Different Approaches for PG Isolation Sophisticated commercial devices versus simple-crafted, custom-built units Gradient Maker Peristalic Pump Column Fraction Collector M Yanagishita et al, Methods Enzymol, 138:279-289, 1987 22
Anion Exchange Chromatography: Different Approaches for PG Isolation Anion exchange as a means to concentrate PGs Samples must be diluted (or dialyzed) to lower salt concentration Also provides additional clean-up step to purify PGs PGs bound in a small volume at the top of an anion exchange medium Remove the PG-bound beads from the top of the column (e.g., cut the column top and separate from the bottom portion) M Yanagishita et al, Methods Enzymol, 138:279-289, 1987 Elute PGs from the anion exchange beads using a small volume of 4 M GnHCl, 0.5% CHAPS buffer 23
Anion Exchange Chromatography: Isolation of PGs M Yanagishita et al, Methods Enzymol, 138:279-289, 1987 24
Anion Exchange Chromatography: Examples of PG Isolation RJ Midura et al, JBC 264:1423-1430, 1989 M Yanagishita et al, Methods Enzymol, 138:279-289, 1987 25
Affinity Chromatography: Isolation of PGs M Yanagishita et al, Methods Enzymol, 138:279-289, 1987 26
Types of Chromatography (continued) Affinity Chromatography This is the most selective type of chromatography employed. It utilizes the specific interaction between one kind of solute molecule and a second molecule that is immobilized on a stationary phase. For example, the immobilized molecule may be an antibody to some specific protein. When solute containing a mixture of proteins are passed by this molecule, only the specific protein is reacted to this antibody, binding it to the stationary phase. This protein is later extracted by changing the ionic strength or ph. 1997 Kevin Yip - Introduction to Biochemical Engineering 27
Hydrophobic Interaction Chromatography: A Form of Affinity Chromatography Support Bead Proteins separate according to their hydrophobicity index in organic or aqueous detergent mobile phases 28
Critical Micelle Concentration of TX-100 TX-100 90,000 Da TX-100 Inflection on Octyl-Sepharose in 4 M GnHCl Approximates TX-100 CMC 650 Da M Yanagishita et al, Methods Enzymol, 138:279-289, 1987 Midura & Yanagishita, Anal Biochem, 228;318-322, 1995 29
Hydrophobic Interaction Chromatography Separation of Different PGs M Yanagishita et al, Methods Enzymol, 138:279-289, 1987 30
Hydrophobic Interaction Chromatography Separation of Different PGs M Yanagishita et al, Methods Enzymol, 138:279-289, 1987 31
Hydrophobic Interaction Chromatography Separation of Different PGs Corneal PGs Midura & Hascall, J Biol Chem 264:1423-1430, 1989 32
Hydrophobic Interaction Chromatography Separation of Different PGs HU Choi et al, J Biol Chem 264:2876-2884, 1989 33
Hydrophobic Interaction Chromatography Separation of Different PGs HU Choi et al, J Biol Chem 264:2876-2884, 1989 34
Other Types of Affinity Chromatography Separation of Different PGs Lectin binding chromatography Antibody binding chromatography Heparin binding chromatography Reporter-tag binding chromatography Recombinant expressed molecules 35
Heparin-Sepharose Affinity Chromatography Separation of Different PGs 36