extraction of proteoglycans (pgs) and...

Extraction of Proteoglycans (PGs) and Glycosaminoglycans (GAGs) from cells and tissues

PEG Trainee LectureJuly 9, 2012

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Development of Extraction Procedures

• Extraction of intact PGs at high yield– Disruptive Extraction

• High speed homogenization in low salt buffers– Dissociative Extraction

• Solvents of high ionic strength and strong denaturing effect (e.g., 4 M guanidine-HCl)

– Dissociative Extraction followed by Associative ionic strength

• 4 M guanidine-HCl followed by dialysis allows HA- binding PGs to re-form aggregate super-structures

Hascall & Kimura, PGs Isolation & characterization, Methods Enzymol. 82;769-800, 1982.

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Hyaluronan binding domain

Cartilage PG: Aggrecan

Monomer

500 nm

Aggrecan

Cartilage proteoglycan

~100 CS chains, ~100 KS chains (“bottle brush”)

Mr

~2.5 x 106

Da

Length ~ 0.5 m

PG preparation by isopycnic density gradient centrifugation in Cesium Chloride (CsCl) and 4 M GnHCl

Pucci M et al. J. Biol. Chem. 2001;276:4756-4765©2001 by American Society for Biochemistry and Molecular Biology

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Aggregate: 1 hyaluronan + ~180 aggrecan

monomersSupramacromolecular

complexes (5-8 m length)

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Aggrecan

Aggregate: 1 hyaluronan + ~100 aggrecan

monomersGlycerol spraying & rotary shadowing Electron Microscopy

Produced by Matthias Morgelin

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Dissociative Extraction followed by Associative ionic strength (0.5 M GnHCl)

4 M guanidine-HCl followed by dialysis allows HA-binding PGs to re- form aggregate super-structures

monomer

aggregate

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Development of Extraction Procedures

• Extraction of intact PGs at high yield– Low temperature extraction in the presence of

a combination of protease inhibitors– Use of select detergents to enhance the

yields of PGs having a higher hydrophobic content (e.g., cell membrane or intracellular PGs)

• Triton X-100, deoxycholate, CHAPS– SDS is not compatible with guanidine-HCl


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Tissue Preparation

• Preference for freshly isolated tissue but rapidly frozen (liquid N2 ) tissue stored at ultra-low temperatures can be used– Frozen tissue should be pulverized and

thawed in the extraction solution• Larger tissues (> 1 cm3) can be

homogenized in the extraction solution at ice-cold temperature to yield smaller tissue pieces thereby increasing extraction yields


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Tissue Preparation• Need to preserve protein’s primary structure

during extraction – pH of extraction solution should be 6.0

• Above acid protease and below neutral protease pH optima

– Add protease inhibitors to the extraction solution• 6-aminohexanoic acid inhibits cathepsin D• Benzamidine HCl inhibits trypsin class• EDTA inhibits metalloproteases• Phenylmethylsulfonyl fluoride (PMSF) inhibits serine-

dependent proteases (AEBSF can also be used)• N-ethylmaleimide inhibits thiol-dependent proteases and

prevents nonspecific disulfide exchange• Pepstatin inhibits cathepsin D


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Tissue Preparation

• There may be the need to add inhibitors of other enzymes if one suspects the presence of other post-translational modifications– Any anticipated post-translational

modifications need to be preserved in native structure

• For example, inhibitors of phosphatases that will cleave organophosphate groups


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Most Common Extraction Procedure for PGs

• 4 M Guanidine-HCl– Typically extracts 70-90% of total PG from tissues– Recovery of the remaining GAGs from the tissue

residue require exhaustive proteolysis or alkaline hydrolysis (at the cost of losing core protein structure)

• Chaotropic agents such as 6 M LiCl2 , 2 M CaCl2 , 3 M MgCl2 or 0.5 M LaCl2 can be used instead of 4 M GnHCl

• What are chaotropes?


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Chaotropes• ‘Chaotropes’ (disorder-maker) unfold proteins,

destabilize hydrophobic aggregates and increase the solubility of hydrophobes– Guanidinium chloride; Urea; Sodium dodecyl sulfate; Phenol;

Butanol; Ethanol; Propanol; Lithium perchlorate; Magnesium chloride; Thiourea

Urea FormamideGuanidine HCl

Sodium dodecyl sulfate

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Water Structure• Water forms liquid-crystal lattices at room

temperature and pressure– Extensive H-bonding between water molecules

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Water Structure• Water solvates ionic groups in proteins,

nucleic acids and carbohydrates

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Solute Interaction with Water Molecules

• Miscible solutes interact with the liquid crystal water lattice – ‘Kosmotropes' (order-maker) stabilize proteins and

hydrophobic aggregates in solution and reduce the solubility of hydrophobes

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Solute Interaction with Water Molecules

• Miscible solutes interact with the liquid crystal water lattice – Chaotropes decrease the polar nature of a water

lattice

Urea – H2 O Guanidinium – H2 O

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• The Hofmeister mechanism involves specific interactions between ions and proteins and ions and the water molecules directly contacting the proteins– Anions appear to have a larger effect than cations, and are usually

ordered:

• Early members of the series increase solvent surface tension and decrease the solubility of nonpolar molecules ("salting out")– they strengthen the hydrophobic interaction.

• Later salts in the series increase the solubility of nonpolar molecules ("salting in") & decrease the order in water– they weaken the hydrophobic effect

• Ions that have a strong 'salting in' effect such as guanidinium and SCN- are strong denaturants and interact much more strongly with the unfolded form of a protein than with its native form. – Consequently, they shift the chemical equilibrium of the unfolding

reaction towards unfolded protein.

Hofmeister series

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Hydrophilicity vs. Hydrophobicity• Water organizes an interface boundary

with lipids

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Hydrophobicity• Hydrophobic (‘water fearing’) molecules

self organize to exclude water molecules– Triacylglycerols (TAGs) vs. Phospholipids (PLs)

Triacylglycerols (‘triglycerides’)

Schematic diagram of a phospholipid bilayer vesicle with bound nanoparticles.

Wang B et al. PNAS 2008;105:18171-18175

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Critical Micelle Concentration (CMC)

• Phospholipids self-organize in water and physiological solutions– Concentration dependent phenomenonPhospholipid monomers Phospholipid multimers

Lower concentration Higher concentration

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Critical Micelle Concentration (CMC)

• Detergents self-organize in physiological solutions– Concentration dependent

phenomenon

Lower

Higher

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Triton X-100


Detergents• Detergents are polar lipids that are soluble in

water and increase the solubility of hydrophobes– Triton X-100; Sodium dodecyl sulfate; CHAPS; CHAPSO;

Deoxycholate

Deoxycholate

CHAPS

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Detergent Micelle Size

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Gel Filtration Chromatography• Simple column-based means to separate

varying sized molecules in solution

29Midura & Yanagishita, Chaotropes increase CMC of Detergents, Anal Biochem, 228;318-322, 1995

Vo Vt

Triton X-100

Phenyl ring absorbs light at 280 nm wavelength

M.W., Da

90,000

650

Vo: Void Volume (totally excluded)Vt: Totally included volume

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Centrifugal Ultrafiltration

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90,000 Da

650 Da

TX-10018,000 Da

288 Da

SDS

6,150 Da

615 Da

CHAPS

Midura & Yanagishita, Chaotropes increase CMC of Detergents, Anal Biochem, 228;318-322, 1995

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NaClin H2 O

GnHClUrea

Formamidein H2 O

Midura & Yanagishita, Chaotropes increase CMC of Detergents, Anal Biochem, 228;318-322, 1995

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PG Migration Behavior in SDS-PAGE

MW Standards stained with Coomaisse Blue

Corneal PGs

extraction of proteoglycans (pgs) and...

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