DETERGENTS AND THEIR PROPERTIES BEN …

1 DETERGENTS AND THEIR PROPERTIESBEN HANKAMERINSTITUTE FOR MOLECULARBIOSCIENCEUNIVERSITY OF TISSUE TO PROTEINSEIBERT ET AL 1987 J CELL BIOL 105:2257 DETERGENT DEFINITIONDETERGENTS ARE AMPHIPATHIC MOLECULES THAT CONTAIN BOTH APOLAR (HEADGROUP) AND HYDROPHOBIC REGIONS (TAIL). PURIFICATION STABILIZATION CRYSTALLIZATIONSEIBERT ET AL 1987 J CELL BIOL105:2257 PLAY AN IMPORTANT ROLE HEADGROUPS FORM HYDROGEN BONDS WITH TAILS DO NOT FORM H-BONDS AGGREGATE INTO REPLACE LIPIDS DURING SOLUBILIZATION ANDSTABILIZE TRANS-MEMBRANE DOMAINS IN PROTEIN SOLUBILIZATIONPROTEIN: DETERGENT MICELLEBICELLE:PROTEIN COMPLEXPROTEIN:DETERGENTCOMPLEX(NATIVE LIPID)PROTEIN:DETERGENTCOMPLEX(ADDED LIPID)MP EMBEDDED IN A PLANAR LIPIDDETERGENT BILAYERSMALL VESICLESDENATURED PROTEINMAIN CLASSES OF DETERGENTSIONIC DETERGENTS1. -VE CHARGE = ANIONIC ( SDS)2. +VE CHARGE = CATIONIC (CTAB)3. EFFECTIVE AT BREAKING PROTEIN: PROTEIN INTERACTIONS4.

2 THEREFORE OFTEN DENATURING5. SIMILARLY CHARGED HEADGROUPS REPEL6. THEREFORE MICELLE SIZE DEPENDENT ON: A. COUNTERION TYPE/CONC- NEUTRALIZATION = REDUCED CURVATURE = INCREASED MICELLE SIZE. B. CHAIN LENGTH & SHAPE OF HYDROPHOBIC REGIONBILE ACID DETERGENTS CONTAINING RIGID STEROIDAL CLEARLY DEFINED HEADGROUP (POLAR/APOLAR FACE)DUE TO HYDROXYLATION OF THE STEROIDAL SHAPED MICELLES-SIZE DEPENDENT TO LOW pKa (5-6) AND LOW SOLUBILITY OFUNCONJUGATED BILE SALTS ONLY USEFUL IN ALKALINERANGE. CONJUGATED BILE SALTS CAN BE USED OVER ABROADER pH DETERGENTS1. NON-IONIC DETERGENTS ARE GOOD AT BREAKING LIPID:LIPIDAND LIPID:PROTEIN CONTACTS2. NOT SO GOOD AT BREAKING PROTEIN:PROTEIN CONTACTS3. THEREFORE PROTECT PROTEIN FUNCTION4. MICELLE SIZE NOT GREATLY EFFECTED BY COUNTERIONS5, MICELLE SIZE IS EFFECTED BY SALT HEADGROUPS ( BRIJ)- CAN FORM RANDOM COIL STRUCTURES- SHORT CHAIN POLYOXYETHELENES FORM AGGREGATES AND VISCOUS SOLUTIONS IN WATER AT ROOM TEMPERATURE-LONG CHAIN POLYOXYETHELENES DO NOT AGGREGATE2.

3 POLYOXYETHELENE HEADGROUPS COUPLED TO PHENOL RING( TRITON)- HIGH ABSORPTION AT 280NM- TRITON CAN OFTEN CONTAIN PEROXIDE CONTAMINATION- OFTEN USED DURING PURIFICATION (CHEAP, LOW CMC)3. SUGAR HEADGROUPS ( MALTOSIDES, GLUCOSIDES)- HOMOGENEOUS STRUCTURE (but check and conformations)- SELECTION OF HYDROPHOBIC CHAIN LENGTHS (C6-C12)STRAIGHT, AS WELL AS CYCLIC) AND HEADGROUPS (MALTOSE,SUCROSE, GLUCOSE)IMPORTANT PROPERTIES OF NONIONIC DETERGENTS : PURE, WELL DEFINED MICELLES, PROTECT AGAINST DENATURATIONZWITTERIONIC DETERGENTSOFFER COMBINED PROPERTIES OF IONIC AND NET CHARGE = LACK CONDUCTIVITY, ELECTROPHORETICMOBILITY AND ABILITY TO BIND TO ION EXCHANGE LIKE IONIC DETERGENTS GOOD AT BREAKINGPROTEIN:PROTEIN STEROID RING OF CHAPS THOUGHT TO MAKE IT LESSDENATURINGBHAIRI, (2001) A GUIDE TO THE PROPERTIES AND USES OF DETERGENTS IN BIOLOGY WEBSITEBHAIRI, (2001) A GUIDE TO THE PROPERTIES AND USES OF DETERGENTS IN BIOLOGY ANDBIOCHEMISTRY.

4 CALBIOCHEM WEBSITEBHAIRI, (2001) A GUIDE TO THE PROPERTIES AND USES OF DETERGENTS IN BIOLOGY ANDBIOCHEMISTRY. CALBIOCHEM WEBSITEGLUCOPYRANOSIDESNON-IONIC DETERGENTS IN MORE DETAILALKYL GLUCOPYRANOSIDES (THIO-GLYCOSIDES ARE LESS POLAR)CMC=250mMLow solubilityALKYL MALTOPYRANOSIDESOFTEN MILDER AND MORE EFFECTIVE THAN GLUCS. INCREASED SOLUBILITYSUCROSE BASED DETERGENTSMILD AND USEFUL FOR EXTRACTION BUT ESTERS ..EASILY HYDROLYSED,ALSO A MIXTURE OF ISOMERSANATRACE CATALOGUENONIONIC DETERGENTS IN MORE DETAILMEGA SERIES: GLUCOSE DERIVATIVES WITH AMIDE LINKAGECHEAP, LOW SOLUBILITY EXCEPT FOR MEGA 8 ANATRACE CATALOGUEHEGA SERIES: GLUCOSE DERIVATIVES WITH AMIDE LINKAGE, PLUS ADDITIONAL OH277mM CMC108mM CMC 35 mM CMC 11 mM CMCINCREASED SOLUBILITYCYMALCYMAL: CYCLIC MALTOSIDEHIGHER CMCCYCLOHEXYL TAILS PACK MORE HYDROPHOBICITY INTO A SHORTER EFFECTIVE CHAIN , GARAVITOANATRACE CATALOGUEHIGER CMC VALUES THAN CORRESPONDING LINEAR DETERGENTALTERNATIVE AMPHIPATHIC MOLECULESP.

5 Nollert 2005 Progress in Biophysics and molecular Biology 88:339 357 SCHAFMEISTER ET AL 1993 A 24-AMINO ACID PEPTIDE DESIGNED TO SOLUBILIZE INTEGRAL MEMBRANE PROTEINS. DESIGNED AS AN AMPHIPATHIC ALPHA HELIX WITH A "FLAT" HYDROPHOBIC SURFACETHAT INTERACTS WITH A TRANSMEMBRANE DOMAIN AS A DETERGENT. WHEN MIXED WITH PEPTIDE, 85 PERCENT OF BACTERIORHODOPSIN AND 60 PERCENT OF RHODOPSIN REMAINED IN SOLUTION OVER A PERIOD OF 2 DAYSIN THEIR NATIVE ET AL 1996 PNAS 93 = AMPHIPHILIC HYDROPHOBIC PHASE TRANSITIONS FOR CRYSTALLIZATIONALTERNATIVE AMPHIPATHIC MOLECULESREVIEW: P. Nollert 2005 Progress in Biophysics and molecular Biology 88:339 357articlesYU ET AL 2000 PROT SCI 9 PORTION CONTAIN AMIDE AND AMINE TO DISPLAY GREATER RIGIDITY (TETRA-SUBSTITUTED C). RIGIDITY PROPOSED TO INCREASE ABILITY TO CRYSTALLIZE articlesMCGREGOR ET AL 2003 NAT BIOTECH 21 LIPOPROTEINS. BILAYER ASSEMBLE INTO A CYLINDRICAL OUTER SHELL (PROTECTIVE) INNER LIPID PHOSPHLIPID (PRESERVE MEMBRANE PROTEIN STRUCTURES)INTRINSIC CURVATURE HYPOTHESIS (Gruner 1985) According to the intrinsic curvature hypothesis (Gruner, 1985) amphiphiles form supramolecularstructures in water due to the hydrophobic effect (Tanford, 1980) and THEIR (1991)INTERMOLECULAR AND SURFACEFORCES (2/E)2.

6 ISRAELACHVILI, MITCHELL & NINHAM1977 BBA470, , 1985. INTRINSIC CURVATUREHYPOTHESIS FOR BIOMEMBRANELIPID COMPOSITION: A ROLE FORNONBILAYER LIPIDS. PNAS 82 , C., 1980. THEHYDROPHOBIC EFFECT. WILEY, forms a highly ordered network of intermolecular hydrogen It is the strength of all of the hydrogen bonds combined that give water its liquid PROPERTIES . 3. Polar or hydrophilic substances dissolve in water because they form hydrogen bonds and electrostatic interactions with water Non-polar or hydrophobic substances are unable to form these interactions and consequently are immiscible with Addition of non-polar substances to water disrupts intermolecular hydrogen bonds of water molecules and creates a cavity which is devoid of water. At the surface of the cavity, water molecules rearrange in an orderly , AMPHIPHILES AND HYDROPHOBIC INTERACTIONS6. This results in a thermodynamicallyunfavourable decrease in entropy(decrease in disorder).

7 7. To compensate for this loss of entropy,water molecules force the hydrophobicmolecules to cluster and thus occupythe minimum This effect is known as the hydrophobiceffect and the forces betweenhydrophobic regions ( membranespanning domains of membraneproteins) are called PHASE LOW TEMPERATURE, DETERGENTS LARGELY IN CRYSTALLINE PHASE2. AS TEMPERATURE RISES DETERGENT REACHES AN EQUILIBRIUM BETWEEN CRYSTALLINE AND MONOMER PHASE3. AT THE KRAFT POINT, SOLID, MONOMER AND MICELLES ARE IN EQUILIBRIUM4. CRITICAL MICELLE CONCENTRATION (CMC) THE LOWEST DETERGENT CONCENTRATION ABOVE WHICH MONOMERS CLUSTER INTO CRITICAL MICELLE TEMPERATURE (CMT) THE TEMPERATURE AT WHICH MICELLES FORM FROM PHASE TO BE IN: FOR PURIFICATION,STABILIZATION AND AT THE START OF 2D CRYSTALLIZATION. A PARTICULAR TEMPERATURE ABOVE THE CMT, AT WHICH NON-IONICDETERGENTS UNDERGO PHASE SEPARATION TO YIELD A DETERGENT RICH AND ADETERGENT POOR AQUEOUS BY DECREASED HYDRATION OF THE X-100 CLOUD POINT = 64oCTRITON X-114 CLOUD POINT = 22oCUSES: MEMBRANE PROTEINS CAN BE SOLUBILIZED AT 0oC, BEFORE BEINGPURIFIED FROM SOLUBLE PROTEINS BY WARMING TO 30oC, SO THAT TWO PHASESARE FORMED, WITH THE MP BEING ENRICHED IN THE DETERGENT RICH (mM) (mM)CHAINLENGTH GLUCOSIDESMALTOSIDESEFFECT OF CHAIN LENGTH ON MALTOSIDE CMC051015202589101112 CHAIN LENGTHCMC (mM)EFFECT OF CHAIN LENGTH ON GLUCOSIDE CMC0501001502002506789101112 CHAIN LENGTHCMC (mM)EFFECT OF CHAIN LENGTH ON CMC1.

8 CMC DECREASES WITH INCREASEDCHAIN LENGTH2. CMC INCREASES WITH DESATURATION3. ADDITIVES THAT BREAK UP WATERSTRUCTURE (EG. UREA) INCREASE LOW CMC DESIREABLE FOR REDUCEDCOST OF PURIFICATION5. HIGH CMC DESIRABLE FORDETERGENT REMOVAL6. LONG CHAIN LENGTHS = PROTECTIVEBUT STERIC HINDRANCE IN 3D CMC DOES VARY: USEFUL TO DEFINE IT ACCURATELYAGGREGATION NUMBER = MICELLAR molecular WEIGHT MONOMER molecular WEIGHTAGGREGATION NUMBERDYE SOLUBILIZATION (DS) ABSORBANCELIGHT SCATTERING (LS) SURFACE TENSION (DYNE/CM)DETERGENT CONCENTRATION (mM)DETERMINING CRITICAL MICELLE CONCENTRATION[MICELLE] = ([DET]-CMC)/AGG. , (2001) A GUIDE TO THE PROPERTIES ANDUSES OF DETERGENTS IN BIOLOGY AND WEBSITEA NOVEL METHOD FOR DETERGENT CONCENTRATION DETERMINATIONKAUFMANN ET AL 2006 BIOPHYS J. 90:1310-317 ($US 2500-4000) DROPLET ON MOLECULES AT SURFACE LESSENERGETICALLY FAVOURABLE THANTHOSE IN SYSTEM TRIES TO REDUCESURFACE OF DET TO SURFACEDISTURBS THE ORDERING OF LEADS TO A DROP IN SURFACETENSION AND A SPREADING OF THEDROPLET (20 l).

9 STABLE AFTER SPREADING IS MEASURED BY THEYOUNG CONTACT ACTIVE COMPOUNDS (GLYCEROL/PEG/LIPIDS) INTERFERE:CALIBRATEA NOVEL METHOD FOR DETERGENT CONCENTRATION DETERMINATIONKAUFMANN ET AL 2006 BIOPHYS J. 90:1310-317 RAWIMAGESTHRESHOLDAPPLIEDDROPLETCONTOURS CONTACTANGLES &MEANVOLUMEACCURACY OF CMC DETERMINATIONSURFACE TENSION RELATED TO DETERGENTS REDUCESURFACE TENSION MORE EFFECTIVELYTHAN CHARGED DETERGENTS HAVE A LIMITEDCAPACITY TO BIND TO THE AIR:WATERINTERFACE (CHARGE REPULSION) LDAO AND OTHER IONIC DETERGENTSCMC IS DEPENDENT ON IONIC STRENGTH(AND pH). ELECTROSTATIC INTERACTIONOF BULKIER THE HEADGROUP, THE LESSTHEY REDUCE SURFACE CHAIN REDUCES CMCCMCCMC USUALLY PURIFIED IN EXCESS WITH LARGE MICELLES ( DDM) ENRICHED DURING EXPOSURE REDUCES CONCENTRATIONS INCREASE CRYSTALLIZATION MICELLES CAN INTERFERE WITH CRYSTAL CONTACTSOPTIMIZING LIPID:PROTEIN RATIOPROTEIN PURIFICATION: SUCROSE GRADIENT CENTRIFUGATIONIMPORTANT PROPERTIES OF DETERGENTSTO CONSIDER FOR ( and DDM) TO SOLUBILIZE AGAINST CONCENTRATIONS (COST)

10 TO EXCHANGE AND REMOVE1 Fos-Fenth2 Nonopol-Fos3 Fos-choline4 Fos-choline5 Fos-choline6 Fos-choline7 C8- -D-thioglucoside8 C7- -D-glucoside9 C8- -D-glucoside10 C9- -D-glucoside11 C10- -D-glucoside12 C8- -D-thiomaltoside13 C8- -D-maltoside14 C10- -D-maltoside15 C11- -D-maltoside16 C12- -D-maltoside17 HEGA818 HEGA919 HEGA1020 HEGA1121 CYMAL322 CYMAL423 CYMAL524 CYMAL625 C8E426 C8E527 C10E428 C10E529 C12E430 C12E531 C12E1032 Brij 3533 Brij 7634 Brij 7835 Brij 9336 Brij 9637 CHAPS38 Big CHAPS39 DDAO 2640 Sodium cholate41 Sarkosyl42 Digitonin43 C7- -D-thioglucoside45 C9- -D-thioglucoside46 C10- -D-thioglucoside48 C9- -D-thiomaltoside49 C10- -D-thiomaltoside50 C12- -D-thiomaltoside51 Surfonyl 48552 Triton X-30553 Triton X-40554 Triton X-70555 Tween 2056 Brij 70057 PEG 4000 distearate58 PEG 6000 distearateRelating Surfactant PROPERTIES to Activity and Solubilization of theHuman Adenosine A3 Receptor Berger BW et al 2005 Biophys J. 89: 452-462 Relating Surfactant PROPERTIES to Activity and Solubilization of theHuman Adenosine A3 Receptor Berger BW et al 2005 Biophys J.