HIC media shown in Fig.3 are all based on the glycidyl ether coupling procedure b-p interactions.The glycidyl-ether coupling technique will introduce a short spacer but the effect of this will be very limited since the short hydrophobic chain is neutralized"with the hydrophilic OH-group. Fig.3. OH oph obic A -O-CH-CH-CH-O-(CH)CH3 dt hyd cross-linked agarose Butyl Sepharose 4 Fast Flow matrices. OH B -O-CH-CH-CH-O-(CH)/CH Octyl Sepharose CL-4B OH -0-CH CH-CH0○》 Phe Phenyl Sepharose CL-4B Phenyl Sepnarose 6 OH O-CH-CH-CH-O-CH-C(CH)3 Alkyl Superose Degree of substitution The protein binding capacities of HIC adsorbents increase with increased degree of substitution of immobilized ligand.At a sufficiently high degree of ligand substitution, the apparent binding capacity of the adsorbent ons (plateau is reached) but the strength of the interaction increases(31-33,35)(Fig.2B).Solutes bound under such circumstances are difficult to elute due to multi-point attachment(34). Type of base matrix It is important not to overlook thec contribution of the ba ase matrix.The two mo widely used types of support are strongly,ecros-inke agarose,or synthetic copolymer materials.The selectivity of a copolymer support will not be exactly the same as for an agarose based support substituted with the same type of ligand. To achieve the same type of results on an agarose-based matrix as on a copolymer support,it may be necessary to modify adsorption and elution conditions. 16
16 HIC media shown in Fig. 3 are all based on the glycidyl ether coupling procedure, which produces gels that are charge free and that should thus only have hydrophobic interactions with proteins. The phenyl group shown in Fig. 3-C also has a potential for þ-þ interactions. The glycidyl-ether coupling technique will introduce a short spacer but the effect of this will be very limited since the short hydrophobic chain is ‘‘neutralized’’ with the hydrophilic OH-group. Degree of substitution The protein binding capacities of HIC adsorbents increase with increased degree of substitution of immobilized ligand. At a sufficiently high degree of ligand substitution, the apparent binding capacity of the adsorbent remains constant (plateau is reached) but the strength of the interaction increases (31–33, 35) (Fig. 2B). Solutes bound under such circumstances are difficult to elute due to multi-point attachment (34). Type of base matrix It is important not to overlook the contribution of the base matrix. The two most widely used types of support are strongly hydrophilic carbohydrates, e.g. cross-linked agarose, or synthetic copolymer materials. The selectivity of a copolymer support will not be exactly the same as for an agarose based support substituted with the same type of ligand. To achieve the same type of results on an agarose-based matrix as on a copolymer support, it may be necessary to modify adsorption and elution conditions. –O–CH –CH–CH –O–(CH ) –CH 2 2 2 3 3 – OH –O–CH –CH–CH –O–(CH ) –CH 2 2 2 7 3 – OH Butyl Sepharose 4 Fast Flow Octyl Sepharose CL-4B –O–CH –CH–CH –O– 2 2 – OH Phenyl Superose Phenyl Sepharose High Performance Phenyl Sepharose CL-4B Phenyl Sepharose 6 Fast Flow (low sub) Phenyl Sepharose 6 Fast Flow (high sub) –O–CH –CH–CH –O–CH –C(CH ) 2 2 2 3 – OH Alkyl Superose 3 A B C D Fig. 3. Different hydrophobic ligands coupled to cross-linked agarose matrices
Type and concentration of salt The addition of various structure-forming(salting ou)saltsto buffer and sample solution promotes ligand-protein interactions in HIC(10,12,36 65,66).As the concentration of such salts is increased,the amount of proteins bound also increases almost linearly up to a specific salt concentration and continues to increase in an exponential manner at still higher concentrations This latter phenomenon is demonstrated in Fig.4 where total binding capacity of Phenyl Sepharose High Performance for a-chymotrypsinogen and RNAse was exami- ned at gradually increasing salt concentrations. Phenyl Sepharose High 80 nce as a tu ion of ■RNA 60 m) 40 3 Initial salt concentration M(NH)SO In this experiment,the column was first equilibrated with buffer containing varying concentrations of salt as indicated in the Figure.The sample was dissolved in buffe including this initial salt concentration prior to application to th umn.However. in those experiments where the protein begins to precipitate at high salt concentration (1.3 Mand2.3 Mammoniumsulphate for a-chymotrypsinogen and RNAse respectively) the sample was dissolved at a slightly lower salt concentration. The samples were loaded on the column until breakthrough could be observed at the column outlet.Then start buffer with initial salt concentration was run through the column until UV-absorption in the eluent returned to the baseline.Finally,the bound proteins were eluted with a decreasing salt gradient. A significant increase in adsorption capacity can be seen when the salt concentratior is increased above the precipitation point. 17
17 Type and concentration of salt The addition of various structure-forming (‘‘salting out’’) salts to the equilibration buffer and sample solution promotes ligand-protein interactions in HIC (10, 12, 36, 65, 66). As the concentration of such salts is increased, the amount of proteins bound also increases almost linearly up to a specific salt concentration and continues to increase in an exponential manner at still higher concentrations. This latter phenomenon is demonstrated in Fig. 4 where total binding capacity of Phenyl Sepharose High Performance for a-chymotrypsinogen and RNAse was examined at gradually increasing salt concentrations. 1 20 3 40 60 80 2 4 Protein capacity mg/ml packed bed Initial salt concentration M (NH ) SO α-chymotrypsinogen RNAse 4 2 4 In this experiment, the column was first equilibrated with buffer containing varying concentrations of salt as indicated in the Figure. The sample was dissolved in buffer including this initial salt concentration prior to application to the column. However, in those experiments where the protein begins to precipitate at high salt concentration (1.3 M and 2.3 M ammonium sulphate for a-chymotrypsinogen and RNAse respectively) the sample was dissolved at a slightly lower salt concentration. The samples were loaded on the column until breakthrough could be observed at the column outlet. Then start buffer with initial salt concentration was run through the column until UV-absorption in the eluent returned to the baseline. Finally, the bound proteins were eluted with a decreasing salt gradient. A significant increase in adsorption capacity can be seen when the salt concentration is increased above the precipitation point. Fig. 4. Protein binding capacity on Phenyl Sepharose High Performance as a function of salt concentration in the column equilibration buffer (Work from Amersham Pharmacia Biotech, Uppsala, Sweden)
This phenomenon is probably due to the precipitation of proteins on the column. It has a concomitant negative effect on the selectivity of the HIC adsorbent. The effects of salts in HICcan be accounted for by reference to the Hofmeister serie for the precipitation of proteins or for their positive influence in increasing the molal surface tension of water (for extensive review,see refs.27,29).These effects are summarized in Tables 1 and 2. -Increasing precipitation("salting -out")effect series on the effect Anions::Po3,SO,2,CH3·CoO,Ct,Br,NO,CLO,上，SCN of some anions and Cations:NH",Rb",K",Na',Cs",Li",Mg2,Ca2,Ba2 tating Increasing chaotropic("salting-in)effect proteins. Kote3eotectsot Na,SO >KSO>(NH),SO >Na,HPO >NaCl>LiCI.>KSCN some salts on the In both instances,sodium,potassium or ammonium sulphates produce relatively higher"salting-out"(precipitation)or molal surface tension increment effects.It is also these salts that effectively promote ligand-protein interactions in HIC.Most of the bound proteins are effectively desorbed by simply washing the HICadsorbent with water or dilute buffer solutions at near neutral pH. Effect of pH The effect of pH in HIC is also not straightforward.In general,an increase in pH weakens hydrophobic interactions(10,41),probably as a result of increased titration of charged groups,thereby leading to ase in the hydrophilicity of the proteins On the other hand,a decrease in pH results in an apparent increase in hydrophobi interactions.Thus,proteins which do not bind to a HIC adsorbent at neutral pH bind at acidic pH(9).Hierten et al.(42)found that the retention of proteins changed more drastically at pH values above85 and/or below 5 than in the range pH5-8.5(Fig5). These findings suggest that pH is an important separation parameter in the optimization of hydrophobic interaction chromatography and it is advisable to check the applicability of these observations to the particular separation problem at hand. 18
18 This phenomenon is probably due to the precipitation of proteins on the column. It has a concomitant negative effect on the selectivity of the HIC adsorbent. The effects of salts in HIC can be accounted for by reference to the Hofmeister series for the precipitation of proteins or for their positive influence in increasing the molal surface tension of water (for extensive review, see refs. 27,29). These effects are summarized in Tables 1 and 2. In both instances, sodium, potassium or ammonium sulphates produce relatively higher ‘‘salting-out’’ (precipitation) or molal surface tension increment effects. It is also these salts that effectively promote ligand-protein interactions in HIC. Most of the bound proteins are effectively desorbed by simply washing the HIC adsorbent with water or dilute buffer solutions at near neutral pH. Effect of pH The effect of pH in HIC is also not straightforward. In general, an increase in pH weakens hydrophobic interactions (10,41), probably as a result of increased titration of charged groups, thereby leading to an increase in the hydrophilicity of the proteins. On the other hand, a decrease in pH results in an apparent increase in hydrophobic interactions. Thus, proteins which do not bind to a HIC adsorbent at neutral pH bind at acidic pH (9). Hjertén et al. (42) found that the retention of proteins changed more drastically at pH values above 8.5 and/or below 5 than in the range pH 5–8.5 (Fig 5). These findings suggest that pH is an important separation parameter in the optimization of hydrophobic interaction chromatography and it is advisable to check the applicability of these observations to the particular separation problem at hand. Increasing precipitation (‘‘salting -out’’) effect Anions: PO4 3– , SO4 2– , CH3 • COO– , Cl– , Br– , NO3 – , CLO4 – , I– , SCN– Cations: NH4 +, Rb+, K+ , Na+ , Cs+ , Li+, Mg2+, Ca2+, Ba2+ Increasing chaotropic (‘‘salting-in’’) effect Table 1. The Hofmeister series on the effect of some anions and cations in precipitating proteins. Table 2. Relative effects of some salts on the molal surface tension of water. t t Na2 SO4 >K2 SO4 >(NH4 )2 SO4 >Na2 HPO4 >NaCl>LiCl. . . >KSCN
Ve/VT 5 2 41 (NH SOa- 4 6 8.5 8.5 10 pH 12 Fig.5. The pH dependence of the interaction between proteins and an octyl agarose gel expressed as V(V is the elut or the dierent pro s and Vr is the t of salt The m del proteins used were ST=soy try psin inhibitor.Ahuman serum albumin,L=lys zyme, T=transferrin,E=enolase,O=ovalbumin,R=ribonuclease, Effect of temperature Based on theories developed for the interaction of hydrophobic solutes in water (22,37),Hjerten (38)propos ed that the binding of f proteins to HIC adsorbents is entropy driven [DG=(DH-TDS)~-TDS],which implies that the interaction increases with an increase in temperature.Experimental evidence to this effect has been presented by Hierten(25)and Jennissen (34).It is interesting to note that the van der rease with increase in temperature(39).However,an opposite effect was reported by Visser Strating(40)indicating that the role of temperature in HIC is of a complex nature. This apparent discrepancy is probably due to the differential effects exerted by temperatur on the conforma al state of different proteins and their solubilities ir aqueous solutions. In practical terms,one should thus be aware that a downstream purification process developed at room temperature might not be reproduced in the cold room,or vice versa. 19
19 Fig. 5. The pH dependence of the interaction between proteins and an octyl agarose gel expressed as Ve/VT (Ve is the elution volume of the different proteins and VT is the elution volume of a non-retarded solute). Elution was by a negative linear gradient of salt. The model proteins used were STI=soy trypsin inhibitor, A=human serum albumin, L=lysozyme, T=transferrin, E=enolase, O=ovalbumin, R=ribonuclease, ETI=egg trypsin inhibitor and C=cytochrome c. (Reproduced with permission, from ref. 42). Effect of temperature Based on theories developed for the interaction of hydrophobic solutes in water (22,37), Hjertén (38) proposed that the binding of proteins to HIC adsorbents is entropy driven [ ÐG = (ÐH-TÐS) ~ -TÐS], which implies that the interaction increases with an increase in temperature. Experimental evidence to this effect has been presented by Hjertén (25) and Jennissen (34). It is interesting to note that the van der Waals attraction forces, which operate in hydrophobic interactions (29), also increase with increase in temperature (39). However, an opposite effect was reported by Visser & Strating (40) indicating that the role of temperature in HIC is of a complex nature. This apparent discrepancy is probably due to the differential effects exerted by temperature on the conformational state of different proteins and their solubilities in aqueous solutions. In practical terms, one should thus be aware that a downstream purification process developed at room temperature might not be reproduced in the cold room, or vice versa
Additives Low concentrations of water-miscible alcohols,detergents and aqueous solutions und solutes.Then n-polar r parts of alcohols and detergents compete effectively with the bound proteins for the adsorption sites on the HIC media resulting in the displacement of the latter.Chaotropic salts affect the ordered structure of water and/or that of the bound proteins.Both types of additives also decrease the surface tension ofwater(see Table 3)thus weakening the hydrophobic interactions to give a subsequent dissociation of the ligand-solute complex. Although additives can be used in the elution buffer to affect selectivity during desorption,there is a risk that proteins could be denatured or inactivated by exposure to high con rations of such chemicas.However,additivescan be very effectivei cleaning up HIC columns that have strongly hydrophobic proteins bound to the gel medium. Viscosity Surface tension Physical properties of some Solvent (dynes/cm) solvents used in HIC(data at 25C). Water 0.89 78.3 72.00 pepnomamde 2n 2371 20
20 Viscosity Dielectric Surface tension Solvent (centipoise) constant (dynes/cm) Water 0.89 78.3 72.00 Ethylene glycol 16.90 40.7 46.70 Dimethyl Sulphoxide 1.96 46.7 43.54 Dimethyl Formamide 0.796 36.71 36.76 n-propanol 2.00 20.33 23.71 Table 3. Physical properties of some solvents used in HIC (data at 25 oC). Additives Low concentrations of water-miscible alcohols, detergents and aqueous solutions of chaotropic (‘‘salting-in’’) salts result in a weakening of the protein-ligand interactions in HIC leading to the desorption of the bound solutes. The non-polar parts of alcohols and detergents compete effectively with the bound proteins for the adsorption sites on the HIC media resulting in the displacement of the latter. Chaotropic salts affect the ordered structure of water and/or that of the bound proteins. Both types of additives also decrease the surface tension of water (see Table 3) thus weakening the hydrophobic interactions to give a subsequent dissociation of the ligand-solute complex. Although additives can be used in the elution buffer to affect selectivity during desorption, there is a risk that proteins could be denatured or inactivated by exposure to high concentrations of such chemicals. However, additives can be very effective in cleaning up HIC columns that have strongly hydrophobic proteins bound to the gel medium