Hydrophobic Interaction Chromatography

Herbert P Jennissen, University of Essen, Essen, Germany

Hydrophobic interaction chromatography involves the separation of protein molecules owing to the differential interaction of these molecules with hydrophobic sites on the surface of a solid support. In the separation process, hydrophobic patches on the protein interact with hydrophobic molecules (e.g. alkyl residues) immobilized on the hydrophilic solid phase surface (agarose).

Secondary article
Article Contents
. Introduction . Essentials of Hydrophobic Interaction Chromatography . Chromatographic Techniques and Applications . Conclusions

Introduction
Two fundamental chromatographic systems employing hydrophobic media have been described: (a) reversedphase and (b) hydrophobic interaction chromatography. In hydrophobic interaction chromatography the solutes (proteins) are adsorbed and separated on a stationary solid phase (i.e. a two-dimensional system) carrying immobilized hydrophobic groups. In reversed-phase chromatography the solutes are absorbed and separated (partitioned) in the apolar stationary liquid phase (i.e. a three-dimensional system) and not on the solid phase. Because of the very dierent scope and methodological details, reversed-phase chromatography will not be treated here. The same holds for hydrophobic forms of liquid liquid partition chromatography. A dierentiation will also not be made between classical chromatographic systems and HPLC (high-pressure or high-performance liquid chromatography) since in essence only the low bead size and monodispersity, not the hydrophobic surface, lead to the higher performance (e.g. throughput and resolution) in the latter method. Noncovalent interactions of the hydrophobic type have been described as the unusually strong attraction between nonpolar molecules and surfaces in water (Israelachvili, 1985). Hydrophobic interactions are driven by the extrusion of a monomolecular layer of ordered water molecules covering two adjacent hydrophobic surfaces into lessordered bulk water with a concomitant increase in entropy. Under the condition of DH ! T DS the Gibbs free energy is negative ( 2 DG % T DS) thus constituting an entropydriven reaction. This entropy-driven attraction between nonpolar groups in water is the basis for hydrophobic interaction chromatography (for review see Jennissen, 2000). In 1972 the group of Yon and the group of Shaltiel independently reported the chromatographic separation of proteins by way of hydrophobic interactions (Er-el et al., 1972; Yon, 1972). In both cases the hydrophobic matrix consisted of agarose (Sepharose 4B, Pharmacia) to which aminoalkane derivatives had been coupled by the CNBr

(cyanogen bromide) method (for reviews see Jennissen, 1995; Shaltiel, 1984; Yon, 1977). The surprising result in Shaltiels experiments (Er-el et al., 1972) was that a normal hydrophilic enzyme, phosphorylase b, could be puried on a C4-hydrocarbon-coated agarose to near homogeneity in one step, implicitly questioning the general doctrine of the time that all hydrophobic amino acids are buried in the interior of proteins. Phosphorylase was adsorbed at low ionic strength on immobilized butyl residues that had no resemblance to the substrates of the enzyme (thus excluding a bioanity type of adsorption) and was eluted by a deforming buer, which led to a limited, reversible unfolding of the enzyme. From this work it was generally concluded that here was a novel method applicable not only to hydrophobic or lipophilic but also to hydrophilic and possibly to all proteins. The name hydrophobic chromatography coined by Shaltiel therefore soon came to widespread use. However, it remained a problem that the positive charges, introduced into the matrix by a side reaction in the CNBr procedure (for review see Jennissen, 1995), into the hydrophobic gels could be inuencing the chromatographic results on hydrocarbon-coated agaroses. In a later paper, Shaltiels group showed conclusively (Halperin et al., 1981) that under their conditions the inuence of charges in his hydrocarbon-coated agaroses had been small to negligible. The charge problem was nally claried by Wilcheks group (Kohn and Wilchek, 1981), who found that, depending on the conditions (i.e. pH), pure charged isourea gels, pure uncharged imidocarbonate/carbamate gels or mixed ionichydrophobic gels could all be obtained by the CNBr procedure. In addition, various other groups n, 1973; Hofstee and Otillio, 1978; Jennissen, (Hjerte 1976a) also showed that neutral salts eectively quenched the charges introduced by the CNBr method, allowing the purication of proteins by hydrophobic interaction criteria. Nevertheless, it became clear in the coming years that uncharged hydrophobic gels would be advantageous to CNBr-type gels. The rst uncharged hydrophobic gels were synthesized in 1973 by Poraths group (Porath et al., 1973), who reacted
1

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Hydrophobic Interaction Chromatography

benzyl chloride with agarose at high temperatures. The synthesis of a graded homologous series of hydrocarboncoated agaroses was, however, not possible by this method. In addition, Porath demonstrated the inverse salt behaviour of proteins adsorbed on such gels. In contrast to ion exchangers, proteins were applied to these gels at high salt concentrations and eluted by decreasing the ionic strength (negative or inverse salt gradients). Interestingly, the protein cytochrome c was adsorbed when 13 mol L 2 1 NaCl was included in the buer, a salt which in itself had very little salting-out potential. In the same year Hjerten n, 1973) showed that high salt concentrations also (Hjerte enhanced the binding of proteins to hydrophobic gels synthesized by the CNBr procedure, thus demonstrating that Shaltiel-type gels had similar properties to the Porathtype gels (see also Jennissen, 1976a). Hjerten also suggested the term hydrophobic interaction chromatography, n, 1973). Finally in 1974 which is popular today (Hjerte n et al., 1974) described the synthesis of Hjerten (Hjerte uncharged hydrophobic alkyl agarose gels via the glycidyl ether method and the preparation of uncharged homologous series of hydrocarbon-coated gels. For other methods of synthesizing homologous series, see Jennissen (2000). In conclusion, although there is no doubt that fully uncharged hydrophobic gels are superior to the CNBrprepared gels, by virtue of displaying a practically pure type of noncovalent, i.e. hydrophobic, interaction, it appears that all groups involved in the discovery and development of hydrophobic (interaction) chromatography observed the binding and fractionation of proteins by predominantly hydrophobic interactions. Concerning the nomenclature, both terms hydrophobic chromatography and hydrophobic interaction chromatography can be used synonymously, the shorter term hydrophobic chromatography being no more a misnomer than anity chromatography.

Pharmacia) contains $ 5 106 beads (Jennissen and Heilmeyer, 1975) and $ 30 mg of dry polysaccharide (Jennissen, 1976b) per mL of packed gel. The agarose beads contain pores for which a pore radius of $ 8090 nm has been calculated (Jennissen and Botzet, 1979; Demiroglou et al., 1989). For the large protein molecule phosphorylase b (197 kDa) the specic surface area of agarose has been calculated as 89 m2 per mL of packed gel (Jennissen, 1981). Agarose can be maximally substituted with alkyl residues to $ 50100 mmol per mL of packed gel. However, under these conditions the gel usually shrinks up to 50% (Demiroglou and Jennissen, 1990). Optimal degrees of substitution without gel contraction lie in the range 130 mmol per mL packed gel. Present data strongly indicate that substitution procedures involving an initial activation step followed by a coupling step, as in the CNBr procedure, lead to a statistically uniform distribution of the immobilized alkyl residues on the gel (Amsterdam et al., 1974). In conclusion, a coupling procedure (see Hermanson et al. (1992) for the alkyl groups should be chosen that is simple, shows a statistical distribution, does not introduce charges, and shows no leakage of residues (see Jennissen, 1979, 2000).

The chain length parameter

The rst systematic approach to the purication of proteins via hydrophobic interactions was reported by Shaltiel (Er-el et al., 1972) who proposed the idea of varying the immobilized alkyl chain length in the form of a homologous series of hydrocarbon-coated agaroses (SephCn, n 5 110; see Figure 1). The major conclusion of these experiments was that an increase of the chain length by CH2 units concomitantly increased the strength of protein binding from retardation to reversible binding up to very tight binding. In addition to this variation in binding anity with the chain length, the gels also changed their specicity towards the adsorbed protein. Thus it was suggested that the properties of hydrophobic agaroses for protein purication could be optimally controlled by variation of the immobilized alkyl chain length and nding the optimal length for adsorbing and eluting the protein.

Essentials of Hydrophobic Interaction Chromatography

The stationary phase
One of the prerequisites for an optimal chromatographic separation of proteins by hydrophobic interactions is an extremely hydrophilic, minimally interactive stationary phase. Agarose optimally meets both of these criteria. Besides being hydrophilic by virtue of its hydrogel structure, it displays an extremely low nonspecic adsorption of proteins. Agarose consists of a hierarchy of structures (for reviews see Demiroglou et al., 1989; Jennissen, 1995) leading to a paracrystalline structure with extensive areas for protein adsorption (Demiroglou et al., 1989). A beaded agarose gel of 4% agarose (Sepharose 4B,
2

The surface concentration parameter

Critical surface concentration of immobilized residues However, in 1975 it was shown that a second requirement is of equal if not greater importance for optimizing hydrophobic supports (Jennissen and Heilmeyer, 1975). It was found that a grading of hydrophobicity could also be produced by simply increasing the degree of substitution (surface concentration) of the agarose with alkyl residues (Figure 2). If, instead of the chain length, the surface concentration of immobilized alkyl groups (i.e. surface concentration series) is varied, protein adsorption is a

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Hydrophobic Interaction Chromatography

CO CO CO CO CO CO CO CO CO CO

NH NH NH NH NH NH NH NH NH NH

CH 3 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2

CH 3 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2

CH 3 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2

CH 3 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2

CH 3 CH 2 CH 2 CH 2 CH 2 CH 2

CH 3 CH 2 CH 2 CH 2 CH 2

CH 3 CH 2 CH 2 CH 2

CH 3 CH 2 CH 2

CH 3 CH 2

CH 3

Seph-C1 Seph-C2 Seph-C3 Seph-C4 Seph-C5 Seph-C6 Seph-C7 Seph-C8 Seph-C9 Seph-C10

Figure 1 Example of the variation of the chain-length parameter in an uncharged homologous series of alkyl agaroses according to the concept of Shaltiel. For an optimal series it is understood that the surface concentration of the immobilized residues is constant, that no charges are introduced and that leakage is low. The uncharged series of the above type is obtained by employing the carbonyl diimidazole (CDI) method for coupling of alkyl amines (see Jennissen and Demiroglou, 1992; Jennissen, 2000).

sigmoidal function of the surface concentration of immobilized alkyl residues (Figure 3). In this case the strength of binding increased from retardation (interaction of few residues with protein) to very tight binding (interaction of many residues with protein) as the surface concentration of hydrophobic groups is increased. The sigmoidal curves are shifted to the left as the chain length is increased. An important nding was that a threshold value of the alkyl surface concentration, a critical hydrophobicity, had to be reached before a protein adsorbed (Jennissen and Heilmeyer, 1975). Sigmoidal adsorption curves and critical hydrophobicities can be obtained at low and at high salt concentrations (Jennissen, 1978; Jennissen and Demiroglou, 1992; Jennissen, 2000). Cooperativity and hysteresis on hydrophobic gels

cooperativity of protein adsorption (Jennissen, 1976b). It became clear that the sigmoidicity and the critical hydrophobicity were due to the multivalence of the interaction (i.e. the necessity for a simultaneous interaction of more than one alkyl residue with the protein moiety). At high salt concentrations, protein binding displayed a positive temperature coecient (Jennissen, 1976b, 1978, Jennissen and Botzet, 1979) in agreement with hydrophobic interactions. A mathematical model of cooperative protein binding to an immobilized alkyl residue lattice was also developed (Jennissen, 1976b, 1981), allowing an

100

Adsorbed protein (%)

A straightforward interpretation of the sigmoidal curves (Figure 3) was provided by the concept of multivalence and
CH3 CH3 CH3

80

60 Seph-Cn 40 Seph-Cn1 Seph-Cn2

(a) CH3 CH3 CH3 CH3 CH3 CH3

20 0

CH

CH

CH

(b) CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3

10

20

30

40

50

Surface concentration of immobilized alkyl residues (mol m2)

Figure 3 Interrelationship between the chain-length parameter and the surface-concentration parameter for the binding of a single protein to various series of alkyl agaroses as shown in model curves. The sigmoidal curves shown here for an idealized system correspond to adsorption isotherms of the lattice-site binding function type (Jennissen, 1981, 1988). This form of the curves is due to the cooperative interaction of multiple alkyl residues with the protein (multivalence). For simplicity the curves were calculated with the same increment although there are indications that the increment can vary (Jennissen and Heilmeyer, 1975). An increase in the chain length of the immobilized alkyl residue shifts the curves from the right to the left. CH indicates the region of critical hydrophobicity, which corresponds to the three different points of origin for the sigmoidal curves on the x-axis. n, number of carbon atoms (n ! 3).

(c)
Figure 2 Example for the variation of the surface-concentration parameter of immobilized alkyl residues of a fixed chain length according to the concept of Jennissen. For an optimal series it is understood that the chain length of the immobilized residues is constant, that no charges are introduced and that leakage is low. The surface concentration shown here as a one-dimensional array of residues increases from (a) to (c). It is easily visualized that the number of alkyl residues capable of interacting with the protein (i.e. the valence) and thus the respective affinity of binding increases as the surface concentration of alkyl residues and concomitantly the hydrophobicity is enhanced. For further details see legend to Figure 1.

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Hydrophobic Interaction Chromatography

Phosphorylase b (Units per mL)

estimation of the minimum number of alkyl residues interacting with the protein. An important consequence of cooperative multivalent protein binding on alkyl-substituted surfaces was protein adsorption hysteresis (Jennissen, 1978, 1985; Jennissen and Botzet, 1979). Protein adsorption hysteresis implies that the adsorption isotherm is not retraced by the desorption isotherm, owing to an increase in binding anity after the protein is adsorbed. The increase in binding anity can be attributed to an increase in the number of interactions (multivalence), resulting from a reorientation of the protein on the surface or from a conformational change in which buried hydrophobic contact sites are exposed due to the surface binding strain on the adsorbed protein. Adsorption hysteresis provided evidence for the concept that protein adsorption to multivalent surfaces in general is thermodynamically irreversible (DiS 4 0) and that a true equilibrium is not reached (Jennissen, 1985). Thermodynamic irreversibility (DiS 4 0) should not be confused with the so-called irreversibility of binding in high-anity systems, where simply a dissociation cannot be detected for technical reasons. Another conclusion from this concept is that protein adsorption in hysteretic systems may be not thermodynamically but kinetically controlled (Jennissen, 1988). Thus adsorption hysteresis has a strong inuence on hydrophobic interaction chromatography by leading to nonlinearity and skewed elution peaks in zonal chromatography (Jennissen, 1981). Hysteresis can be reduced by decreasing the surface concentration of immobilized alkyl residues (Jennissen and Botzet, 1979; Jennissen, 1981, 1985).

gradient of MgCl2 as shown by Raymond et al. (1981). Raibaud reported that the concentration of salts necessary for the inhibition of b-galactosidase adsorption on a Seph C3-column also showed an inverse relationship to the salting-in power of the anions employed (Raibaud et al., 1975). Finally Pahlman showed that the salting-out power of anions, for the adsorption of human serum albumin on uncharged Seph-C5, also followed the order of the Hofmeister series of salts (Pahlman et al., 1977). An example for such a salting-out type of chromato-graphy (Jennissen, 1976a) is shown in Figure 4. All of these experiments clearly indicate that the action of the ions is due not to a pure electrostatic but rather to lyotropic eects (for review see Jennissen, 2000).

20 1 phos.b No salt 2 1 mol L1 NaCl (a)

15

10

5 20

10

15

20

25 (b)

The salt parameter

Salting-out and salting-in on hydrophobically substituted hydrophilic gels The enhancement of hydrophobic interactions by high neutral salt concentrations was rst applied to chromatography on uncharged benzyl ether agarose by Porath (Porath et al., 1973) and to a homologous series of n (Hjerte n, alkyl agaroses of the Shaltiel-type by Hjerte 1973). Further evidence of the mechanism and principle underlying these salt eects came in simultaneous, independent reports that the eect of salts on the adsorption and elution of proteins on alkyl agaroses indeed followed the Hofmeister series of lyotropic salts (Jennissen and Heilmeyer, 1975; Raibaud et al., 1975). It was shown that phosphorylase kinase is eluted (salted-in) from a Seph-C2 column by increasing salt gradients (Jennissen and Heilmeyer, 1975). The ionic strength of the peak fractions eluted was inversely related to the salting-in power of the anions in the gradient, in agreement with the Hofmeister series of salts. Similarly, proteins could also be eluted (salted-in) from uncharged octyl-Sepharose by an increasing salt
4

15

1 phos.b

2 No salt

3 1 mol L1 NaCl

10 1.1 mol L1 (NH4)2SO4 5

10

15

20

25

Fraction number
Figure 4 Inverse salt dependence of the chromatography of purified phosphorylase b (phos. b) on Seph-C1 (30 mmol per mL of packed gel). The equilibration buffer contained 10 mmol L 2 1 sodium b-glycerophosphate, 20 mmol L 2 1 mercaptoethanol, 2 mmol L 2 1 EDTA, 20% sucrose, 0.5 mmol L 2 1 phenylmethylsulfonyl fluoride, pH 7.0 (buffer A), to which either 1.1 mol L 2 1 ammonium sulfate or NaCl was added. Phosphorylase b 6 mg/3 mL was added to 20 ml Seph-C1 in a 2 cm i.d. 17 cm column. Fractions of 6.5 mL were collected. The gel was prepared by the CNBr procedure. (a) Application of enzyme to a column equilibrated with buffer without ammonium sulfate (NH4)2SO4 or NaCl. (1) Application of phosphorylase b in buffer A. (2) Elution with buffer A 1 1 mol L 2 1 NaCl. (b) Application of enzyme to a column equilibrated with buffer with (NH4)2SO4. (1) Application of phosphorylase b in buffer A 1 1.1 mol L 2 1 (NH4)2SO4. (2) Elution with buffer A. (3) Elution with buffer A 1 NaCl. For further details see the text and for the source see Jennissen (1976a).

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Hydrophobic Interaction Chromatography

Chromatographic Techniques and Applications

Although a large number of proteins have successfully been puried by hydrophobic interaction chromatography n, 1981; Hofstee and Otillio, 1978; Oscarsson, 1997; (Hjerte Mohr and Pommerening, 1986; Ochoa, 1978; Shaltiel, 1984; Yon, 1977), this method has not gained the same foothold in the methodological repertoire of protein chemistry as has anity chromatography. This is because the commercially available hydrophobic adsorbents are to a large extent inadequate for an ideal downstream processing because their hydrophobicity is too high (Oscarsson et al., 1995). The major problem encountered with commercial hydrophobic gels is that most proteins can be very eectively adsorbed but an elution in the native state is impossible. Although a similar problem can be encountered in anity chromatography, it is easier to counteract there and it does not show the same generality as in hydrophobic interaction chromatography.

shown for homologous alkyl agarose series of the n et al., 1974; Rosengren et al., 1975) uncharged (Hjerte n, 1973). The and the charge-containing types (Hjerte optimization strategy, which was also utilized by a number of commercial rms, is illustrated in Table 2. In this case the homologous series is tested at high ionic strength. Again the main problem of the procedure was that often the proteins cannot be eluted in the native form by the inverse salt gradient. This procedure or variants of it are still the method of choice for most groups today. However, as illustrated by the paper of Oscarsson et al. (1995), the number of failures is probably very high.

The critical-hydrophobicity method

There are two methods for the synthesis of controlled hydrophobicity gels: (a) via the homologous series procedure (variation of alkyl chain length; see Shaltiel, 1974, 1984) or (b) via the surface concentration series procedure (variation of the alkyl surface concentration (see Jennissen and Heilmeyer, 1975; Jennissen, 2000). The importance of the latter series has been underestimated. Both gel series essentially correspond to members of hydrophobicity gradients. Although the decisive importance of the immobilized alkyl residue concentration for the hydrophobic adsorption of proteins (critical hydrophobicity) was stressed for many years, no hydrophobicity gradient gel series of this type has ever been produced commercially. Against the background of obvious problems in hydrophobic interaction chromatography, a novel rational basis for the optimization and design of low-hydrophobicity chromatographic supports working at the critical point has been suggested as shown in the strategy of Table 3 (see Jennissen, 2000). High yields in hydrophobic interaction chromatography can only be obtained if the protein to be puried is fully excluded from the gel under elution conditions that are as physiological as possible, i.e. at low ionic strength. This means that the gel should be fully nonadsorbing under these conditions. On the other hand, since a purication is only possible if the protein is adsorbed to the gel, the matrix should be constructed in a way that adsorption can be induced easily by other means without denaturing the protein, i.e by neutral salts. Thus working at, or near to, the critical hydrophobicity point should solve both problems. In the synthesis of such critical hydrophobicity gels, the

The homologous series method

The low-salt concentration approach Shaltiels homologous series method of synthesizing hydrocarbon-coated agaroses for protein purication was supplemented by the so-called exploratory kit for choosing the most appropriate column and for optimizing resolution (Shaltiel, 1974). This analytical kit, which was commercially available for some years, contained a homologous series of small columns from Seph-C1 to Seph-C10 with two control columns (Shaltiel, 1974). The principle was to determine the lowest member of the homologous series capable of retaining the desired enzyme or protein at low ionic strength. This column was then selected for the purication of the desired protein. In a second step it was attempted to increase resolution by optimizing the elution procedure, which ranged from salting-in procedures to reversible denaturation steps (see strategy in Table 1). The high-salt concentration approach As discussed above, proteins can also be adsorbed to hydrophobic agaroses at high salt concentrations, to be subsequently eluted by an inverse salt gradient. This was

Table 1 Strategy for optimizing an exploratory kit of Seph-Cn columns (n 5 110) according to Shaltiel. 1. Application of protein sample ( $ 1 mg per mL of packed gel) at low salt concentration ( $ 10100 mmol L 2 1) 2. Selection of column that eciently binds enzyme (i.e. variation of chain-length parameter) 3. Selection of column from which the enzyme can be eluted with the highest specic activity by a deforming buer (reversible denaturation)
The surface concentration parameter is not optimized. An inverse salt gradient is not utilized, since protein is applied at a low salt concentration. For further details see the text, Figure 1 and Shaltiel (1984), Jennissen (2000).

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Hydrophobic Interaction Chromatography

Table 2 Strategy of commercial screening kits of uncharged Seph-Cn columns (n 5 4, 6, 8, 12) according to the work of ns group. Hjerte 1. Application of protein sample ( $ 540 mg per mL of packed gel) at high salt concentration ( $ 5001500 mmol L 2 1). Starting point: e.g. 1.0 mol L 2 1 ammonium sulfate (i.e. variation of salt parameter) 2. Selection of column that eciently binds enzyme (i.e. variation of the chain-length parameter) 3. Elution with inverse salt gradient plus organic solvents (ethylene glycol, glycerol, ethanol, propanol), chaotropic agents (trichloroacetate, urea, guanidine hydrochloride) or detergents (Triton X-100, sodium dodecyl sulfate)
The surface concentration parameter is not optimized. For further details see Rosengren et al. (1975), Pahlman et al. (1977), Jennissen (2000).

charge-free immobilized residues should be restricted to alkane derivatives, to ensure a purity of hydrophobic interactions. As salt, NaCl centrally located in the Hofmeister series appears to be ideal in simplifying the salt selection procedure. The critical hydrophobicity method involves three basic steps: (i) selection of an appropriate alkyl chain length; (ii) determination of the critical surface concentration of alkyl residues (critical hydrophobicity); (iii) determination of the minimal salt concentration (NaCl) necessary for a complete adsorption of the protein. The three parameters can be determined for a certain protein by a form of quantitative hydrophobic interaction chromatography (for review see Jennissen, 2000) in which primarily the high-anity adsorption sites are evaluated. Selection of the appropriate alkyl chain length In the rst step, an experimental setup (e.g. test runs on Seph-C4 Seph-C6) similar to the homologous series method of Shaltiel is employed to gain information on the general hydrophobic binding properties of the protein and columns. However it is essential to quantify the immobilized surface concentration of alkyl groups. The surface concentration is set to $ 20 mmol per mL of packed gel. In general a constant amount of protein ( $ 0.5 mg per mL of packed gel, which can be 100% adsorbed on the column of highest hydrophobicity) is applied at low or physiological salt concentration (0.15 mol L 2 1 NaCl) to each column (12 mL packed gel). One then determines the gel in the homologous series that adsorbs $ 50% of the applied protein. In the case of the example below, $ 50%

of the applied brinogen was adsorbed on an uncharged Seph-C5 gel containing 22 mmol per mL of packed gel, so that Seph-C5 was chosen for the following concentration series. Determination of the critical hydrophobicity As previously dened, the critical hydrophobicity is that degree of substitution at which adsorption of a protein begins (Jennissen and Heilmeyer, 1975). As shown in Figure 5, a strongly sigmoidal adsorption curve of brinogen is obtained on the concentration series of uncharged Seph-C5 gels at a physiological NaCl concentration. The aim is to get as close as possible to the critical hydrophobicity point with a minimum of adsorbed protein. Since there was no measurable adsorption of brinogen at 12 mmol per mL of packed gel and only $ 2% was adsorbed at 13.6 mmol per mL of packed gel (critical hydrophobicity), the ideal juxtacritical hydrophobicity range for brinogen was taken as 1214 mmol per mL of packed gel (Figure 5). Determination of the minimal salt concentration (NaCl) necessary for adsorption In subsequent experiments with NaCl, the nonadsorbing critical hydrophobicity gel (13.6 mmol per mL of packed gel) could be changed to a gel strongly adsorbing brinogen by adding 1.5 mol L 2 1 NaCl to the adsorbing buer (Figure 6). The salt concentration necessary for halfmaximal adsorption of brinogen was $ 0.75 mol L 2 1 NaCl (not shown). Since no (i.e. only 2%) brinogen was adsorbed to this pentyl-Sepharose at low ionic strength

Table 3 Strategy for optimizing hydrophobic gels by the critical hydrophobicity method. 1. Chain-length parameter: Selection of appropriate alkyl chain length from homologous series experiments ( $ 20 mmol per mL of packed gel; protein sample: 0.51 mg per mL of packed gel), i.e. the gel that adsorbs $ 50% of the applied protein at low salt concentration 2. Surface concentration parameter: Determination of critical hydrophobicity for the selected chain length from a surface concentration series 3. Salt parameter: Determination of the optimum high salt concentration (15 mol L 2 1 NaCl) for binding the protein (0.5 1.0 mg per mL of packed gel) on the critical hydrophobicity support 4. Chromatography: Adsorption of protein on critical hydrophobicity support at specied high salt concentration (step 3) and elution by inverse salt gradient
The three decisive parameters are optimized. For further details see the text and Jennissen (2000).

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Hydrophobic Interaction Chromatography

90

70 Adsorbed fibrinogen (%)

multiple forms of brinogen. Maximal yields of brinogen of 60% have been obtained. If blood plasma equilibrated with 1.5 mol L 2 1 NaCl is applied to the gel and eluted by a negative salt gradient, a clottability of 80% is obtained (not shown).

50

Conclusions
30 Critical hydrophobicity

10 0 10 0 10 20 30 40

Hydrophobic interaction chromatography is one of the very basic and key separation methods in classical and the now emerging proteome biochemistry. Present chromatographic techniques on commercially available gels are associated with a high number of failures. On this background it appears that the critical-hydrophobicity method for the optimization of hydrophobic supports poses a comprehensive and rational approach to the successful purication of proteins. A successful separation
1600 1 0.4 ) 2 1200 ) 800 0.2 400 0 40 40 NaCl (mmol L1) (

Immobilized pentyl residue concentration (mol per mL of packed gel)

Figure 5 Determination of the critical surface concentration (critical hydrophobicity) of Seph-C5 for the adsorption of purified fibrinogen. The uncharged pentyl-agaroses were synthesized by the carbonyldiimidazole method. Purified human fibrinogen (1 mg) was applied in 1 mL to a column (0.9 cm i.d. 12 cm) containing 2 mL packed gel in 50 mmol L 2 1 TrisHCl, 150 mmol L 2 1 NaCl, 1 mmol L 2 1 EGTA, pH 7.4. Fractions of 1.5 mL were collected. The column was washed with 15 mL buffer followed by elution either with 7.5 mol L 2 1 urea or, at high hydrophobicity of the gel, with 1% sodium dodecyl sulfate for the determination of the amount of protein bound. 100% equals 1 mg fibrinogen adsorbed to 2 mL packed gel of Seph-C5. The total amount of adsorbed fibrinogen, corrected for the amount adsorbed to unsubstituted control Sepharose 4B, is shown. For further details see the text and for the source see Jennissen (2000).

OD280 nm (

0.0 0 (a)

10

20 Fraction number

30

Clottability (%)

(0.15 mol L 2 1 NaCl), a complete recovery of brinogen adsorbed at high ionic strength (1.5 mol L 2 1 NaCl) was now possible by decreasing the salt concentration alone. Thus the critical hydrophobicity gel together with NaCl constituted a fully reversible hydrophobic adsorption and purication system for brinogen. One-step purification of native fibrinogen from human blood plasma Employing Seph-C5 of critical hydrophobicity equilibrated with 1.5 mol L 2 1 NaCl it was possible to purify brinogen from human plasma in a single step (Figure 6). The procedure was so robust that brinogen could be puried from human blood plasma directly (without dialysis) in spite of a temporary decrease in NaCl concentration (fractions 59) during the run. After extensive washing with 1.5 mol L 2 1 NaCl, $ 20-fold puried pure brinogen (clottability 9399%, Figure 6) was eluted by a negative step gradient from 1.5 to 0.15 mol L 2 1 NaCl (see Jennissen, 2000). The total yield was 25% owing to some loss in the run-through, to errors of the clottability test and possibly to the presence of

100 75 50 25 0 0 (b)

10

20 Fraction number

30

Figure 6 One-step purification of fibrinogen from human blood plasma by hydrophobic interaction chromatography at the critical hydrophobicity point of Seph-C5. (a) 19 mL fresh unclotted human blood plasma was applied (arrow 1) to 20 mL packed Seph-C5 (13.6 mmol per mL of packed gel in a column 1.4 cm i.d. 13 cm) equilibrated with 50 mmol L 2 1 TrisHCl, 1.5 mol L 2 1 NaCl, pH 7.4, at a flow rate of 70 mL h 2 1 and a fraction volume of 6 mL. The nonadsorbed protein was washed out with 200 mL equilibration buffer. Elution (arrow 2) was facilitated by equilibration buffer containing a 10-fold lower salt concentration of 150 mmol L 2 1 NaCl. (b) The fractions 30 32 contain pure fibrinogen with a clottability of 93 100% with a total yield of 25%. For further details see the text and legend to Figure 5 and for the source see Jennissen (2000).

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Hydrophobic Interaction Chromatography

of proteins by this method depends on the optimization of three basic parameters: (i) the chain-length parameter, (ii) the surface-concentration parameter and (iii) the salt parameter. The only drawback is that the necessary gel kits are not commercially available so that the application of this method necessitates experience in the synthesis of alkyl agaroses and the quantication of immobilized alkyl residues.

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