Hydrophobic (interaction) chromatography c*). - Bashan Foundation

BIOCHIMIE, 1978, 60, 1-1_5. 

Revue 

Hydrophobic (interaction) chromatography c*). 

J.-L. OCHOA * * 

lnstitnlo de Quimica. 

Universidad Nacional Autonoma 

de Mexico (UNAM), 

Ciudad Universitaria, 

Mexico, D.F. 

Introduction. 

The isolation and purification of macromolecules 

by biochemical fractionation techniques, 

like ion-exchange chromatography, gel filtration 

(molecular-sieve chromatography), affinity chro- 

Inatography, electrophoresis, etc., is primarily 

dependent on their biological and physicochcmical 

properties [1-5]. 

Based on biospecific interactions [6], affinity 

chromatography has been considered as one of 

the most effective separating methods. However, 

serious disadvantages are found upon its application, 

due mainly to undesirable non-biospecific 

adsorption [7] attributed to the characteristics of 

the matrix ov support, and the nature of the 

ligand and spacer-arm introduced to b~idge the 

ligand from the matrix backbone. Moreover, once 

a protein is attached to an immobilized ligand, 

for which it sho~vs affinity, the properties of the 

support may change and become like those of an 

ion-exchanger, interfering with the chromatographic 

process. 

The belief that such interferences could be 

adequately controlled by eliminating ionic groups 

in the matrix and/or in the spacer-arm, brought 

as a consequence other types of undesirable 

effects closely related to the extent of hydrophobicity 

of the spacer-arm employed [57]. Yon [93, 

and Er-eI et aL [10], reported that by coupling 

different types of spacer-arms (varying in hydrophobicity) 

to an inert matrix, potent adsorbents 

for proteins were obtained. The adsorption mechanism 

seems to be based, fundamentally, on 

hydrophobic interactions between the protein and 

the adsorbent, and it has been demonstrated that 

it can be positively exploited for separation purposes 

[11, 105]. In this way, a novel technique 

which takes advantage of the hydrophobicity of 

* This article is a complementary part to my contribution 

at the Forum des Jeunes, in Lyon, France 

(July 6-8, •977). 

** Present address : Facultd de Mddecine de Strasbourg, 

Institut de Clfimie Biologique, 11 rue 

Humann, 67000 Strasbourg, France. 

biomolecules, has been introduced as a complement 

to the other, routinely employed, separating 

methods mentioned above. 

Since the separation is based on hydrophobic 

interactions, this technique receives a number of 

names 'which intend to describe the principle and 

the parameters involved in the separation process, 

though all of them are entirely or partly dealing 

with the concept of hydrophobicity : 

Hydrophobic chromatography [11] ; Hydrophobic 

(interaction) chromatography [51]; Hydrophobic 

salting-out chromatography [93]; Phosphate-induced 

protein chromatography [54] ; Repulsion 

controlled chromatography [94] ; Detergent 

protein-interaction [95, 961 ; Hydrophobic 

affinity chromatography [128] ; etc. 

It has been shown [12] that a large part of the 

non-polar residues of the amino acids in proteins 

are exposed to ~vater interface, as opposed to the 

expected preferential location of the hydrophobic 

amino acids in the interior of the biomolecule. 

These non-polar amino acids are found in the 

protein surface forming ) of distinct 

hydrophobic character which Can account for the 

biospecific conformation of the protein [13] as 

well as for its ability to complex or aggregate to 

other types of molecules for instance, lipids. Presumably, 

these hydrophobic (< patches >> are randomly 

distributed on the surface of the biomolecule. 

Their number (possible number of interacting 

sites) and the extent of their hydrophobicity 

(type and distribution of the non-polar amino 

acids) should be a characteristic of each macromolecule. 

Therefore, their specific separation 

should be possible with an adequate hydrophobically 

coated support or matrix. 

I. THE BIOLOGICAL ROLE OF THE HYDROPHOBICITY 

IN BIOMOLECULES. 

There is no doubt that the hydrophobie interactions 

play an important role in biological systems. 

The membranes in the living cell are made 

1

- - An 

2 J.-L. Ochoa. 

up of mainly hydrophobically interacting lipidlipids 

and lipid-proteins. The sub-units of large 

proteins are often held together by hydrophobic 

bonds, and it is well accepted that they are important 

in supporting the tertiary protein structure 

[13]. 

Functionally, hydrophobic interactions seem to 

be involved in recognition processes, as for example 

in the case of some enzyme-substrate complexes 

and antigen-antibodies associations, etc. 

[14-15, 27-30]. Except for the case of membranes, 

in which an exclusive structural function has 

been attributed [31], there is not much work in 

correlating the hydrophobic properties of the 

biomolecules with their functions [32, 99-100]. 

Therefore, it could be interesting to find out 

whether these hydrophobic regions in the biomolecules 

are related to their intrinsic biological 

properties, and/or to their location in the cell. 

Perhaps one of the reasons why fe"w systematic 

studies of hydrophobic effects have been made, 

is that the hydrophobic compounds possess lo"w 

solubility in water. If they are made soluble by 

the introduction of polar groups, they tend to 

form micelles as exemplified by the case of soaps. 

Although molecular dispersion may be obtained 

through the addition of polarity reducing agents 

to the medium, such agents would also reduce the 

interaction of the hydrophobic compounds with 

proteins and even alter the protein structure, 

which generally depends on the integrity of the 

hydrophobic core [13]. 

A means to obtain molecular dispersion of a 

hydrophobic ligand in aqueous milieu, without the 

addition of polarity reducing agents, is to attach 

the ligand to a hydrophilic but insoluble polymer 

such as agarose. Consequently, hydrophobic supports 

can be used not only for separation purposes, 

but also to study the hydrophobicity of the 

biomolecules and their modes of interaction. In 

turn, this may lead to a clearer understanding of 

their functions. 

II. THE PHYSICOCHEMICAL CONCEPT 

OF HYDROPHOBICITY. 

Perrin [16] `was the first to utilize the terms 

> and ¢ hydrophobic >> when studying 

colloids. He considered them hydrophilic 

if their stability was relatively insensitive to the 

addition of electrolytes, or hydrophobic if they 

exhibit extreme sensitivity to added electrolytes. 

Later, Langmuir [17], in a classical experiment, 

discussed polar molecules (fatty acids) in terms of 

BIOCHIMIE, 1978, 60, n ° 1. 

hydrophilic and hydrophobic groups. Since then, 

these terms have been in every day use. 

In general, "when an apolar group (or hydrophobic) 

is inserted into `water, several effects can be 

observed (for review see ref. 18) : 

-- A negative unitary entropy change (--AS), 

`which implies an overall increase in the degree 

of order. The entropy distribution becomes relatively 

important as the molecular weight of the 

apolar group increases. 

--A small enthalpy change, usually negative 

(--AH), which reflects an energetic component 

in the interaction between 'water and an apolar 

group and opposes the negative unitary effect 

favouring mixing of the hydrocarbon in water. 

increase in heat capacity (+ ACp), which 

implies either an increased degree of freedom of 

the vibrational and rotational motion within the 

existing structure, or a progressive alteration of 

an existing structure as the temperature is raised. 

-- A decrease in volume (--AV), which indicates 

that the apolar groups and the water molecules 

have packed together with a contraction of 

some structure, or that the apolar groups are 

accomodated into open spaces or voids preexisting 

`within the water structure itself. The former 

is unlikely in view of the small enthalpy change 

involved. 

--Finally, the presence of apolar groups in 

water results in an increase in the number 

or strength of hydrogen bonds within the water 

molecules, as has been demonstrated by spin lattice 

relaxation studies and further supported by 

Raman measurements [60]. Its probable reason is 

that the hydrocarbon chains restrict the mobility 

of water molecules in such a `way that the covalent 

character of the hydrogen bound is increased 

[92]. 

On the other hand, the molecular picture of the 

hydrophobic interaction is the reverse of that obtained 

when introducing apolar groups into water 

(see mechanism below). The withdrawal of the 

apolar groups from the aqueous phase removes 

restrictions on hydrogen-bond bending and thus, 

achieves a positive unitary entropy. This supports 

the observation that the hydrophobic interaction 

is a spontaneous process. This assumption was 

substantially demonstrated by Frank and Evans 

in 1945 [20]. Some years later, it has been shown 

[19] that the free energy values of the transfer of 

aliphatic hydrocarbons from an apolar medium 

to a polar one, like water, increases linearly with 

increasing numbers of (CH 2) methylene groups, 

and thus the hydrophobicity of the molecule.

Hydrophobic (interaction) chromatography. 3 

III. DETERMINATION OF THE HYDROPHOBICITY OF 

BIOMOLECULES. 

It is well kno~vn that most proteins contain a 

relatively high proportion of amino acids ~dth 

non-polar chains (table I). Tanford [21], and 

TABLE I. 

Non-polar (hydrophobic) amino acids commonly 

found in proteins. 

AI anine CH 3 -R 

Leucine 

Isoleucine 

Valine 

Proline 

Phenylalanine 

Tryptophane 

Methionine 

CH3x 

/ CH-CH2-R 

CH 3 

CH3-CH2-CH-R 

I 

CH 3 

CH3 x 

CH-R 

CH3 / 

CH CH2\ CH_CO0tl 

CH , / 

2XNH 

d CH:CH\ 

CH ,,,CH-CH _ -R 

"~ CH-CH >" z 

/1 CH-CH \\ 

C -- CH-CH ~-R 

CH % CH=C ~ Ixrfl "~CI-I " 

CH3-S-CH2-CH2-R 

R 

-CH-CO0- 

! 

+NH 3 

Nozaki and Tanford [22], formulated an experimental 

procedure based more or less on the Kauzmann 

[13] conception which permits the estimation 

of the amino acid hydrophobicity. The method 

consisted, essentially, in determining the 

solubilities of the amino acids in water as ~vell 

as in progressively increasing concentration of 

some organic solvents, such as ethanol in water. 

The solubilities of the amino acids were extrapolated 

to pure organic solvents and then the free 

BIOCHIMIE, 1978, 60, n ° 1. 

energy value of the transfer for the amino acid 

from pure organic solvent to ~vater was calculated. 

Using as a reference glycine, and substracring 

its free energy transfer value from that of all 

the other amino acids, it was possible to formulate 

a hydrophobicity scale for amino acid residues 

~vhere their free energy transfer values 

become more and more positive as the hydrophobic 

character of the compounds increases [18]. 

Other attempts in scaling the amino acid hydrophobicity 

can be illustrated by the ~vorks of Bull 

and Breese [23] and Bigelov¢ [24]. The former 

studied the effect of the amino acid on the surface 

tension of water and the latter calculated the 

average hydrophobicity of several proteins by 

their amino acid composition according to data 

of Tanford and of Bull and Breese. 

A different approach ~vas done in terms of the 

frequency of non-polar side chains in proteins 

[2~], in which yeas found a variation from 0.21 to 

0.47. Separately, Fisher [26] employed the ratio 

of the volume of the polar groups to those of the 

non-polar as a hydrophobicity degree, but this 

idea has the inconvenience, like the one mentioned 

above [25], to consider that a group is 

either polar or non-polar, ~without any gradation 

between these t~o extremes. 

Recently, a method for studying the magnitude 

of the interaction bet~veen protein and aliphatic 

hydrocarbon chains, and thus indirectly the hydrophobicity 

, ~vas reported [~0]. It is based on 

the partition of proteins in an aqueous two phase 

system containing dextran and polyethylene glycol 

and different:fatty esters of polyethylene glycol. 

However, the measurements depend largely 

on a critical chain length which, by this technique, 

should be greater than 8 carbons. 

Finally, an approach to localize the hydrophobic 

sites on the surface of the proteins by means 

of interacting with small molecules, has been 

attempted using fluorescent probes [27]. It is not 

difficult to speculate that ~vith this set of information, 

a better comprehension of many biological 

phenomena is near. We do not know yet 

how the complex enzymatic systems are organized, 

nor how the recognition bet~veen proteins 

occurs to constitute such enzymatic complexes 

after protein synthesis. Neither do we have a good 

explanation for the transport of many substances, 

including proteins, through the hydrophobic core 

of the membrane. And furthermore, it is possible 

to believe that hydrophobic ((patches >> in the 

membrane (either out or inside) are needed to 

make possible many of the most common biolo-


gical phenomena like the recognition of non-polar 

substances by membrane receptors and even cellcell 

interactions. 

IV. THE MECHANISM OF THE HYDROPHOBIC INTER- 

ACTION. 

The hydrophobic interaction is the result of the 

adherence of two non-polar groups. The case of 

detergents can be considered as an example where 

negative enthalpy changes are observed during 

the micelle formation in aqueous solvents [33!. 

If the adsorption of proteins to hydrophobic matrices 

is considered to be a process of limited micelle 

formation, a negative change in the enthalpy 

value ~vould not preclude the hydrophobic nature 

of the binding. In addition, this change must be 

negligible as compared to the value of increasing 

entropy of the system I20]. Furthermore, before 

the interaction the water molecules are forced to 

keep in orde~ around the hydrophobic entities 

(fig. 1) as compared to the order in the bulk. When 

the hydrophobic sticks come in contact with each 

ooooooooooo 

o o o 

oeOOooeeoeooo o~ o • • 

o o o o 

::~ii........oj_ 

oooooooooooo 

• 

ooooooooooo O O0000QeO 

0 0 0 000 O • 0 

D • 

a) b) 

F[~. 1. -- The mechanism of hydrophobic interaction. 

The water molecules around the hydrophobie 

> are doted, for simplicity, in order to 

distinguish them from those in the bul~. Notice. that 

the difference bet'ween a) and b) is only the improved 

degree of disorder (-I-AS) of vcater molecules. 

other, the ordered water molecules will be excluded 

and 'will adopt the less ordered buIk water 

state which is equivalent to an increase in entropy. 

This hypothesis is supported experimentally 

by the study of antigen-antibody complex formation 

[27] where a relative insensitivity to dissolution 

of a preformed antigen-antibody precipitate 

is observed, suggesting that once the complex is 

formed, the solvent is largely excluded in lhe 

regions of contact. 

Thermodynamically, the free energy value AG 

of a hydrophobic interaction is a function of AH 

and AS, according to equation : 

AG ~ AH -- TAS ..................... (N ° 1) 

Since AH is small as compared to TAS value, 

the process is fundamentally determined by the 

BIOCHIMIE, 1978, 60, n ° 1. 

change in entropy. In these conditions the reaction 

proceeds spontaneously [51]. In other words, 

the input of energy or chemical work is not necessary 

to make possible the interaction between two 

hydrophobic molecules in aqueous solutions. 

The contact between two different molecules, 

like the sub-units in the case of some proteins, 

is largely dependent on the surface areas occupied 

by the residues which participate in the interaction. 

The concept of accessible surface area [80j 

describes the extent to ~vhich protein atoms can 

form contacts with water, and is related to hydrophobic 

free energies [81]. In any case, the association 

of protein sub-units, whether by van der 

Waals contacts, electrostatic forces and/or hydrophobic 

interactions, leads to a reduction of the 

surface area accessible to the solvent ~vhen the 

two molecules associate. Evidence against the 

hydrogen bond as a major contribution to the 

free energy of the protein-protein interaction has 

been obtained lhermodynamically [82]. On the 

other hand, van der Waals interactions, though 

they are more numerous as they involve all the 

pair of neighbouring atoms, are much less energetic 

and their overall contribution is small. The 

hydrophobic contribution is largely dependent 

upon the entropy gained by water due to the 

smaller accessible protein surface area when the 

protein molecules form a complex. The hydrophobic 

interaction seems to be entirely unspecific 

as compared to the complementarity of the surfaces 

involving hydrogen bonds and van der 

Waals contacts ; ho~,ever, they decide which proteins 

can recognize each other. 

V. THE SPECIFICITY OF THE ADSORPTION 

OF BIOMOLECULES ON HYDROPHOBIC SUPPORTS. 

Biospecific affinity, whether involving an 

> or not, presumably depends to a 

large extent on the complementarity of the contours 

of the interacting molecules. In the absence 

of any specific effect, the binding of proteins to 

substituted agaroses is greatly affected by the 

overall charge of the biomolecule [105]. 

The lack of activity in the adsorbed state of 

some proteolytic enzymes [90], indicates that the 

binding site is occupied by the hydrophobic 

group of the substituted agarose. Whether the 

hydrophobic group interacts with the hydrophobic 

poc1~ets of the active sites of the enzymes or 

acts by a less specific ~way can be questioned. In 

any event, both cases may contribute to the 

adsorption phenomena.

Hydrophobic (interaction) chromatography. 

In other examples, the addition of the specific 

substrate accompanying the enzyme during the 

chromatography (to mask the specific binding 

sites) did not alter the binding of the enzyme to 

the hydrophobic matrix, indicating that the 

adsorption takes place through sites other than 

the specific substrate binding site [971. 

It is interesting to note that glutamine synthetase 

and other three proteins involved in the regulation 

of glutamine metabolism are all retained by 

the same amino-aIkyl agarose derivatives [99~ (see 

table II). Although, as Shaltiel and coworkers 

have signaled, this could be fortuitous, it might 

reflect a mutual hiospecific affinity among these 

proteins since they must interact with each other 

in order to effect their regulatory functions in the 

highly integrated glutamine synthetase cascade 

system. 

The models proposed by Shaltiel [11] and 

Jennissen [78] explain in a different manner the 

mechanism by which the interaction between the 

protein and the adsorbent occurs. For neutral 

supports, Jennissen [86~ has presented evidence 

in favor of the idea that the adsorption of proteins 

to aIkyl-agarose derivatives takes place at a 

critical alkyl group density and is a function of 

its hydrophobicity. In other ~vords, this hypothesis 

considers that the protein needs to present 

multiple attachment points in order to be adsorbed 

by a determinate member of the series of 

alkyl-agarose derivatives. This is in agreement 

with an earlier observation made by Hjert6n el 

al. [521. Shaltiel Ill on the other hand, suggests 

that the adsorption of the protein is due to the 

inter.action of an allkyl residue of specific length 

(¢ yard-stick ~) with a hydrophobic pocket of the 

protein. Evidently, and as compared to Jennissen's 

model, the tatter implies a more specific mechanism. 

Ho~vever, it has been demonstrated [583 that 

at high chain length, when the interactions are 

stronger, the binding is also less specific. 

The [841 

through the multivalent binding, may explain the 

increased free energy value of adsorption of a 

determinate member of the alkyl-agarose series. 

That is, a given protein may be adsorbed by a 

certain member of agarose-substituted series only 

if the number of ¢ contacts ~> is big enough to 

effect its retention, and this can be done by variation 

of the degree of substitution. Comparatively, 

amino-alkyl agaroses present a > effect to,wards the adsorption of phosphorylase 

b, possibly due to a different mechanism of 

interaction 'which does not necessarily exclude 

the multivalent attachment E84]. 

BIOCHIMIE, 1978, 60, n ° 1. 

It has been observed, in the case of some hydrophobic 

aIkyl- and alkylamino supports, that certain 

> binding sites are occupied first, 

and that others of decreasing affinity become 

occupied when more protein is fed to the column. 

This is shown by the fact that only part of the 

protein can be eluted by increasing the salt concentration, 

whereas the remained is dislodged by 

the addition to the eluant of a polarity reducing 

{a) 

(c) 

(b) 

(d) 

Fro. 2. -- Models of adsorption o[ proteins on hydrophobic 

matrices : a} The model suggested by Shaltiel 

[11] ; b) and c) Represents the two possibilities of multi-attachment 

adsorption : on the matrix surface (b) 

and on a cavity in the matrix (c) ; d) The irregularity 

of the surface (attributed to the nature of the agarose 

structure) is the probable cause for 'which binding 

sites are not identical, and consequently the forces 

involved in the binding are different. 

agent such as ethylene-glycol. It should be emphasized 

that this problem is mostly related to those 

hydrophobic supports which possess both hydrophobic 

and ionic groups, resulting from the coupling 

[70, 471 procedure or the nature of the 

ligand [96], like in the case of amino-alkyl derivatives. 

This apparent inhomogeneity of both 

matrix and protein can lead to possible wrong 

interpretations about the purity and characteristics 

of many proteins [98, 105]. For instance, 

the recovery of rechromatographed materials is 

improved up to 95 per cent, as compared to that 

of 70 per cent obtained when the crude extract 

is applied into the column [98]. In other cases, 

desorption of purified material requires the variation 

of the eluant, as pointed above. It seems


TABLE II. 

Hydrophobic matrices. 

Type Coupling procedure Bepresentation References 

I. Agarose derivatives 

1. Un-substituted 

2. Amino-alkyl- 

3, Alkyl- 

4. Amino acid 

5. Other derivatives 

Fatty acids 

AcetyI-N-amino-alkyl 

Aniline 

Benzyl-ether 

Phcnyl-N-butyl-amine- 

NAD +- 

Hydroxy- alky 1 

Diverse aromatic-alkyl 

derivatives 

BrCN 

BrCN 

glycidyl ether 

BrCN 

BrCN (azide) 

BrCN (cvrbodiimide) 

(acetylation of aminoalkyl-(A)) 

BrCN and glycidyl ether 

glycidyl ether 

(A) 

(A)-CH-N H-(CH~)n-N H,_, 

I 

NH.~ 

+ 

(A)-CH-NH-( CH.~)-CH:~ 

I 

NH~ 

+ 

(A)-O-CH~-CH-CH~-O- 

[ (CH'2)n-CH3 

OH 

(A)-glycine 

(A)-valine 

(A)-leucine 

(A)-tyrosine 

(A)-phenylalanine 

(A)-tryptophane 

(A)-(CHs)n-OH 

41, 47, li3] 

[9-11, 94, li3, Ill, 

li4, 58] 

[II,94, I13, li4] 

[52, li2l 

[97, I15] 

[54, 68, 97, li6, 77] 

[77, 97] 

[97] 

[35, 40, 42] 

[I051 

llo6] 

179, 58] 

[57, 94, li7] 

[40] 

[93, 90] 

[105l 

[57, 58] 

[58, li2] 

[I12, 55] 

II. Dextran derivatives 

(Sephadex) 

(s) 

1. Acetyl-Sephadex 

2. Methyl ether-(S) 

3. Hydroxy-propyl- ether- 

(S) 

4. Hydroxy-alkyL(S) 

5. LH-20-(S) 

acetylation 

acetylation 

from commercial sources 

CH3-CO-(S) 

CH3-O-(S) 

HO-CHs-CHs-CH.2-O-(S) 

HO-(CH~)n-O-(S) 

H3ol 

[91] 

[9t] 

[91] 

[9i] 

III. Cellulose derivatives 

(C) 

1. Diethyl amino ethyl 

cellulose 

2. Carboxy methyl-(C) 

3. Benzoylated-DEAE-(C) 

4. Esters of alkyl and aryl- 

(C) (paper, cotton) 



benzoyl chloride 

phenoxy acetylation 

DEAE-(C) 

CM-(C) 

BD-(C) 

[47, 71] 

[47, 71] 

[I03] 

[101] 

IV. Glass derivatives 

(G) 

1. Alkyl-silated-(G) 

2. Propyl_lipoamide-(G) 

BIOCHIMIE, 1978, 60, n ° 1. 

amino-silanization and 

amide bond formation 

with the corresponding 

alkyl chloride 

[ii8] 

[i07]

Hydrophobic (inleraction) chromatography. 

TABLE II. 

Type Coupling procedure Representation Relerences 

V. Others supports 

1. Polyamino methylstyrene 

(polystyrene, 

polyamide, Dowex 

I-X8) 

2. Alkyl-amine of polyacrylic 

resins (butyl, 

capryl, lauryl, palmityl, 

steatyl, oleyl, linoleyl) 


17t1 

chlorination for amide [761 

bond formation 

that this discrepancy depends on the hydrophobicity 

of the protein and the adsorbent, and moreover, 

that the binding sites are not identical in 

a given carrier (which is true for most of the 

adsorbents employed in chromatography in general). 

The multiple point attachment binding is most 

likely to occur in a cavity of the adsorbent than 

on its surface, particularly when the protein molecule 

fits into the cavity (fig. 2). Conversely, at 

points on the matrix ~vhere the bound ligands are 

distributed over a protruding area, binding would 

he less strong. Since the surface of the adsorbent 

may be assumed to be irregular, many different 

situations in addition to these two hypothetical 

cases ~vould be obtained. Additionally, small 

amounts of protein bind more homogeneously on 

a particular column than a large amount. This 

could mean that by reducing the amount of 

applied protein, the stronger binding sites might 

predominate in the binding. 

If multiple point attachment were one of the 

reasons for strong non-specific adsorption, one 

~vay to reduce it ~vould be to louver the degree of 

substitution to the point where the distance 

between the substitution groups is larger than the 

diameter of the protein molecule. This ~vould not 

affect the specific ¢ one-to-one >> interaction as 

that between an enzyme active site and an immobilized 

subslrate analogue. 

VI. THE FACTORS INVOLVED IN HYDROPHOBIC 

(INTERACTION) CHROMATOGRAPHY. 

A. The matrix hydrophobicity. 

1. Matrices and supports. 

Proteins differ in their hydrophobicity as a 

function of their primary structure, that is, in the 

BIOCHIMIE, 1978, 60, n ° 1. 

sequence and amino acid composition. Hence, it 

is not surprising that the first type of hydrophobic 

supports ever employed were derivated from 

coupling various non-polar amino acids to an 

inert support or matrix like agarose [54]. Other 

supports have also been employed: cellulose, 

glass, dextran, etc. (see table II), but the ideal 

matrix 'without secondary non-biospecific adsorption 

effects has not been found. 

All the different types of supports and matrices 

utilized at the present possess undesirable interferences 

attributed to their chemical composition. 

Additional effects arc obtained in such away that 

is difficult to obtain a pure hydrophobic interaction 

chromatography after the introduction of the 

spacer-arm or hydrophobic ligand. 

In table II, the various types of matrices 

employed in HIC, as well as the different types 

of ligands coupled, have been summarized. References 

are given to illustrate specific applications. 

As has been demonstrated, un-substituted agarose 

is sufficiently non-polar presumably due to 

the 3-6 methylene diether bridges present in every 

second galactose residue of the polysaccharide 

chain [67] with respect to lhe retention of halophilie 

proteins [47] and nucleic acids [41, 119], at 

high salt concentrations (2.5 M ammonium sulfate). 

Though the halophilic proteins are highly 

negatively charged [48], as a result of an excess 

of acidic groups [49], it is believed that the high 

salt concentration eliminates the possible ionic 

interactions. 

When DEAE- and CM-derivatives, either of 

cellulose or agarose, have been used, the elution 

pattern shifts to higher or louver salt concentrations 

than those required 'when tile neutral derivative 

is used as a support. This is explained by


electrostatic attraction-repulsion forces between 

the negatively charged haloproteins and the nature 

of the charge on the matrices. 

With few exceptions, agarose is the type of 

matrix most commonly used (table II), and the 

kind of ligands or spacer-arm introduced are in 

a wide range of hydrophobicities and structures. 

Consequently, the hydrophobicity of the nmtrix 

depends on the type of the ligand E78], and on the 

degree of substitution [5,6, 86]. In this respect, the 

properties of the ligand should be discussed first. 

2. The role of the ligand in the hydrophobicity 

of the matrix. 

As pointed out above, HIC was born (in many 

cases) as a consequence of non-biospecific adsorption 

found in affinity chromatography systems 

and attributed to the type of spacer-arm used to 

bridge the ligand to the matrix. Nevertheless, evidences 

exist that prove that HIC was conceived 

by Hjerl6n and his group in a different way, 

while studying the solubilization of membrane 

proteins. They attempted their separation using 

a hydrophobic support to 'which a detergent 

(SDS) was coupled. In this case, however, the 

adsorption was so strong that the proteins could 

not be desorbed by any non-denaturing system 

(unpublished results, personal communication). 

When Cuatrecasas [87] sho~,ed that ~-galactosidase 

could only be fractionated if the ligand was 

sufficiently separated from the matrix, and thus 

avoiding sterical hindrance, the idea of separating 

ligands from the matrix backbone by use of 

spacer-arms rapidly spread. Very soon, it became 

clear, that many proteins were adsorbed nonspecifically 

because the presence of the arm introduced 

non-biospecific adsorption centers. For 

instance, a spacer-arm carrying charged groups 

gives origin to electrostatic interactions [106]. The 

substitution of the charged arms by other neutral 

chemical analogs was thought to be an excellent 

alternative but then, other type of non-specific 

interactions, hydrophobic in nature, turned out 

to be important [7]. 

Yon [9], Er-el et al. [10] found that protein 

could bind substituted agarose matrices without 

any specific ligand. The adsorption ~ras attributed 

to the presence of the diamino-alkyl group 

employed as a spacer. This observations were 

further supported by O'Carra and coworkers [57, 

88], Who found that the presence of the ligand 

did not al'ways account for the ratardation effect 

and again the spacer-arm was considered as 

responsible. Yon [9] suggested that it was possible 

to take advantage of such non-biospecific adsorp- 

BIOCHIMIE, 1978, 60, n ° 1. 

tions and showed that certain proteins could be 

selectively adsorbed on decyl-agarose derivatives 

differing in the ionic character of the spacer-arm. 

Later, Shaltiel and his group [10, 11] reported 

purification of several enzymes on a series of 

alkyl and amino-alkyl agarose derivatives. Almost 

simultaneously, Hjert6n and his group [521 reported, 

for the first time, the preparation of different 

substituted agaroses 'with uncharged groups 

that could illustrate an exclusive hydrophobic 

mechanism of adsorption. 

The effect of the hydrophobicity of the spacerarm 

in the purification of various proteins [8-11] 

has been possible thanks to the development of 

the (< mock affinity systems >>. Steers [87 demonstrated 

that the adsorption of ~-galactosidase 

was strongly related to the length of the spacerarm. 

The series of aIkyl and amino-alkyl derivatives 

of agarose prepared by Shaltiel have also 

been successfully applied in many cases [11]. 

The difference of a single C-atom in agarose 

bound N-aIkyl groups may have a large effect in 

the hydrophobic binding of a particular protein 

[75]. Therefore intermediary hydrophobic compounds 

between two consecutive members of a 

homologous series of ligauds are needed. Such 

intermediate hydrophobicities can be obtained, 

for instance, through the introduction of a 

charged or other hydrophilic group, e.g., hydroxyl 

group. The introduction of double bonds or 

of branching of the chain, reduces also the hydrophobicity 

as compared to the corresponding saturated 

straight chains [13]. Weiss and Bucher 

['76] have prepared some alkyl agarose derivatives 

of increasing insaturation for this purpose. In 

addition aromatic derivatives may possess intermediate 

hydrophobicities between t~wo consecutive 

members of the alkyl-agarose series. Benzene, 

for example, is equivalent to that of 3-4 straight 

chain hydrocarbon [19]. 

Although Tanford [1O] has showed that the 

hydrophobicity of a linear aliphatic carbon 

increases linearly with increasing number of CH 2- 

groups, Shanbhag [50] considers that the effective 

hydrophobicity depends also on the flexibility 

of the hydrocarbon chain and consequently 

on the degree of interaction within such chains, 

specially for long chains. Hofstee [75], on the 

other hand, assures that neither the difference in 

molecular shape of the N-alkyl ligands and the 

side chain of aromatic compounds like phenylalanine 

or tryptophan, nor the difference in net 

charge of the adsorbent is a determining factor 

for protein binding. It seems then, that the me-

Hydrophobic (interaction) chromatography. 

chanism of the adsorption of proteins to hydrophobic 

matrices follows some very complex rules. 

This problem can be exemplified by the case of 

the adsorption of erythrocytes on a series of 

aIkyl-agarose derivatives [110] in ~which case a 

decrease in adsorption bet'ween C6-C s is noticed 

to occur without a reasonable explanation. 

The combination of the principles of ionicexchange 

chromatography and HIC has been successfully 

applied in some cases [9, 41]. The use of 

ligands containing both ionic and hydrophobic 

characters have been recommended in order to 

avoid the denaturing effect detected in the use of 

alkyl-agaroses [108]. It was also argued that the 

elution procedure might be milder than in the 

case of pure hydrophobic spacer-arms or ligands 

[9] and furthermore, that the use of charged 

arms was capable of finer discrimination between 

lipophilic proteins. The protein, in this case, 

interacts hydrophobically, involving the alkyl 

chains, and electrostatically, involving the terminal 

ionic groups. With this type of adsorbents, 

if the chromatography is carried out at a pH 

equivalent to the isoelectric point (IP) of the protein, 

the adsorption will be due to hydrophobic 

binding alone. By changing the pH to introduce 

in the protein a net charge of the same sign as the 

charge of the adsorbent, the repulsion effect will 

decrease the adsorptive force due to hydrophobie 

bonding, making possible the desorption. Obviously, 

a dra~vback in using this type of adsorbent 

arises from the problem that frequently the IP of 

the biomolecule of interest is unknown, and that 

many proteins precipitate at their IP. Moreover, 

the simultaneous presence of extraneous ionic 

and hydrophobic groups in affinity adsorbents, 

has been found to cause a substantial non-specific 

protein binding thus resulting in a reduced biospecificity 

of these materials [106]. 

3. The influence of the degree of substitution 

on the hydrophobicity of the matrix. 

The degree of substitution on Sepharose 4B 

with aIkyl-amines of different hydrophobicities 

is a critical parameter in the adsorption of proteins. 

About 1012 alkyl residues per Sepharose 

sphere appears to be a critical degree of substitution 

for the adsorption of enzymes like phosphorylase 

kinase, phosphorylase phosphatase, 

3'5'-cAMP dependent protein kinase, glycogen 

synthetase, and phosphorylase b which are successively 

adsorbed when the hydrophobicity of 

the Sepharose is increased. In addition, the 

degree of substitution determines the capacity of 

the gel [78]. 

The critical hydrophobicity needed to adsorb 

proteins can be obtained by either increasing the 

degree of substitution or by elongating the 

employed alkyl-amine chain at a constant degree 

of substitution. Consequently, as the hydrophobicity 

of the gel is increased, higher binding affinities 

result and the desorption requires more 

and more severe conditions. In the case of neutral 

alkyl-agaroses [3~1], elution of proteins from the 

hydrophobic matrix can be described in terms of 

salting- in phenomena, since desorption is dependent 

on the type of salt employed and not on the 

ionic strength alone. 

In all cases, a minimal chain length seems to be 

required in order to obtain the adsorption of a 

given protein. This fact has been interpreted as 

fitting a hydrophobic group into a hydrophobic 

pocket of the protein. By variation of the concentration 

of cyanogen bromide in the activation 

mixture, the amount of hydrophobic residues 

may be varied. Thus, the corresponding hydrophobicity 

may be increased such that the amount 

of adsorbed material increases exponentially 

when the degree of substitution of the gel is 

enhanced. Control experiments with methyl-agarose 

show that similarly increased degrees of 

substitution, and thus similar numbers of positive 

charges introduced by the coupling procedure 

[117], do not affect the adsorption of the assayed 

protein. Therefore, the adsorption of larger 

amounts of material, determined by increasing 

the degree of substitution, is not a function of 

the additional number of charges [78]. One may 

conclude that, if in a series of gels of different 

hydrophobicities a crude extract containing hydrophobically 

differing proteins is applied, the 

one vcith the highest hydrophobicity will be adsorbed 

by the gel of the lowest degree of substitution. 

Then as the number of alkyl residues increases 

on the matrix, proteins of lower hydrophobicities 

are adsorbed. 

A direct method of determining the degree of 

substitution for charged hydrophobie groups has 

been proposed by Hofstee [89]. The method describes 

the use of Ponceau S, a dye which carries 

hydrophobic groups in conjunction with an overall 

negative charge bound in an irreversible 

fashion to the ligand. Under a given set of conditions, 

and after application of a saturating amount 

of dye, a certain amount will remain bound even 

after extensive washing. Eighty-five percent of the 

Ponceau S binding capacity is lost after a period 

of almost 5 months indicating that the degree of 

substitution decreases gradually upon storage. In 

some cases, 40 per cent was lost in only 40 days. 

BIOCHIMIE, 1978, 60, n ° 1.

1 0 J.-L. Ochoa. 

The data also suggest that the adsorbents with the 

highest degree of substitution are the least stable 

[89]. 

An estimation of the relative degree of substitution 

can be obtained from the acid-base treatment 

of the adsorbent [89], by a nucleophilic attack 

of N-substituted isourea (derivated from the 

coupling procedure 'with BrCN) with an active 

chromogenic substance [70] ; or more indirectly, 

by measuring the capacity of the adsorbent with 

a coloured protein like cytochrome C [90] or phycoerythrin 

[56]. A inuch more sophisticated but 

accurate method utilizes NMR spectra [56]. 

Finally, it is important to mention that a consequence 

of the degree of substitution is the shrinkage 

of the gel owing to a decrease in hydrophilicity 

due to the hydrophobic character of the 

ligand. Depending on the special structure of the 

agarose gel, the shrinkage seems to be much less 

important than with other gels like Sephadex or 

polyacrylamide [90]. 

B. The influence of salt. 

The influence of salt on the adsorption of proteins 

to hydrophobic supports is probably due 

to a number of factors acting on the protein as 

well as on the matrix. It has been demonstrated 

that neutral salts induce conformational and 

structural changes in biomolecules [34]. These 

studies have been carried out by circular dichroism 

spectra of the protein at cons,taut ionic 

strength. It has been concluded that salting-out 

ions cause conformational but not structural changes 

whereas salting-in ions cause sometimes 

severe structural changes which may be one reason 

of their denaturing effect [34]. 

The > properties of saltingout 

ions enhance intramolecular, as well as intermolecular, 

hydrophobic bonding as reflected by a 

stabilization of the hydrophobic core of the biomolecule 

[35, 43']. The effect of specific ions on 

macromolecules was first noticed by Hofmeister 

[36]. He found that salts differ greatly in their 

ability to salt-out proteins at a given salt concentration. 

At high concentrations of salting-out ions, 

the solubility of the protein is adversely affected 

by decreasing the availability of ~vater molecules 

in the bulk and increasing the surface tension of 

water, resulting in an enhancement of the hydrophobic 

interactions [31, 371. Accordingly, the influence 

of neutral salts on the adsorption phenomena 

determines to a large extent the degree of 

adsorption and correlates closely with the Hofmeister 

series [63, 58]. Salting-in ions, on the other 

BIOCHIMIE, 1978, 60, n ° 1. 

hand, are > ions, and thus 

do not favour hydrophobic interactions. This 

effect can be regarded as an inevitable consequence 

of the new order imposed by the ion 

orienting water molecules in such a way that 

water cannot undergo further positive entropy 

change, as it is required for the formation of hydrophobic 

bonds [18]. These ions have also been 

termed ¢ chaotropic >> [44] because they provoke 

unfolding, extension and dissociation of the macromolecules. 

In this respect, they might be used 

in the elution of strongly adsorbed materials on 

hydrophobic supports. 

Already in 1948, Tiselius [38] noticed that proteins 

and other substances (e.g., dyes) that could 

be precipitated by high concentrations of neutral 

salts could be adsorbed (at much lo'wer salt concentration) 

to common adsorbents, whereas in the 

absence of salts those adsorbents showed no affinity 

for the substance. In the years afler~vards, 

some attempts have been made to purify proteins 

on solid supports using gradients of ammonium 

sulfate [39, 40, 54]. In general, proteins which precipitate 

at low ammonium sulfate concentrations 

should likewise be retained on hydrophobic supports 

at low salt concentrations ; and similarly, 

proteins ~vhich precipitate at high concentrations 

of ammonium sulfate would require relatively 

higher concentrations of salt to obtain their retention 

[40]. 

The purification of nucleic acids [41] has also 

been reported using unsubstituted agarose with 

high concentrations of ammonium sulfate. It 

should be emphasized that tbe adsorption occurs 

at concentrations below which the macromolecules 

precipitate out of solution. Since the adsorption 

is controlled by salt concentration, rather 

than by the hydrophobicity of the column, the 

names of > and hydrophobic (salting-out) chromatography 

have been employed elsewhere [42, 54. 

67]. Hovccver, the hydrophobicity of the gel and 

the effect of the salt concentration may be combined 

efficiently to improve the adsorption phenomena 

and/or the purification [64J, especially of 

proteins which may be affected by the high salt 

concentration required for its adsorption on the 

given gel [46]. If the use of high salt concentrations 

is limited by the enzyme activity or stability 

for instance, a comparatively longer alkyl-chain 

must be employed in order to obtain sufficient 

capacity. Because a similar salting-out/salting-in 

effect has been noticed in the case of high concentrations 

of sugars, namely sugaring-out and 

sugaring-in effect [45], the possibility in using


sugars instead of salts should be considered. However, 

this effect is probably not general. 

C. Effect of temperature and pH. 

From the equation N ° 1, it is clear that the temperature 

may influence, in a positive way, the 

adsorption of proteins on hydrophobic matrices 

~52, 551. However, temperature may also provoke 

other effects, such as increased solvation and decreased 

surface tension, ~vhich may withstand the 

ability of molecules like proteins to interact xvith 

hydrophobic supports [53J. 

The protein molecules retain their biological 

ac.fivity or capacity to function only within a 

limited range of temperature and pH. Their exposure 

to extremes of pH and temperature causes 

them to undergo denaturation ; the most visible 

effect is a decreased solubility of globular proteins. 

Since the covalent chemical bonds in the 

peptide backbone of the protein are not broken 

during denaturation, it has been concluded that 

it is due to the unfolding of the characteristical 

conformation of the native form of the protein 

molecule. The refolding of a denatured protein 

does not require the input of chemical work from 

outside. It proceeds spontaneously, provided that 

the conditions of temperature and pH are adjusted 

to be compatible with the stability of the native 

conformation of the protein. In this respect, the 

process is similar to the one in hydrophobic bond 

formation. 

The strength of hydrophobic bonds should increase 

with rising temperature up to above 60°C, 

at which point the additional stability arising 

from hydrogen bonding, electrostatic forces between 

charges or dipoles, van der Waals interactions 

and disulfide bridges disappear, and turn 

against the favoured hydrophobic interaction [18, 

30, 50] of the protein. 

A pH effect on hydrophobic bonding is observed 

in the case, for example, of bovine serum albumin 

binding alkanes at pH 4. It 'was observed 

that at low pH the site of hydrophobic bonding is 

disrupted [61J, and that the degree of aggregation 

of the globulin does not affect the degree of binding. 

The conclusion ~vas that the sites associated 

with aggregation are relatively non-hydrophobic 

and that any conformational changes, resulting 

from the polymerization do not exert any effect 

at the hydrophobic binding site of alkanes [62]. 

Decreasing pH belo'w pK value of the amino 

group of adenylic and cytidylic acid residues of 

tRNA changes the negative charge of the molecule, 

BIOCHIMIE, 1978, 60, n ° 1. 

altering its tertiary structure [65] and provoking 

its desorption from the charged alkyl amine agarose 

columns. On the other hand, binding at 

pH 7.5 does not occur probably due to other conformational 

changes in the molecule, which could 

mask key sites involved in the binding E41_~. 

Lysozyme, a basic protein, becomes more and 

more retarded on Clo (aIkyl Sepharose) as the pH 

of the eluant is lowered, and binds to this column 

at pH 1.5 [661. Cytochrome C is also highly pHdependent 

in its adsorption to neutral ethers of 

agarose derivatives [67J. 

No influence of pH has been observed on the 

adsorption of polysaccharides to substituted agaroses 

[68~. In this case, the retention is mostly 

determined by the hydrophobicity of the column 

and by the size of the polymer. 

Naturally, pH plays an important role in the 

adsorption of biomolecules on hydrophobic matrices 

carrying charged groups. With this type of 

adsorbents, pH may modify the charge either 

of the biomolecule or of the adsorbent. By 

changing the pH, a net charge may be introduced 

in protein, which can be of the same sign, 

or opposite of the charge on the adsorbent. In 

the first case, the repulsion effect ~vill affect negatively 

the adsorption phenomena. As was pointed 

out previously, a maximal hydrophobic adsorption 

will thus occur at the isoelectric point of the 

protein E9!. 

VII. METHODS OF DESORPTIOX on ELUTION 

OF ADSORBED MATERIALS 

ON HYDROPHOBICS MATRICES. 

The elution of adsorbed materials from hydrophobic 

gels can be obtained in a number of ~vays 

depending on the type of adsorbent employed, 

the conditions in 'which the adsorption occurred, 

and the properties of the biomolecule. 

When proteins have been adsorbed at high salt 

concentrations, a decreasing ionic strength of the 

eluant ~vill usually result in the removal of the 

material from neutral adsorbents [71, 791. If the 

support carries charges introduced either by the 

coupling procedure [70], or by the nature of the 

ligand, electrostatic interactions will become 

important at low ionic strength, and the elution 

will not be possible [47, 1031. In such cases, 

changes in the pH [9, 65], buffer composition 

[47], temperature, or the addition of nonpolar 

organie substances may be quite useful 

procedures.


The fact that in many cases elution is possible 

by raising salt concentration, demonstrates the 

presence of cooperative electrostatic interactions 

in the overall adsorption phenomena in the case 

of charged-hydrophobic supports. Nevertheless, it 

has been shaw that in those situations, the mechanism 

of hydrophobic adsorption at high salt 

concentration prevents the electrostatic interactions 

of being the main driving force involved 

in the process. 

The use of denaturing agents, such as urea, 

guanidine-HC1 etc, has been successfully applied 

in desorption procedures. Their mechanism of 

action appears to be involved in both direct interaction 

'with the side-chains and peptide groups 

and disruption of hydrophobic interactions between 

side chains [23, 69, 77, 109]. 

Chaotropie ions, on the other hand, are known 

for their ability to impart lipophilic properties to 

water [72] and to alter the structure of biomolecules 

[44]. Their application for the elution of 

proteins from high members of alkyl-agarose derivatives 

has been reported to be successful [73] ; 

ho'wever, it is important to bear in mind their 

property of dissolving agarose gels and denaturating 

proteins. In this case, cross-linked agaroses, 

which are kno'~vn to support more severe conditions 

(pH, temperature, ionic strength etc) than 

the normal agarose gel, are to be employed. 

As has been mentionned before, by increasing 

the hydrophobicity of the gel tighter binding resuits, 

and desorption of proteins requires ever 

more drastic conditions [78]. The combination of 

salt gradients with > agents 

(e.g., ethylene glycol):, variation in pH or temperature, 

etc., may give an increased selectivity of 

the elution. As an illustration, it can be mentionned 

the case of serum albumin, where elution 

occurs at pH 3 with 50 per cent ethanol in the 

buffer [791. Similarly, the use of mixed gradients 

of buffer solution with high salt concentration, 

and buffer without salt but containing 50 per cent 

of ethylene glycol, have been successfully applied 

to desorb enzymes such as chymotrypsin and 

trypsin from hydrophobic matrices [90]. 

A simple pure salt gradient may result in a 

strongly diluted peak in the elution because the 

cond,itions in 'which the equilibrium for the 

hydrophobic interaction is attained are slob,. 

Only lower flow rates could provide more favourable 

results. In addition, it should be emphasized 

that in some cases the elution with ethylene 

glycol, in the absence of salts, may be inefficient 

due to the presence of electroslatie interactions 

BIOCHIMIE, 1978, 60, n ° 1. 

[98] when adsorbents involving mixed effects are 

employed. 

The use of detergents [127] is usually the last 

resource, when the elution by other milder conditions 

has not been possible. The problem in using 

detergents is their intrinsic denaturing effect and 

the difficulty of their removal from the columns 

during the regeneration step [127]. A wide range 

of different detergents has been used varying in 

their efficiency to eliminate strongly adsorbed 

materials E127]. In this respect, the more ionic 

ones are to be preferred for the reasons discussed 

above. 

Finally, flat curves are obtained by gradient 

elution [75], presumably due to a postulated 

irregularity of the matrix (see fig. 2) and the 

occurence of a wide range of binding sites of 

varied strengths [74]. For this reason, attempts 

should be made to fractionate through > [123] (as opposed to differential-elulion) 

on a series of adsorbents of 

increasing hydrophobicities. In this way, each 

protein tends to be adsorbed or bound to the 

column that provides the minimum degree of 

hydrophobicity required for binding, and complete 

elution of the materials can be accomplished 

with mild eluants. Another alternative consists 

of the use of a high member of the alkyl agarose 

series, which may retain a high percentage of the 

protein content of a particular mixture, and 

allows the elution of the molecule of interest 

under mild conditions nvhile most of the other 

protein remain adsorbed on the column [123]. 

Obviously, this possibility can be utilized only 

when the biomolecule in question has a lo~,- 

hydrophobicity. One should note that in principle 

it should be possible to achieve purification by 

HIC, not only by a selective retention of a given 

protein, but also by exclusion of a desired protein 

when most of the other proteins in the mixture 

are adsorbed [114]. 

VIII. APPLICATIONS, FUTURE AND IMPLICATIONS 

OF HIC. 

The relevance of HIC lies in the fact that it is 

perhaps the first technique which takes advantage 

of the hydrophobic properties of the biomolecules. 

The hydrophobic character of biomolecules 

should be a specific property, similar to the ionic 

characler, since this is a function of the primary 

structure in the case of proteins and nucleic 

acids. So, it is not unrealistic to consider that


their selective separations are feasible. As an 

example, the purification of two interconvertible 

forms of glycogen-phosphorylase, which differ in 

a unique serine residue, has been successfully 

achieved through their ability to adsorb on two 

distinct members of the alkyl-agarose series [129]. 

Unfortunately, the types and nature of the 

hydrophobic adsorbents available to date are probably 

not well systematized, and the lack of intermediate 

hydrophobic matrices limits the applicability 

of the method. To provide for a larger 

variety in the types of ligands, including the 

introduction of aromatic structures, it might be 

expedient to attach these ligands to (< arms )~ that 

by themselves are not hydTophobic, e.g., carbohydrates 

instead of hydrocarbons [96]. At first 

sight, hydrophobically coated supports may be 

used as concentrating systems as well [52]. In 

fact, the protein 'which is going to be purified 

does not require a pre-concentration step if the 

conditions under 'which the chromatography is 

performed allow its adsorption. 

The application of HIC to the purification of 

nucleic acids using matrices of varied hydrophobicities 

according to the type of ligand attached 

or to its degree of subslitution awaits for further 

studies which may greatly improve the application 

of this technique. This aspect is especially 

interesting when the molecules are not stable or 

soluble at either too high or too low salt concentrations. 

The separation of particles, like sub-cellular 

fractions or entire cells [110, 121] has been reported, 

and it seems to be a very promising application 

of HIC, since membranes can be considered 

as aggregates of molecules of varied hydrophobicities. 

A similar application in a quite different 

field of biochemistry is preparation of enzymatic 

reactors through the immobilization of enzymes 

on hydrophobic supports [32, 101, 122, 126]. The 

basic idea is the possibility to have a reactor 

which can be periodically recycled, when the 

adsorbed material loses its activity by elution 

and adsorption of freshly active substances 

(which may be of the same or of another biological 

activity, but similarly adsorbed on the 

hydrophobic support). Thus, the hydrophobic 

matrix may function as an universal support of 

easier handling than those systems in which the 

immobilization necessitates a covalent linkage 

between the biomolecule and the adsorbent, with 

their corresponding limitations in function and 

utility, 

It should be reminded that some hydrophobic 

ligands denature proteins through a > action [96]. This effect can be reduced 

by employing hydrophobic ligands of milder 

influence, or by introducing additional polar or 

ionic groups in the hydrocarbon chain [9]. Finally, 

the problem of >, which 

constitutes another important drawback in protein 

separation by chromatography on hydrophobic 

columns, must be considered. If inhomogeneity 

occurs in the adsorbent, that is if the interacting 

sites on a given member of the suhstituted-agarose 

series are different in their strength of interaction 

with a particular protein (fig. 2), elution by 

decreasing salt concentration or increasing concentration 

of > agents, will 

never result in a narrow peak but rather in flat 

curves, which increase contamination of the sample 

by other proteins. Some suggestions to avoid 

this phenomenon have already been mentioned 

taking advantage of the hydrophobicity of the 

matrix. 

In conclusion, the correct application of hydrophobic 

chromatography implies the consideration 

of three main factors : 

-- the nature and hydrophobicity of the adsorbent 

and of the protein, 

-- the conditions of the adsorption, 

-- the conditions of the elution of the adsorbed 

material. 

It may be expected that as the mellmd will be 

more and more aplied wi'.h various biomolecules, 

in different conditions, our understanding will be 

enriched so about many biological events in 

which a hydrophobic interaction is involved. 

Acknowledgments. 

The author expresses his gratitude to Dr. Jean-Marc 

Egly for his encouragement in the preparation of this 

article. Special thanks are due to Professor Shmuel 

Shaltiel and Professor Stellan Hjertdn for their criticism 

and suggestions, and to Professor Barbarin 

Arreguin for his continuous interest on my ,work. My 

appreciation to Dr. Dana Fawlkes for the linguistic 

reoision and to Mrs. Inga Johansson and Mr. GSsta 

ForMing for typing the manuscrip and drawing the 

figures, respectioely. 

To the National University of Mexico and the National 

Council of Sciences and Technology of Mexico 

many thanks for their financial support. 

REFERENCES. 

1. Hirs, C. W. W. (1955) Methods in Enzymol., Colowick 

S. P. and Kaplan, eds, Academic Press, 

New York° 1, p. 113. 

2. Colowick, S. P., ibid, p. 90. 

3. Porath, J. (1968) Nature (London), 218, 834.


4. Green, A. A. a Hughes, W. L. (1955) Methods in 

Enzymol., 5, 67. 

5. Cuatreeasas, P. ~ Anfinsen, C. B. (1971) Ann. Reo. 

Biochem., 40, 259. 

6. Cuatreeasas, P., Wilehek, M. a Anfinsen, C. B. 

(196~) Proc. Nat. Acad. Sci. U. S., 61, 636. 

7. O'Carra, P..a Griffin, T. (1974) Methods in Enzytool., 

34, 108. 

8. Steers, Jr., E.. Cuatreeasas, P. ~ Pollard, H. B. 

(1971) J. Biol. Chem., 2~6, 196. 

9. Yon, R. J. (1972) Biochem. J., 126, 765. 

10. Er-el, Z., Zaindenzaig, Y. & Shaltiel, S. (1972) 

Biochem. Bioohys. Res. Commun., 49, 383. 

11. Shaltiel, S. (1974) Methods in Enzymol., 34, 126. 

12. !Klotz, I. M. (1970) Arch. Biochem. Biophys., 138, 

704. 

13. Kauzmann, W. (1959) Adv. Prol. Chem., 14, 1. 

14. Vol'kenshtein, M. V. (1970) Molecules and Life, 

Plenum Press. N. Y., pp. 189 and 357. 

15. Didkerson, R. E. ,~ Gets, I. (1969) The structure 

and action of proteins, Haper ~ Roxv Publ., 

N. Y., D. 92. 

16. Perrin, J. (1905) J. Chem. Phtls., 3~ 50. 

17. Langmuir, I. (1917) J. Am. Chem. Soc., 39, 1848. 

18. Dandliker, W. B. a de Saussure, V. A. (1971) The 

Chemistry of Biosnrfaces, Vol. I (Hair, M. L., 

Ed.), Marcel D~kker, Inc., N. Y. 

19. Tanford, C. (1972) J. Mol. Biol., 67. 59. 

20. Frank, H. S. ~ Evans, M. W. (1945) J. Chem. Phys., 

13, 5O7. 

21. Tanford, C. (19.62) J. Am. Chem. Soc., 84, 4240. 

22. Noz~ki, Y. ~ Tanford, C. (19.71) J. Biol. Chem., 

'246, 2211. 

23. Bull, H. B. ~ Bresse, K. (1974) Arch. Bioehem., 161, 

665. 

24. Bigelo,w, C. C. (1967) J. Theor. Biol., 16, 187. 

25. Waugh, D. F. (1954) Adv. Prof. Chem., 9. 325. 

26. Fisher, H. F. (1964) Proc. Nat. Aead. Sci. U. S., 51, 

1285. 

27. Dandliker. W. B.. Alonso, R., de Saussnre. V. A., 

Kierszenb.~um. F.. Levison. S. A. ~ Schapiro, H. 

C. (1967) P;ocbemistry, 6, 1460. 

28. Davey, M. W., Huan~', J. W.. Sulkowski. E. & 

Carter. 'W. A. (1974) .L Biol. Chem., '2A9, 635~. 

29. Boldt. D. H., Spee~rart. S. F- Richards. R. 1,. 

Alving, C. R. (1977) Biochem. Biophys. Res. 

Comrnun., 74, 208. 

30. Barry, A. ~ Briningar, W. S. (1975) J. Biochem., 

250, 8856. 

31. Tanford, C. (1973) The h udrophobic effect : Formation 

of micelles and biological membranes, 

Wiley, N. Y., p. 9. 

32. Davis, M. A. F.. Hanser. H.. Leslie. R. B. 

Phillil)s, M. C. (1973) Bioehim. Biophys. Aeta, 

3]7, 214. 

33. Brandts, J. F. (1969) Biol. MacromoL, 2, 213. 

34. P~hlman, S., Rosengren, J. ~ Hjert~n, S. (I977) 

J. Chromatoqr.. I.-~1. 99. 

35. Hofstee, B. H..L (I975) Biochem. Biophys. Bes. 

Commnn., ~;~. 618 

36. Hofmeister. F. (1888) Arch. Exp. Pathol. Pharmakol., 

24, 247. 

37. Tanford, C. (1968) Adv. Prof. Chem., 2.~, 121. 

38. Tiselius, A. (1948) Ark. Kemi Min. Geol., B 26. 

39. King, T. P. (1972) Biochemistry, ll, 367. 

40. Memoly, V. A. ~ Doellgast, G. J. (1975) Biochem. 

Biophys. Res. Commun., 66, 1011. 

41. Holmes, W. M., Hurd, R. E., Reid. B. R., Rimerman, 

R. A. ~ Hattie]d, G. Vv'. (1975) Proc. Nat. 

Acad. Sci. U. S., 72, 1068. 

42. Doellgast, G. J. &Plaut, A. G. (1976) Immunochem., 

1;3, 135. 

43. Von Hippel, P. H. ~ Schlieh, T. (1969) Structure 

and stability of macromolecules (Timascheff, S. 

N. and Fasman, G. D., Eds), Marcel Dekker, N. 

Y., p. 41. 

44. Hamaguchi, K. & Geiduschek, E. P. (1962) .L Am. 

Chem. Soc., 84, 1329. 

45. Lakshmi, T. S. ,& Nandi, P. K. (1976) Y. Chromatoyr., 

116, 177. 

BIOCHIMIE, 1978, 60, n ° 1. 

46. Hamar, L., P~hlman, S..~ Hjert6n, S. (1975) Biochim. 

Biophys. Acta, 403, 554~ 

47. Mevarech, M., Leicht, W. & Werler, M. M. (1976) 

Biochemistry, 15, 2383. 

48. Larsen, H. (1967) Advances in Microbiolotlical 

Physiology (Rose~ A. H. ~ Wilkinson, J. F., Eds.), 

Aead. Press, N. Y., p. 97. 

49. Reistad, R. (1970) Arch. Mikrobiol., 71, 353. 

50. Shanbhag, V. P. ~ Axelsson, C. C. (1975) Eur. J. 

Biochem., 60, 17. 

51. Hjert6n, S. (1976) Meth. Prof. Sep., 2, 233. 

52. Hjert~n, S., Rosengren, J. ~ Phhlman, S. (1974) J. 

Chromatoar., 101. 281. 

53. Jennissen, H. P. (1976) Biochemistry, 15, 5683. 

54. Rimerman, R. A. ~ Hatfield, G. W. (1973) Science, 

182, 1268. 

55. Hjert~n, S. (1973) J. Chromatoar., 87, 325. 

56. Rosengren, J., Phhlman, S., Glad, M. & Hjert6n, 

S. (1975) Biochim. Biophys. Acta. 412, 51. 

57. O'Carra, P.. Barry, S..& Griffin, T. (1974) FEBS 

Letters, 43, 169. 

58. Visser, J. • Strating, M. (1975) Biochim. Biophys. 

Acta, 384, 69. 

59. Scheraga. H. A., Nemethy, G. ~ Steinberg, I. Z. 

(1962) J. Biol. Chem., ~7, 2506. 

60. Hertz, H. G. • Zeidler, M. D. (1964) Z. Electrochem., 

68, 821. 

61. Wishnia, A..& Pinder, Jr., T. W. (1964) Biochemistry, 

3, 1377. 

62. Wishnia, A. &Pinder, Jr., T. W. (1966) Biochemistry, 

5, 1534. 

63. Raibaud, O., H6gberg-Raibaud, A. ~ Goldberg, M. 

(1975) FEBS Letters, ;50, 130. 

64. Visser, J. ~ Strating, M. (1975) FEBS Letters, 57, 

183. 

65. Dziegiele~vski, T. & Jakubo~wski, H. (1975) J. Chromatogr., 

10g, 364. 

66. Shaltied, S. (1975) in Enzymes (Desnuelle, P., Ed.), 

North Holland, Amsterdam, pp. 117-127. 

67. Jennissen, H. P. (197'5) Biochemistry, 14, 754. 

68. Busey, H., Rimerman, R. A. ,~ Hatfield. G. W. 

(1975) Anal. Biochem., 64, 380. 

69. Oakenfull, D. G. (1974) Proc. Aust. Bioehem. Soc, 

7, 20. 

70. Weber, M. M. (1976) Anal. Biochem., 76, 177. 

71. Sch6p,. W., Meinert, S., Thyfronitov, J. ~ Anrich, 

M. (1975) J. Chromatogr.. 10d, 99. 

72. Hatefi. Y. • Hanstein, V~'. H. (1969) Proc. Nat. 

Acad. Sci. U. S., 62, 1129. 

73. Neurath. A. R.. Lerman. S.. Chen, M. ~ Prince, A. 

M. (1975) J. Gen. Virol., 28. 251. 

74. Hofstee, B. H. J. (1974) in > (Dunlop, R. B., 

Ed.), Plenum Pub]. Corr., N. Y., op. 43-59. 

75. Hofstee, B. H. J. (1975) Prep. Biochem., 5, 7. 

76. Weiss. H. a Bueher, T. (I970) Eur. J. Biochem., 

17, 561. 

77. Bizelis, R..a Umbarger, H. E. (1975) J. Biol. 

Chem., ~5~. 4315. 

78. Jennissen, H. P. ,~ Heilmeyer, Jr., L. M. G. (1975) 

Biochemistry, 14, 754. 

79. Peters. Jr., T~, Taniuehi, H. a Anfinsen, Jr., C. B. 

(1973) J. Biol. Chem., ~4'8. 9447. 

80. Lee, B. a Richards, F. M. (1971) J. Mol. Biol., 55, 

379. 

81. Chothia, C. H. (1974) Nature (London), 248, 338. 

82. Klotz, I. M. ,¢ Franzen, D. S. (1945) J. Am. Chem. 

Soc., 67, 1003. 

83. Chothia, C. ~ Janin, J. L. (1975) Nature (London), 

256, 705. 

84. Jennissen, H. P. (1976) Hoppe-Seyler's Z. Physiol. 

Chem., ,357, 1201. 

85. Arnott, S., Fulmer, A. Scott, E. E., Dea, I. C., 

Moorhouse, R. ~ Reis, D. A. (1970) J. Mol. Biol., 

90, 269. 

86. Jennissen, H. P. (1976) in Prof. Biol. Fluids, Proc. 

Colloq., 1975 (Peeters, L. H., Ed.), Vol. 23, Pergamon 

Press, Oxford ~ N.Y., pp. 675-679. 

87. Cuatrecasas, P. (1970) J. Biol. Chem., 245, 3059. 

88. Barry, S. ~ O'Carra, P. (1973) Biochem. J., 135, 

595.


89. Hofstee, B. H. J. (1974) Adv. Exp. Med. Biol., 42, 

43. 

90. L/~/ls, T. (1975) J. Chromatogr., 111, 373. 

91. Ellingboe, J., Nystr6m, E. ,¢ Sj(ivall, J. (1970) J. 

Lipzd Res., 11, 266. 

92. Clifford, J., Pethica. B. A. ~ Senior, W. A. (1965) 

Ann. N. Y. Acad. Sei., 125, 458. 

93. Porath, J., Sundberg, L., Fornstedt, N. ,~ Olson, I. 

(1973) Nature (London), 245, 465. 

94. Yon, R. J. ~ Simmonds, R. J. (1975) Bioehem. J., 

161, 281. 

95. Jost, R., Miron, T. ,¢ Wilchek, M. (1974) Biochim. 

Biophys. Acta, 362, 75. 

96. Hofstee, B. H. J. (1973) Biochem. Biophys. Res. 

Commun., 50, 751. 

97. Geren, C. R., Magee, S. C. ,~ Ebner, K. E. (1976) 

Arch. Biochem. Biophys., 172, 149. 

98. Ho~ksc, H. (1974) Carbollyd. Res., 37, 390. 

99. Shaltiel, S., Adler, S. P., Purich, D., Caban, C., 

Senior, P. ~ Stadtman, E. R. (1975) Proe. Nat. 

Acad. Sci. U. S., 72, 3397. 

100. Palade, G. E. (1959) in

Hydrophobic (interaction) chromatography c*). - Bashan Foundation

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