Hydrophobic (interaction) chromatography c*). - Bashan Foundation
Hydrophobic (interaction) chromatography c*). - Bashan Foundation
Hydrophobic (interaction) chromatography c*). - Bashan Foundation
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BIOCHIMIE, 1978, 60, 1-1_5.<br />
Revue<br />
<strong>Hydrophobic</strong> (<strong>interaction</strong>) <strong>chromatography</strong> <strong>c*</strong>).<br />
J.-L. OCHOA * *<br />
lnstitnlo de Quimica.<br />
Universidad Nacional Autonoma<br />
de Mexico (UNAM),<br />
Ciudad Universitaria,<br />
Mexico, D.F.<br />
Introduction.<br />
The isolation and purification of macromolecules<br />
by biochemical fractionation techniques,<br />
like ion-exchange <strong>chromatography</strong>, gel filtration<br />
(molecular-sieve <strong>chromatography</strong>), affinity chro-<br />
Inatography, electrophoresis, etc., is primarily<br />
dependent on their biological and physicochcmical<br />
properties [1-5].<br />
Based on biospecific <strong>interaction</strong>s [6], affinity<br />
<strong>chromatography</strong> has been considered as one of<br />
the most effective separating methods. However,<br />
serious disadvantages are found upon its application,<br />
due mainly to undesirable non-biospecific<br />
adsorption [7] attributed to the characteristics of<br />
the matrix ov support, and the nature of the<br />
ligand and spacer-arm introduced to b~idge the<br />
ligand from the matrix backbone. Moreover, once<br />
a protein is attached to an immobilized ligand,<br />
for which it sho~vs affinity, the properties of the<br />
support may change and become like those of an<br />
ion-exchanger, interfering with the chromatographic<br />
process.<br />
The belief that such interferences could be<br />
adequately controlled by eliminating ionic groups<br />
in the matrix and/or in the spacer-arm, brought<br />
as a consequence other types of undesirable<br />
effects closely related to the extent of hydrophobicity<br />
of the spacer-arm employed [57]. Yon [93,<br />
and Er-eI et aL [10], reported that by coupling<br />
different types of spacer-arms (varying in hydrophobicity)<br />
to an inert matrix, potent adsorbents<br />
for proteins were obtained. The adsorption mechanism<br />
seems to be based, fundamentally, on<br />
hydrophobic <strong>interaction</strong>s between the protein and<br />
the adsorbent, and it has been demonstrated that<br />
it can be positively exploited for separation purposes<br />
[11, 105]. In this way, a novel technique<br />
which takes advantage of the hydrophobicity of<br />
* This article is a complementary part to my contribution<br />
at the Forum des Jeunes, in Lyon, France<br />
(July 6-8, •977).<br />
** Present address : Facultd de Mddecine de Strasbourg,<br />
Institut de Clfimie Biologique, 11 rue<br />
Humann, 67000 Strasbourg, France.<br />
biomolecules, has been introduced as a complement<br />
to the other, routinely employed, separating<br />
methods mentioned above.<br />
Since the separation is based on hydrophobic<br />
<strong>interaction</strong>s, this technique receives a number of<br />
names 'which intend to describe the principle and<br />
the parameters involved in the separation process,<br />
though all of them are entirely or partly dealing<br />
with the concept of hydrophobicity :<br />
<strong>Hydrophobic</strong> <strong>chromatography</strong> [11] ; <strong>Hydrophobic</strong><br />
(<strong>interaction</strong>) <strong>chromatography</strong> [51]; <strong>Hydrophobic</strong><br />
salting-out <strong>chromatography</strong> [93]; Phosphate-induced<br />
protein <strong>chromatography</strong> [54] ; Repulsion<br />
controlled <strong>chromatography</strong> [94] ; Detergent<br />
protein-<strong>interaction</strong> [95, 961 ; <strong>Hydrophobic</strong><br />
affinity <strong>chromatography</strong> [128] ; etc.<br />
It has been shown [12] that a large part of the<br />
non-polar residues of the amino acids in proteins<br />
are exposed to ~vater interface, as opposed to the<br />
expected preferential location of the hydrophobic<br />
amino acids in the interior of the biomolecule.<br />
These non-polar amino acids are found in the<br />
protein surface forming ) of distinct<br />
hydrophobic character which Can account for the<br />
biospecific conformation of the protein [13] as<br />
well as for its ability to complex or aggregate to<br />
other types of molecules for instance, lipids. Presumably,<br />
these hydrophobic (< patches >> are randomly<br />
distributed on the surface of the biomolecule.<br />
Their number (possible number of interacting<br />
sites) and the extent of their hydrophobicity<br />
(type and distribution of the non-polar amino<br />
acids) should be a characteristic of each macromolecule.<br />
Therefore, their specific separation<br />
should be possible with an adequate hydrophobically<br />
coated support or matrix.<br />
I. THE BIOLOGICAL ROLE OF THE HYDROPHOBICITY<br />
IN BIOMOLECULES.<br />
There is no doubt that the hydrophobie <strong>interaction</strong>s<br />
play an important role in biological systems.<br />
The membranes in the living cell are made<br />
1
- - An<br />
2 J.-L. Ochoa.<br />
up of mainly hydrophobically interacting lipidlipids<br />
and lipid-proteins. The sub-units of large<br />
proteins are often held together by hydrophobic<br />
bonds, and it is well accepted that they are important<br />
in supporting the tertiary protein structure<br />
[13].<br />
Functionally, hydrophobic <strong>interaction</strong>s seem to<br />
be involved in recognition processes, as for example<br />
in the case of some enzyme-substrate complexes<br />
and antigen-antibodies associations, etc.<br />
[14-15, 27-30]. Except for the case of membranes,<br />
in which an exclusive structural function has<br />
been attributed [31], there is not much work in<br />
correlating the hydrophobic properties of the<br />
biomolecules with their functions [32, 99-100].<br />
Therefore, it could be interesting to find out<br />
whether these hydrophobic regions in the biomolecules<br />
are related to their intrinsic biological<br />
properties, and/or to their location in the cell.<br />
Perhaps one of the reasons why fe"w systematic<br />
studies of hydrophobic effects have been made,<br />
is that the hydrophobic compounds possess lo"w<br />
solubility in water. If they are made soluble by<br />
the introduction of polar groups, they tend to<br />
form micelles as exemplified by the case of soaps.<br />
Although molecular dispersion may be obtained<br />
through the addition of polarity reducing agents<br />
to the medium, such agents would also reduce the<br />
<strong>interaction</strong> of the hydrophobic compounds with<br />
proteins and even alter the protein structure,<br />
which generally depends on the integrity of the<br />
hydrophobic core [13].<br />
A means to obtain molecular dispersion of a<br />
hydrophobic ligand in aqueous milieu, without the<br />
addition of polarity reducing agents, is to attach<br />
the ligand to a hydrophilic but insoluble polymer<br />
such as agarose. Consequently, hydrophobic supports<br />
can be used not only for separation purposes,<br />
but also to study the hydrophobicity of the<br />
biomolecules and their modes of <strong>interaction</strong>. In<br />
turn, this may lead to a clearer understanding of<br />
their functions.<br />
II. THE PHYSICOCHEMICAL CONCEPT<br />
OF HYDROPHOBICITY.<br />
Perrin [16] `was the first to utilize the terms<br />
> and ¢ hydrophobic >> when studying<br />
colloids. He considered them hydrophilic<br />
if their stability was relatively insensitive to the<br />
addition of electrolytes, or hydrophobic if they<br />
exhibit extreme sensitivity to added electrolytes.<br />
Later, Langmuir [17], in a classical experiment,<br />
discussed polar molecules (fatty acids) in terms of<br />
BIOCHIMIE, 1978, 60, n ° 1.<br />
hydrophilic and hydrophobic groups. Since then,<br />
these terms have been in every day use.<br />
In general, "when an apolar group (or hydrophobic)<br />
is inserted into `water, several effects can be<br />
observed (for review see ref. 18) :<br />
-- A negative unitary entropy change (--AS),<br />
`which implies an overall increase in the degree<br />
of order. The entropy distribution becomes relatively<br />
important as the molecular weight of the<br />
apolar group increases.<br />
--A small enthalpy change, usually negative<br />
(--AH), which reflects an energetic component<br />
in the <strong>interaction</strong> between 'water and an apolar<br />
group and opposes the negative unitary effect<br />
favouring mixing of the hydrocarbon in water.<br />
increase in heat capacity (+ ACp), which<br />
implies either an increased degree of freedom of<br />
the vibrational and rotational motion within the<br />
existing structure, or a progressive alteration of<br />
an existing structure as the temperature is raised.<br />
-- A decrease in volume (--AV), which indicates<br />
that the apolar groups and the water molecules<br />
have packed together with a contraction of<br />
some structure, or that the apolar groups are<br />
accomodated into open spaces or voids preexisting<br />
`within the water structure itself. The former<br />
is unlikely in view of the small enthalpy change<br />
involved.<br />
--Finally, the presence of apolar groups in<br />
water results in an increase in the number<br />
or strength of hydrogen bonds within the water<br />
molecules, as has been demonstrated by spin lattice<br />
relaxation studies and further supported by<br />
Raman measurements [60]. Its probable reason is<br />
that the hydrocarbon chains restrict the mobility<br />
of water molecules in such a `way that the covalent<br />
character of the hydrogen bound is increased<br />
[92].<br />
On the other hand, the molecular picture of the<br />
hydrophobic <strong>interaction</strong> is the reverse of that obtained<br />
when introducing apolar groups into water<br />
(see mechanism below). The withdrawal of the<br />
apolar groups from the aqueous phase removes<br />
restrictions on hydrogen-bond bending and thus,<br />
achieves a positive unitary entropy. This supports<br />
the observation that the hydrophobic <strong>interaction</strong><br />
is a spontaneous process. This assumption was<br />
substantially demonstrated by Frank and Evans<br />
in 1945 [20]. Some years later, it has been shown<br />
[19] that the free energy values of the transfer of<br />
aliphatic hydrocarbons from an apolar medium<br />
to a polar one, like water, increases linearly with<br />
increasing numbers of (CH 2) methylene groups,<br />
and thus the hydrophobicity of the molecule.
<strong>Hydrophobic</strong> (<strong>interaction</strong>) <strong>chromatography</strong>. 3<br />
III. DETERMINATION OF THE HYDROPHOBICITY OF<br />
BIOMOLECULES.<br />
It is well kno~vn that most proteins contain a<br />
relatively high proportion of amino acids ~dth<br />
non-polar chains (table I). Tanford [21], and<br />
TABLE I.<br />
Non-polar (hydrophobic) amino acids commonly<br />
found in proteins.<br />
AI anine CH 3 -R<br />
Leucine<br />
Isoleucine<br />
Valine<br />
Proline<br />
Phenylalanine<br />
Tryptophane<br />
Methionine<br />
CH3x<br />
/ CH-CH2-R<br />
CH 3<br />
CH3-CH2-CH-R<br />
I<br />
CH 3<br />
CH3 x<br />
CH-R<br />
CH3 /<br />
CH CH2\ CH_CO0tl<br />
CH , /<br />
2XNH<br />
d CH:CH\<br />
CH ,,,CH-CH _ -R<br />
"~ CH-CH >" z<br />
/1 CH-CH \\<br />
C -- CH-CH ~-R<br />
CH % CH=C ~ Ixrfl "~CI-I "<br />
CH3-S-CH2-CH2-R<br />
R<br />
-CH-CO0-<br />
!<br />
+NH 3<br />
Nozaki and Tanford [22], formulated an experimental<br />
procedure based more or less on the Kauzmann<br />
[13] conception which permits the estimation<br />
of the amino acid hydrophobicity. The method<br />
consisted, essentially, in determining the<br />
solubilities of the amino acids in water as ~vell<br />
as in progressively increasing concentration of<br />
some organic solvents, such as ethanol in water.<br />
The solubilities of the amino acids were extrapolated<br />
to pure organic solvents and then the free<br />
BIOCHIMIE, 1978, 60, n ° 1.<br />
energy value of the transfer for the amino acid<br />
from pure organic solvent to ~vater was calculated.<br />
Using as a reference glycine, and substracring<br />
its free energy transfer value from that of all<br />
the other amino acids, it was possible to formulate<br />
a hydrophobicity scale for amino acid residues<br />
~vhere their free energy transfer values<br />
become more and more positive as the hydrophobic<br />
character of the compounds increases [18].<br />
Other attempts in scaling the amino acid hydrophobicity<br />
can be illustrated by the ~vorks of Bull<br />
and Breese [23] and Bigelov¢ [24]. The former<br />
studied the effect of the amino acid on the surface<br />
tension of water and the latter calculated the<br />
average hydrophobicity of several proteins by<br />
their amino acid composition according to data<br />
of Tanford and of Bull and Breese.<br />
A different approach ~vas done in terms of the<br />
frequency of non-polar side chains in proteins<br />
[2~], in which yeas found a variation from 0.21 to<br />
0.47. Separately, Fisher [26] employed the ratio<br />
of the volume of the polar groups to those of the<br />
non-polar as a hydrophobicity degree, but this<br />
idea has the inconvenience, like the one mentioned<br />
above [25], to consider that a group is<br />
either polar or non-polar, ~without any gradation<br />
between these t~o extremes.<br />
Recently, a method for studying the magnitude<br />
of the <strong>interaction</strong> bet~veen protein and aliphatic<br />
hydrocarbon chains, and thus indirectly the hydrophobicity<br />
, ~vas reported [~0]. It is based on<br />
the partition of proteins in an aqueous two phase<br />
system containing dextran and polyethylene glycol<br />
and different:fatty esters of polyethylene glycol.<br />
However, the measurements depend largely<br />
on a critical chain length which, by this technique,<br />
should be greater than 8 carbons.<br />
Finally, an approach to localize the hydrophobic<br />
sites on the surface of the proteins by means<br />
of interacting with small molecules, has been<br />
attempted using fluorescent probes [27]. It is not<br />
difficult to speculate that ~vith this set of information,<br />
a better comprehension of many biological<br />
phenomena is near. We do not know yet<br />
how the complex enzymatic systems are organized,<br />
nor how the recognition bet~veen proteins<br />
occurs to constitute such enzymatic complexes<br />
after protein synthesis. Neither do we have a good<br />
explanation for the transport of many substances,<br />
including proteins, through the hydrophobic core<br />
of the membrane. And furthermore, it is possible<br />
to believe that hydrophobic ((patches >> in the<br />
membrane (either out or inside) are needed to<br />
make possible many of the most common biolo-
4 J.-L. Ochoa.<br />
gical phenomena like the recognition of non-polar<br />
substances by membrane receptors and even cellcell<br />
<strong>interaction</strong>s.<br />
IV. THE MECHANISM OF THE HYDROPHOBIC INTER-<br />
ACTION.<br />
The hydrophobic <strong>interaction</strong> is the result of the<br />
adherence of two non-polar groups. The case of<br />
detergents can be considered as an example where<br />
negative enthalpy changes are observed during<br />
the micelle formation in aqueous solvents [33!.<br />
If the adsorption of proteins to hydrophobic matrices<br />
is considered to be a process of limited micelle<br />
formation, a negative change in the enthalpy<br />
value ~vould not preclude the hydrophobic nature<br />
of the binding. In addition, this change must be<br />
negligible as compared to the value of increasing<br />
entropy of the system I20]. Furthermore, before<br />
the <strong>interaction</strong> the water molecules are forced to<br />
keep in orde~ around the hydrophobic entities<br />
(fig. 1) as compared to the order in the bulk. When<br />
the hydrophobic sticks come in contact with each<br />
ooooooooooo<br />
o o o<br />
oeOOooeeoeooo o~ o • •<br />
o o o o<br />
::~ii........oj_<br />
oooooooooooo<br />
•<br />
ooooooooooo O O0000QeO<br />
0 0 0 000 O • 0<br />
D •<br />
a) b)<br />
F[~. 1. -- The mechanism of hydrophobic <strong>interaction</strong>.<br />
The water molecules around the hydrophobie<br />
> are doted, for simplicity, in order to<br />
distinguish them from those in the bul~. Notice. that<br />
the difference bet'ween a) and b) is only the improved<br />
degree of disorder (-I-AS) of vcater molecules.<br />
other, the ordered water molecules will be excluded<br />
and 'will adopt the less ordered buIk water<br />
state which is equivalent to an increase in entropy.<br />
This hypothesis is supported experimentally<br />
by the study of antigen-antibody complex formation<br />
[27] where a relative insensitivity to dissolution<br />
of a preformed antigen-antibody precipitate<br />
is observed, suggesting that once the complex is<br />
formed, the solvent is largely excluded in lhe<br />
regions of contact.<br />
Thermodynamically, the free energy value AG<br />
of a hydrophobic <strong>interaction</strong> is a function of AH<br />
and AS, according to equation :<br />
AG ~ AH -- TAS ..................... (N ° 1)<br />
Since AH is small as compared to TAS value,<br />
the process is fundamentally determined by the<br />
BIOCHIMIE, 1978, 60, n ° 1.<br />
change in entropy. In these conditions the reaction<br />
proceeds spontaneously [51]. In other words,<br />
the input of energy or chemical work is not necessary<br />
to make possible the <strong>interaction</strong> between two<br />
hydrophobic molecules in aqueous solutions.<br />
The contact between two different molecules,<br />
like the sub-units in the case of some proteins,<br />
is largely dependent on the surface areas occupied<br />
by the residues which participate in the <strong>interaction</strong>.<br />
The concept of accessible surface area [80j<br />
describes the extent to ~vhich protein atoms can<br />
form contacts with water, and is related to hydrophobic<br />
free energies [81]. In any case, the association<br />
of protein sub-units, whether by van der<br />
Waals contacts, electrostatic forces and/or hydrophobic<br />
<strong>interaction</strong>s, leads to a reduction of the<br />
surface area accessible to the solvent ~vhen the<br />
two molecules associate. Evidence against the<br />
hydrogen bond as a major contribution to the<br />
free energy of the protein-protein <strong>interaction</strong> has<br />
been obtained lhermodynamically [82]. On the<br />
other hand, van der Waals <strong>interaction</strong>s, though<br />
they are more numerous as they involve all the<br />
pair of neighbouring atoms, are much less energetic<br />
and their overall contribution is small. The<br />
hydrophobic contribution is largely dependent<br />
upon the entropy gained by water due to the<br />
smaller accessible protein surface area when the<br />
protein molecules form a complex. The hydrophobic<br />
<strong>interaction</strong> seems to be entirely unspecific<br />
as compared to the complementarity of the surfaces<br />
involving hydrogen bonds and van der<br />
Waals contacts ; ho~,ever, they decide which proteins<br />
can recognize each other.<br />
V. THE SPECIFICITY OF THE ADSORPTION<br />
OF BIOMOLECULES ON HYDROPHOBIC SUPPORTS.<br />
Biospecific affinity, whether involving an<br />
> or not, presumably depends to a<br />
large extent on the complementarity of the contours<br />
of the interacting molecules. In the absence<br />
of any specific effect, the binding of proteins to<br />
substituted agaroses is greatly affected by the<br />
overall charge of the biomolecule [105].<br />
The lack of activity in the adsorbed state of<br />
some proteolytic enzymes [90], indicates that the<br />
binding site is occupied by the hydrophobic<br />
group of the substituted agarose. Whether the<br />
hydrophobic group interacts with the hydrophobic<br />
poc1~ets of the active sites of the enzymes or<br />
acts by a less specific ~way can be questioned. In<br />
any event, both cases may contribute to the<br />
adsorption phenomena.
<strong>Hydrophobic</strong> (<strong>interaction</strong>) <strong>chromatography</strong>.<br />
In other examples, the addition of the specific<br />
substrate accompanying the enzyme during the<br />
<strong>chromatography</strong> (to mask the specific binding<br />
sites) did not alter the binding of the enzyme to<br />
the hydrophobic matrix, indicating that the<br />
adsorption takes place through sites other than<br />
the specific substrate binding site [971.<br />
It is interesting to note that glutamine synthetase<br />
and other three proteins involved in the regulation<br />
of glutamine metabolism are all retained by<br />
the same amino-aIkyl agarose derivatives [99~ (see<br />
table II). Although, as Shaltiel and coworkers<br />
have signaled, this could be fortuitous, it might<br />
reflect a mutual hiospecific affinity among these<br />
proteins since they must interact with each other<br />
in order to effect their regulatory functions in the<br />
highly integrated glutamine synthetase cascade<br />
system.<br />
The models proposed by Shaltiel [11] and<br />
Jennissen [78] explain in a different manner the<br />
mechanism by which the <strong>interaction</strong> between the<br />
protein and the adsorbent occurs. For neutral<br />
supports, Jennissen [86~ has presented evidence<br />
in favor of the idea that the adsorption of proteins<br />
to aIkyl-agarose derivatives takes place at a<br />
critical alkyl group density and is a function of<br />
its hydrophobicity. In other ~vords, this hypothesis<br />
considers that the protein needs to present<br />
multiple attachment points in order to be adsorbed<br />
by a determinate member of the series of<br />
alkyl-agarose derivatives. This is in agreement<br />
with an earlier observation made by Hjert6n el<br />
al. [521. Shaltiel Ill on the other hand, suggests<br />
that the adsorption of the protein is due to the<br />
inter.action of an allkyl residue of specific length<br />
(¢ yard-stick ~) with a hydrophobic pocket of the<br />
protein. Evidently, and as compared to Jennissen's<br />
model, the tatter implies a more specific mechanism.<br />
Ho~vever, it has been demonstrated [583 that<br />
at high chain length, when the <strong>interaction</strong>s are<br />
stronger, the binding is also less specific.<br />
The [841<br />
through the multivalent binding, may explain the<br />
increased free energy value of adsorption of a<br />
determinate member of the alkyl-agarose series.<br />
That is, a given protein may be adsorbed by a<br />
certain member of agarose-substituted series only<br />
if the number of ¢ contacts ~> is big enough to<br />
effect its retention, and this can be done by variation<br />
of the degree of substitution. Comparatively,<br />
amino-alkyl agaroses present a > effect to,wards the adsorption of phosphorylase<br />
b, possibly due to a different mechanism of<br />
<strong>interaction</strong> 'which does not necessarily exclude<br />
the multivalent attachment E84].<br />
BIOCHIMIE, 1978, 60, n ° 1.<br />
It has been observed, in the case of some hydrophobic<br />
aIkyl- and alkylamino supports, that certain<br />
> binding sites are occupied first,<br />
and that others of decreasing affinity become<br />
occupied when more protein is fed to the column.<br />
This is shown by the fact that only part of the<br />
protein can be eluted by increasing the salt concentration,<br />
whereas the remained is dislodged by<br />
the addition to the eluant of a polarity reducing<br />
{a)<br />
(c)<br />
(b)<br />
(d)<br />
Fro. 2. -- Models of adsorption o[ proteins on hydrophobic<br />
matrices : a} The model suggested by Shaltiel<br />
[11] ; b) and c) Represents the two possibilities of multi-attachment<br />
adsorption : on the matrix surface (b)<br />
and on a cavity in the matrix (c) ; d) The irregularity<br />
of the surface (attributed to the nature of the agarose<br />
structure) is the probable cause for 'which binding<br />
sites are not identical, and consequently the forces<br />
involved in the binding are different.<br />
agent such as ethylene-glycol. It should be emphasized<br />
that this problem is mostly related to those<br />
hydrophobic supports which possess both hydrophobic<br />
and ionic groups, resulting from the coupling<br />
[70, 471 procedure or the nature of the<br />
ligand [96], like in the case of amino-alkyl derivatives.<br />
This apparent inhomogeneity of both<br />
matrix and protein can lead to possible wrong<br />
interpretations about the purity and characteristics<br />
of many proteins [98, 105]. For instance,<br />
the recovery of rechromatographed materials is<br />
improved up to 95 per cent, as compared to that<br />
of 70 per cent obtained when the crude extract<br />
is applied into the column [98]. In other cases,<br />
desorption of purified material requires the variation<br />
of the eluant, as pointed above. It seems
6 J.-L. Ochoa.<br />
TABLE II.<br />
<strong>Hydrophobic</strong> matrices.<br />
Type Coupling procedure Bepresentation References<br />
I. Agarose derivatives<br />
1. Un-substituted<br />
2. Amino-alkyl-<br />
3, Alkyl-<br />
4. Amino acid<br />
5. Other derivatives<br />
Fatty acids<br />
AcetyI-N-amino-alkyl<br />
Aniline<br />
Benzyl-ether<br />
Phcnyl-N-butyl-amine-<br />
NAD +-<br />
Hydroxy- alky 1<br />
Diverse aromatic-alkyl<br />
derivatives<br />
BrCN<br />
BrCN<br />
glycidyl ether<br />
BrCN<br />
BrCN (azide)<br />
BrCN (cvrbodiimide)<br />
(acetylation of aminoalkyl-(A))<br />
BrCN and glycidyl ether<br />
glycidyl ether<br />
(A)<br />
(A)-CH-N H-(CH~)n-N H,_,<br />
I<br />
NH.~<br />
+<br />
(A)-CH-NH-( CH.~)-CH:~<br />
I<br />
NH~<br />
+<br />
(A)-O-CH~-CH-CH~-O-<br />
[ (CH'2)n-CH3<br />
OH<br />
(A)-glycine<br />
(A)-valine<br />
(A)-leucine<br />
(A)-tyrosine<br />
(A)-phenylalanine<br />
(A)-tryptophane<br />
(A)-(CHs)n-OH<br />
41, 47, li3]<br />
[9-11, 94, li3, Ill,<br />
li4, 58]<br />
[II,94, I13, li4]<br />
[52, li2l<br />
[97, I15]<br />
[54, 68, 97, li6, 77]<br />
[77, 97]<br />
[97]<br />
[35, 40, 42]<br />
[I051<br />
llo6]<br />
179, 58]<br />
[57, 94, li7]<br />
[40]<br />
[93, 90]<br />
[105l<br />
[57, 58]<br />
[58, li2]<br />
[I12, 55]<br />
II. Dextran derivatives<br />
(Sephadex)<br />
(s)<br />
1. Acetyl-Sephadex<br />
2. Methyl ether-(S)<br />
3. Hydroxy-propyl- ether-<br />
(S)<br />
4. Hydroxy-alkyL(S)<br />
5. LH-20-(S)<br />
acetylation<br />
acetylation<br />
from commercial sources<br />
CH3-CO-(S)<br />
CH3-O-(S)<br />
HO-CHs-CHs-CH.2-O-(S)<br />
HO-(CH~)n-O-(S)<br />
H3ol<br />
[91]<br />
[9t]<br />
[91]<br />
[9i]<br />
III. Cellulose derivatives<br />
(C)<br />
1. Diethyl amino ethyl<br />
cellulose<br />
2. Carboxy methyl-(C)<br />
3. Benzoylated-DEAE-(C)<br />
4. Esters of alkyl and aryl-<br />
(C) (paper, cotton)<br />
from commercial sources<br />
from commercial sources<br />
benzoyl chloride<br />
phenoxy acetylation<br />
DEAE-(C)<br />
CM-(C)<br />
BD-(C)<br />
[47, 71]<br />
[47, 71]<br />
[I03]<br />
[101]<br />
IV. Glass derivatives<br />
(G)<br />
1. Alkyl-silated-(G)<br />
2. Propyl_lipoamide-(G)<br />
BIOCHIMIE, 1978, 60, n ° 1.<br />
amino-silanization and<br />
amide bond formation<br />
with the corresponding<br />
alkyl chloride<br />
[ii8]<br />
[i07]
<strong>Hydrophobic</strong> (inleraction) <strong>chromatography</strong>.<br />
TABLE II.<br />
Type Coupling procedure Representation Relerences<br />
V. Others supports<br />
1. Polyamino methylstyrene<br />
(polystyrene,<br />
polyamide, Dowex<br />
I-X8)<br />
2. Alkyl-amine of polyacrylic<br />
resins (butyl,<br />
capryl, lauryl, palmityl,<br />
steatyl, oleyl, linoleyl)<br />
from commercial sources<br />
17t1<br />
chlorination for amide [761<br />
bond formation<br />
that this discrepancy depends on the hydrophobicity<br />
of the protein and the adsorbent, and moreover,<br />
that the binding sites are not identical in<br />
a given carrier (which is true for most of the<br />
adsorbents employed in <strong>chromatography</strong> in general).<br />
The multiple point attachment binding is most<br />
likely to occur in a cavity of the adsorbent than<br />
on its surface, particularly when the protein molecule<br />
fits into the cavity (fig. 2). Conversely, at<br />
points on the matrix ~vhere the bound ligands are<br />
distributed over a protruding area, binding would<br />
he less strong. Since the surface of the adsorbent<br />
may be assumed to be irregular, many different<br />
situations in addition to these two hypothetical<br />
cases ~vould be obtained. Additionally, small<br />
amounts of protein bind more homogeneously on<br />
a particular column than a large amount. This<br />
could mean that by reducing the amount of<br />
applied protein, the stronger binding sites might<br />
predominate in the binding.<br />
If multiple point attachment were one of the<br />
reasons for strong non-specific adsorption, one<br />
~vay to reduce it ~vould be to louver the degree of<br />
substitution to the point where the distance<br />
between the substitution groups is larger than the<br />
diameter of the protein molecule. This ~vould not<br />
affect the specific ¢ one-to-one >> <strong>interaction</strong> as<br />
that between an enzyme active site and an immobilized<br />
subslrate analogue.<br />
VI. THE FACTORS INVOLVED IN HYDROPHOBIC<br />
(INTERACTION) CHROMATOGRAPHY.<br />
A. The matrix hydrophobicity.<br />
1. Matrices and supports.<br />
Proteins differ in their hydrophobicity as a<br />
function of their primary structure, that is, in the<br />
BIOCHIMIE, 1978, 60, n ° 1.<br />
sequence and amino acid composition. Hence, it<br />
is not surprising that the first type of hydrophobic<br />
supports ever employed were derivated from<br />
coupling various non-polar amino acids to an<br />
inert support or matrix like agarose [54]. Other<br />
supports have also been employed: cellulose,<br />
glass, dextran, etc. (see table II), but the ideal<br />
matrix 'without secondary non-biospecific adsorption<br />
effects has not been found.<br />
All the different types of supports and matrices<br />
utilized at the present possess undesirable interferences<br />
attributed to their chemical composition.<br />
Additional effects arc obtained in such away that<br />
is difficult to obtain a pure hydrophobic <strong>interaction</strong><br />
<strong>chromatography</strong> after the introduction of the<br />
spacer-arm or hydrophobic ligand.<br />
In table II, the various types of matrices<br />
employed in HIC, as well as the different types<br />
of ligands coupled, have been summarized. References<br />
are given to illustrate specific applications.<br />
As has been demonstrated, un-substituted agarose<br />
is sufficiently non-polar presumably due to<br />
the 3-6 methylene diether bridges present in every<br />
second galactose residue of the polysaccharide<br />
chain [67] with respect to lhe retention of halophilie<br />
proteins [47] and nucleic acids [41, 119], at<br />
high salt concentrations (2.5 M ammonium sulfate).<br />
Though the halophilic proteins are highly<br />
negatively charged [48], as a result of an excess<br />
of acidic groups [49], it is believed that the high<br />
salt concentration eliminates the possible ionic<br />
<strong>interaction</strong>s.<br />
When DEAE- and CM-derivatives, either of<br />
cellulose or agarose, have been used, the elution<br />
pattern shifts to higher or louver salt concentrations<br />
than those required 'when tile neutral derivative<br />
is used as a support. This is explained by
8 J.-L. Ochoa.<br />
electrostatic attraction-repulsion forces between<br />
the negatively charged haloproteins and the nature<br />
of the charge on the matrices.<br />
With few exceptions, agarose is the type of<br />
matrix most commonly used (table II), and the<br />
kind of ligands or spacer-arm introduced are in<br />
a wide range of hydrophobicities and structures.<br />
Consequently, the hydrophobicity of the nmtrix<br />
depends on the type of the ligand E78], and on the<br />
degree of substitution [5,6, 86]. In this respect, the<br />
properties of the ligand should be discussed first.<br />
2. The role of the ligand in the hydrophobicity<br />
of the matrix.<br />
As pointed out above, HIC was born (in many<br />
cases) as a consequence of non-biospecific adsorption<br />
found in affinity <strong>chromatography</strong> systems<br />
and attributed to the type of spacer-arm used to<br />
bridge the ligand to the matrix. Nevertheless, evidences<br />
exist that prove that HIC was conceived<br />
by Hjerl6n and his group in a different way,<br />
while studying the solubilization of membrane<br />
proteins. They attempted their separation using<br />
a hydrophobic support to 'which a detergent<br />
(SDS) was coupled. In this case, however, the<br />
adsorption was so strong that the proteins could<br />
not be desorbed by any non-denaturing system<br />
(unpublished results, personal communication).<br />
When Cuatrecasas [87] sho~,ed that ~-galactosidase<br />
could only be fractionated if the ligand was<br />
sufficiently separated from the matrix, and thus<br />
avoiding sterical hindrance, the idea of separating<br />
ligands from the matrix backbone by use of<br />
spacer-arms rapidly spread. Very soon, it became<br />
clear, that many proteins were adsorbed nonspecifically<br />
because the presence of the arm introduced<br />
non-biospecific adsorption centers. For<br />
instance, a spacer-arm carrying charged groups<br />
gives origin to electrostatic <strong>interaction</strong>s [106]. The<br />
substitution of the charged arms by other neutral<br />
chemical analogs was thought to be an excellent<br />
alternative but then, other type of non-specific<br />
<strong>interaction</strong>s, hydrophobic in nature, turned out<br />
to be important [7].<br />
Yon [9], Er-el et al. [10] found that protein<br />
could bind substituted agarose matrices without<br />
any specific ligand. The adsorption ~ras attributed<br />
to the presence of the diamino-alkyl group<br />
employed as a spacer. This observations were<br />
further supported by O'Carra and coworkers [57,<br />
88], Who found that the presence of the ligand<br />
did not al'ways account for the ratardation effect<br />
and again the spacer-arm was considered as<br />
responsible. Yon [9] suggested that it was possible<br />
to take advantage of such non-biospecific adsorp-<br />
BIOCHIMIE, 1978, 60, n ° 1.<br />
tions and showed that certain proteins could be<br />
selectively adsorbed on decyl-agarose derivatives<br />
differing in the ionic character of the spacer-arm.<br />
Later, Shaltiel and his group [10, 11] reported<br />
purification of several enzymes on a series of<br />
alkyl and amino-alkyl agarose derivatives. Almost<br />
simultaneously, Hjert6n and his group [521 reported,<br />
for the first time, the preparation of different<br />
substituted agaroses 'with uncharged groups<br />
that could illustrate an exclusive hydrophobic<br />
mechanism of adsorption.<br />
The effect of the hydrophobicity of the spacerarm<br />
in the purification of various proteins [8-11]<br />
has been possible thanks to the development of<br />
the (< mock affinity systems >>. Steers [87 demonstrated<br />
that the adsorption of ~-galactosidase<br />
was strongly related to the length of the spacerarm.<br />
The series of aIkyl and amino-alkyl derivatives<br />
of agarose prepared by Shaltiel have also<br />
been successfully applied in many cases [11].<br />
The difference of a single C-atom in agarose<br />
bound N-aIkyl groups may have a large effect in<br />
the hydrophobic binding of a particular protein<br />
[75]. Therefore intermediary hydrophobic compounds<br />
between two consecutive members of a<br />
homologous series of ligauds are needed. Such<br />
intermediate hydrophobicities can be obtained,<br />
for instance, through the introduction of a<br />
charged or other hydrophilic group, e.g., hydroxyl<br />
group. The introduction of double bonds or<br />
of branching of the chain, reduces also the hydrophobicity<br />
as compared to the corresponding saturated<br />
straight chains [13]. Weiss and Bucher<br />
['76] have prepared some alkyl agarose derivatives<br />
of increasing insaturation for this purpose. In<br />
addition aromatic derivatives may possess intermediate<br />
hydrophobicities between t~wo consecutive<br />
members of the alkyl-agarose series. Benzene,<br />
for example, is equivalent to that of 3-4 straight<br />
chain hydrocarbon [19].<br />
Although Tanford [1O] has showed that the<br />
hydrophobicity of a linear aliphatic carbon<br />
increases linearly with increasing number of CH 2-<br />
groups, Shanbhag [50] considers that the effective<br />
hydrophobicity depends also on the flexibility<br />
of the hydrocarbon chain and consequently<br />
on the degree of <strong>interaction</strong> within such chains,<br />
specially for long chains. Hofstee [75], on the<br />
other hand, assures that neither the difference in<br />
molecular shape of the N-alkyl ligands and the<br />
side chain of aromatic compounds like phenylalanine<br />
or tryptophan, nor the difference in net<br />
charge of the adsorbent is a determining factor<br />
for protein binding. It seems then, that the me-
<strong>Hydrophobic</strong> (<strong>interaction</strong>) <strong>chromatography</strong>.<br />
chanism of the adsorption of proteins to hydrophobic<br />
matrices follows some very complex rules.<br />
This problem can be exemplified by the case of<br />
the adsorption of erythrocytes on a series of<br />
aIkyl-agarose derivatives [110] in ~which case a<br />
decrease in adsorption bet'ween C6-C s is noticed<br />
to occur without a reasonable explanation.<br />
The combination of the principles of ionicexchange<br />
<strong>chromatography</strong> and HIC has been successfully<br />
applied in some cases [9, 41]. The use of<br />
ligands containing both ionic and hydrophobic<br />
characters have been recommended in order to<br />
avoid the denaturing effect detected in the use of<br />
alkyl-agaroses [108]. It was also argued that the<br />
elution procedure might be milder than in the<br />
case of pure hydrophobic spacer-arms or ligands<br />
[9] and furthermore, that the use of charged<br />
arms was capable of finer discrimination between<br />
lipophilic proteins. The protein, in this case,<br />
interacts hydrophobically, involving the alkyl<br />
chains, and electrostatically, involving the terminal<br />
ionic groups. With this type of adsorbents,<br />
if the <strong>chromatography</strong> is carried out at a pH<br />
equivalent to the isoelectric point (IP) of the protein,<br />
the adsorption will be due to hydrophobic<br />
binding alone. By changing the pH to introduce<br />
in the protein a net charge of the same sign as the<br />
charge of the adsorbent, the repulsion effect will<br />
decrease the adsorptive force due to hydrophobie<br />
bonding, making possible the desorption. Obviously,<br />
a dra~vback in using this type of adsorbent<br />
arises from the problem that frequently the IP of<br />
the biomolecule of interest is unknown, and that<br />
many proteins precipitate at their IP. Moreover,<br />
the simultaneous presence of extraneous ionic<br />
and hydrophobic groups in affinity adsorbents,<br />
has been found to cause a substantial non-specific<br />
protein binding thus resulting in a reduced biospecificity<br />
of these materials [106].<br />
3. The influence of the degree of substitution<br />
on the hydrophobicity of the matrix.<br />
The degree of substitution on Sepharose 4B<br />
with aIkyl-amines of different hydrophobicities<br />
is a critical parameter in the adsorption of proteins.<br />
About 1012 alkyl residues per Sepharose<br />
sphere appears to be a critical degree of substitution<br />
for the adsorption of enzymes like phosphorylase<br />
kinase, phosphorylase phosphatase,<br />
3'5'-cAMP dependent protein kinase, glycogen<br />
synthetase, and phosphorylase b which are successively<br />
adsorbed when the hydrophobicity of<br />
the Sepharose is increased. In addition, the<br />
degree of substitution determines the capacity of<br />
the gel [78].<br />
The critical hydrophobicity needed to adsorb<br />
proteins can be obtained by either increasing the<br />
degree of substitution or by elongating the<br />
employed alkyl-amine chain at a constant degree<br />
of substitution. Consequently, as the hydrophobicity<br />
of the gel is increased, higher binding affinities<br />
result and the desorption requires more<br />
and more severe conditions. In the case of neutral<br />
alkyl-agaroses [3~1], elution of proteins from the<br />
hydrophobic matrix can be described in terms of<br />
salting- in phenomena, since desorption is dependent<br />
on the type of salt employed and not on the<br />
ionic strength alone.<br />
In all cases, a minimal chain length seems to be<br />
required in order to obtain the adsorption of a<br />
given protein. This fact has been interpreted as<br />
fitting a hydrophobic group into a hydrophobic<br />
pocket of the protein. By variation of the concentration<br />
of cyanogen bromide in the activation<br />
mixture, the amount of hydrophobic residues<br />
may be varied. Thus, the corresponding hydrophobicity<br />
may be increased such that the amount<br />
of adsorbed material increases exponentially<br />
when the degree of substitution of the gel is<br />
enhanced. Control experiments with methyl-agarose<br />
show that similarly increased degrees of<br />
substitution, and thus similar numbers of positive<br />
charges introduced by the coupling procedure<br />
[117], do not affect the adsorption of the assayed<br />
protein. Therefore, the adsorption of larger<br />
amounts of material, determined by increasing<br />
the degree of substitution, is not a function of<br />
the additional number of charges [78]. One may<br />
conclude that, if in a series of gels of different<br />
hydrophobicities a crude extract containing hydrophobically<br />
differing proteins is applied, the<br />
one vcith the highest hydrophobicity will be adsorbed<br />
by the gel of the lowest degree of substitution.<br />
Then as the number of alkyl residues increases<br />
on the matrix, proteins of lower hydrophobicities<br />
are adsorbed.<br />
A direct method of determining the degree of<br />
substitution for charged hydrophobie groups has<br />
been proposed by Hofstee [89]. The method describes<br />
the use of Ponceau S, a dye which carries<br />
hydrophobic groups in conjunction with an overall<br />
negative charge bound in an irreversible<br />
fashion to the ligand. Under a given set of conditions,<br />
and after application of a saturating amount<br />
of dye, a certain amount will remain bound even<br />
after extensive washing. Eighty-five percent of the<br />
Ponceau S binding capacity is lost after a period<br />
of almost 5 months indicating that the degree of<br />
substitution decreases gradually upon storage. In<br />
some cases, 40 per cent was lost in only 40 days.<br />
BIOCHIMIE, 1978, 60, n ° 1.
1 0 J.-L. Ochoa.<br />
The data also suggest that the adsorbents with the<br />
highest degree of substitution are the least stable<br />
[89].<br />
An estimation of the relative degree of substitution<br />
can be obtained from the acid-base treatment<br />
of the adsorbent [89], by a nucleophilic attack<br />
of N-substituted isourea (derivated from the<br />
coupling procedure 'with BrCN) with an active<br />
chromogenic substance [70] ; or more indirectly,<br />
by measuring the capacity of the adsorbent with<br />
a coloured protein like cytochrome C [90] or phycoerythrin<br />
[56]. A inuch more sophisticated but<br />
accurate method utilizes NMR spectra [56].<br />
Finally, it is important to mention that a consequence<br />
of the degree of substitution is the shrinkage<br />
of the gel owing to a decrease in hydrophilicity<br />
due to the hydrophobic character of the<br />
ligand. Depending on the special structure of the<br />
agarose gel, the shrinkage seems to be much less<br />
important than with other gels like Sephadex or<br />
polyacrylamide [90].<br />
B. The influence of salt.<br />
The influence of salt on the adsorption of proteins<br />
to hydrophobic supports is probably due<br />
to a number of factors acting on the protein as<br />
well as on the matrix. It has been demonstrated<br />
that neutral salts induce conformational and<br />
structural changes in biomolecules [34]. These<br />
studies have been carried out by circular dichroism<br />
spectra of the protein at cons,taut ionic<br />
strength. It has been concluded that salting-out<br />
ions cause conformational but not structural changes<br />
whereas salting-in ions cause sometimes<br />
severe structural changes which may be one reason<br />
of their denaturing effect [34].<br />
The > properties of saltingout<br />
ions enhance intramolecular, as well as intermolecular,<br />
hydrophobic bonding as reflected by a<br />
stabilization of the hydrophobic core of the biomolecule<br />
[35, 43']. The effect of specific ions on<br />
macromolecules was first noticed by Hofmeister<br />
[36]. He found that salts differ greatly in their<br />
ability to salt-out proteins at a given salt concentration.<br />
At high concentrations of salting-out ions,<br />
the solubility of the protein is adversely affected<br />
by decreasing the availability of ~vater molecules<br />
in the bulk and increasing the surface tension of<br />
water, resulting in an enhancement of the hydrophobic<br />
<strong>interaction</strong>s [31, 371. Accordingly, the influence<br />
of neutral salts on the adsorption phenomena<br />
determines to a large extent the degree of<br />
adsorption and correlates closely with the Hofmeister<br />
series [63, 58]. Salting-in ions, on the other<br />
BIOCHIMIE, 1978, 60, n ° 1.<br />
hand, are > ions, and thus<br />
do not favour hydrophobic <strong>interaction</strong>s. This<br />
effect can be regarded as an inevitable consequence<br />
of the new order imposed by the ion<br />
orienting water molecules in such a way that<br />
water cannot undergo further positive entropy<br />
change, as it is required for the formation of hydrophobic<br />
bonds [18]. These ions have also been<br />
termed ¢ chaotropic >> [44] because they provoke<br />
unfolding, extension and dissociation of the macromolecules.<br />
In this respect, they might be used<br />
in the elution of strongly adsorbed materials on<br />
hydrophobic supports.<br />
Already in 1948, Tiselius [38] noticed that proteins<br />
and other substances (e.g., dyes) that could<br />
be precipitated by high concentrations of neutral<br />
salts could be adsorbed (at much lo'wer salt concentration)<br />
to common adsorbents, whereas in the<br />
absence of salts those adsorbents showed no affinity<br />
for the substance. In the years afler~vards,<br />
some attempts have been made to purify proteins<br />
on solid supports using gradients of ammonium<br />
sulfate [39, 40, 54]. In general, proteins which precipitate<br />
at low ammonium sulfate concentrations<br />
should likewise be retained on hydrophobic supports<br />
at low salt concentrations ; and similarly,<br />
proteins ~vhich precipitate at high concentrations<br />
of ammonium sulfate would require relatively<br />
higher concentrations of salt to obtain their retention<br />
[40].<br />
The purification of nucleic acids [41] has also<br />
been reported using unsubstituted agarose with<br />
high concentrations of ammonium sulfate. It<br />
should be emphasized that tbe adsorption occurs<br />
at concentrations below which the macromolecules<br />
precipitate out of solution. Since the adsorption<br />
is controlled by salt concentration, rather<br />
than by the hydrophobicity of the column, the<br />
names of > and hydrophobic (salting-out) <strong>chromatography</strong><br />
have been employed elsewhere [42, 54.<br />
67]. Hovccver, the hydrophobicity of the gel and<br />
the effect of the salt concentration may be combined<br />
efficiently to improve the adsorption phenomena<br />
and/or the purification [64J, especially of<br />
proteins which may be affected by the high salt<br />
concentration required for its adsorption on the<br />
given gel [46]. If the use of high salt concentrations<br />
is limited by the enzyme activity or stability<br />
for instance, a comparatively longer alkyl-chain<br />
must be employed in order to obtain sufficient<br />
capacity. Because a similar salting-out/salting-in<br />
effect has been noticed in the case of high concentrations<br />
of sugars, namely sugaring-out and<br />
sugaring-in effect [45], the possibility in using
<strong>Hydrophobic</strong> (<strong>interaction</strong>) <strong>chromatography</strong>. 11<br />
sugars instead of salts should be considered. However,<br />
this effect is probably not general.<br />
C. Effect of temperature and pH.<br />
From the equation N ° 1, it is clear that the temperature<br />
may influence, in a positive way, the<br />
adsorption of proteins on hydrophobic matrices<br />
~52, 551. However, temperature may also provoke<br />
other effects, such as increased solvation and decreased<br />
surface tension, ~vhich may withstand the<br />
ability of molecules like proteins to interact xvith<br />
hydrophobic supports [53J.<br />
The protein molecules retain their biological<br />
ac.fivity or capacity to function only within a<br />
limited range of temperature and pH. Their exposure<br />
to extremes of pH and temperature causes<br />
them to undergo denaturation ; the most visible<br />
effect is a decreased solubility of globular proteins.<br />
Since the covalent chemical bonds in the<br />
peptide backbone of the protein are not broken<br />
during denaturation, it has been concluded that<br />
it is due to the unfolding of the characteristical<br />
conformation of the native form of the protein<br />
molecule. The refolding of a denatured protein<br />
does not require the input of chemical work from<br />
outside. It proceeds spontaneously, provided that<br />
the conditions of temperature and pH are adjusted<br />
to be compatible with the stability of the native<br />
conformation of the protein. In this respect, the<br />
process is similar to the one in hydrophobic bond<br />
formation.<br />
The strength of hydrophobic bonds should increase<br />
with rising temperature up to above 60°C,<br />
at which point the additional stability arising<br />
from hydrogen bonding, electrostatic forces between<br />
charges or dipoles, van der Waals <strong>interaction</strong>s<br />
and disulfide bridges disappear, and turn<br />
against the favoured hydrophobic <strong>interaction</strong> [18,<br />
30, 50] of the protein.<br />
A pH effect on hydrophobic bonding is observed<br />
in the case, for example, of bovine serum albumin<br />
binding alkanes at pH 4. It 'was observed<br />
that at low pH the site of hydrophobic bonding is<br />
disrupted [61J, and that the degree of aggregation<br />
of the globulin does not affect the degree of binding.<br />
The conclusion ~vas that the sites associated<br />
with aggregation are relatively non-hydrophobic<br />
and that any conformational changes, resulting<br />
from the polymerization do not exert any effect<br />
at the hydrophobic binding site of alkanes [62].<br />
Decreasing pH belo'w pK value of the amino<br />
group of adenylic and cytidylic acid residues of<br />
tRNA changes the negative charge of the molecule,<br />
BIOCHIMIE, 1978, 60, n ° 1.<br />
altering its tertiary structure [65] and provoking<br />
its desorption from the charged alkyl amine agarose<br />
columns. On the other hand, binding at<br />
pH 7.5 does not occur probably due to other conformational<br />
changes in the molecule, which could<br />
mask key sites involved in the binding E41_~.<br />
Lysozyme, a basic protein, becomes more and<br />
more retarded on Clo (aIkyl Sepharose) as the pH<br />
of the eluant is lowered, and binds to this column<br />
at pH 1.5 [661. Cytochrome C is also highly pHdependent<br />
in its adsorption to neutral ethers of<br />
agarose derivatives [67J.<br />
No influence of pH has been observed on the<br />
adsorption of polysaccharides to substituted agaroses<br />
[68~. In this case, the retention is mostly<br />
determined by the hydrophobicity of the column<br />
and by the size of the polymer.<br />
Naturally, pH plays an important role in the<br />
adsorption of biomolecules on hydrophobic matrices<br />
carrying charged groups. With this type of<br />
adsorbents, pH may modify the charge either<br />
of the biomolecule or of the adsorbent. By<br />
changing the pH, a net charge may be introduced<br />
in protein, which can be of the same sign,<br />
or opposite of the charge on the adsorbent. In<br />
the first case, the repulsion effect ~vill affect negatively<br />
the adsorption phenomena. As was pointed<br />
out previously, a maximal hydrophobic adsorption<br />
will thus occur at the isoelectric point of the<br />
protein E9!.<br />
VII. METHODS OF DESORPTIOX on ELUTION<br />
OF ADSORBED MATERIALS<br />
ON HYDROPHOBICS MATRICES.<br />
The elution of adsorbed materials from hydrophobic<br />
gels can be obtained in a number of ~vays<br />
depending on the type of adsorbent employed,<br />
the conditions in 'which the adsorption occurred,<br />
and the properties of the biomolecule.<br />
When proteins have been adsorbed at high salt<br />
concentrations, a decreasing ionic strength of the<br />
eluant ~vill usually result in the removal of the<br />
material from neutral adsorbents [71, 791. If the<br />
support carries charges introduced either by the<br />
coupling procedure [70], or by the nature of the<br />
ligand, electrostatic <strong>interaction</strong>s will become<br />
important at low ionic strength, and the elution<br />
will not be possible [47, 1031. In such cases,<br />
changes in the pH [9, 65], buffer composition<br />
[47], temperature, or the addition of nonpolar<br />
organie substances may be quite useful<br />
procedures.
12 J.-L. Ochoa.<br />
The fact that in many cases elution is possible<br />
by raising salt concentration, demonstrates the<br />
presence of cooperative electrostatic <strong>interaction</strong>s<br />
in the overall adsorption phenomena in the case<br />
of charged-hydrophobic supports. Nevertheless, it<br />
has been shaw that in those situations, the mechanism<br />
of hydrophobic adsorption at high salt<br />
concentration prevents the electrostatic <strong>interaction</strong>s<br />
of being the main driving force involved<br />
in the process.<br />
The use of denaturing agents, such as urea,<br />
guanidine-HC1 etc, has been successfully applied<br />
in desorption procedures. Their mechanism of<br />
action appears to be involved in both direct <strong>interaction</strong><br />
'with the side-chains and peptide groups<br />
and disruption of hydrophobic <strong>interaction</strong>s between<br />
side chains [23, 69, 77, 109].<br />
Chaotropie ions, on the other hand, are known<br />
for their ability to impart lipophilic properties to<br />
water [72] and to alter the structure of biomolecules<br />
[44]. Their application for the elution of<br />
proteins from high members of alkyl-agarose derivatives<br />
has been reported to be successful [73] ;<br />
ho'wever, it is important to bear in mind their<br />
property of dissolving agarose gels and denaturating<br />
proteins. In this case, cross-linked agaroses,<br />
which are kno'~vn to support more severe conditions<br />
(pH, temperature, ionic strength etc) than<br />
the normal agarose gel, are to be employed.<br />
As has been mentionned before, by increasing<br />
the hydrophobicity of the gel tighter binding resuits,<br />
and desorption of proteins requires ever<br />
more drastic conditions [78]. The combination of<br />
salt gradients with > agents<br />
(e.g., ethylene glycol):, variation in pH or temperature,<br />
etc., may give an increased selectivity of<br />
the elution. As an illustration, it can be mentionned<br />
the case of serum albumin, where elution<br />
occurs at pH 3 with 50 per cent ethanol in the<br />
buffer [791. Similarly, the use of mixed gradients<br />
of buffer solution with high salt concentration,<br />
and buffer without salt but containing 50 per cent<br />
of ethylene glycol, have been successfully applied<br />
to desorb enzymes such as chymotrypsin and<br />
trypsin from hydrophobic matrices [90].<br />
A simple pure salt gradient may result in a<br />
strongly diluted peak in the elution because the<br />
cond,itions in 'which the equilibrium for the<br />
hydrophobic <strong>interaction</strong> is attained are slob,.<br />
Only lower flow rates could provide more favourable<br />
results. In addition, it should be emphasized<br />
that in some cases the elution with ethylene<br />
glycol, in the absence of salts, may be inefficient<br />
due to the presence of electroslatie <strong>interaction</strong>s<br />
BIOCHIMIE, 1978, 60, n ° 1.<br />
[98] when adsorbents involving mixed effects are<br />
employed.<br />
The use of detergents [127] is usually the last<br />
resource, when the elution by other milder conditions<br />
has not been possible. The problem in using<br />
detergents is their intrinsic denaturing effect and<br />
the difficulty of their removal from the columns<br />
during the regeneration step [127]. A wide range<br />
of different detergents has been used varying in<br />
their efficiency to eliminate strongly adsorbed<br />
materials E127]. In this respect, the more ionic<br />
ones are to be preferred for the reasons discussed<br />
above.<br />
Finally, flat curves are obtained by gradient<br />
elution [75], presumably due to a postulated<br />
irregularity of the matrix (see fig. 2) and the<br />
occurence of a wide range of binding sites of<br />
varied strengths [74]. For this reason, attempts<br />
should be made to fractionate through > [123] (as opposed to differential-elulion)<br />
on a series of adsorbents of<br />
increasing hydrophobicities. In this way, each<br />
protein tends to be adsorbed or bound to the<br />
column that provides the minimum degree of<br />
hydrophobicity required for binding, and complete<br />
elution of the materials can be accomplished<br />
with mild eluants. Another alternative consists<br />
of the use of a high member of the alkyl agarose<br />
series, which may retain a high percentage of the<br />
protein content of a particular mixture, and<br />
allows the elution of the molecule of interest<br />
under mild conditions nvhile most of the other<br />
protein remain adsorbed on the column [123].<br />
Obviously, this possibility can be utilized only<br />
when the biomolecule in question has a lo~,-<br />
hydrophobicity. One should note that in principle<br />
it should be possible to achieve purification by<br />
HIC, not only by a selective retention of a given<br />
protein, but also by exclusion of a desired protein<br />
when most of the other proteins in the mixture<br />
are adsorbed [114].<br />
VIII. APPLICATIONS, FUTURE AND IMPLICATIONS<br />
OF HIC.<br />
The relevance of HIC lies in the fact that it is<br />
perhaps the first technique which takes advantage<br />
of the hydrophobic properties of the biomolecules.<br />
The hydrophobic character of biomolecules<br />
should be a specific property, similar to the ionic<br />
characler, since this is a function of the primary<br />
structure in the case of proteins and nucleic<br />
acids. So, it is not unrealistic to consider that
<strong>Hydrophobic</strong> (<strong>interaction</strong>) <strong>chromatography</strong>. 13<br />
their selective separations are feasible. As an<br />
example, the purification of two interconvertible<br />
forms of glycogen-phosphorylase, which differ in<br />
a unique serine residue, has been successfully<br />
achieved through their ability to adsorb on two<br />
distinct members of the alkyl-agarose series [129].<br />
Unfortunately, the types and nature of the<br />
hydrophobic adsorbents available to date are probably<br />
not well systematized, and the lack of intermediate<br />
hydrophobic matrices limits the applicability<br />
of the method. To provide for a larger<br />
variety in the types of ligands, including the<br />
introduction of aromatic structures, it might be<br />
expedient to attach these ligands to (< arms )~ that<br />
by themselves are not hydTophobic, e.g., carbohydrates<br />
instead of hydrocarbons [96]. At first<br />
sight, hydrophobically coated supports may be<br />
used as concentrating systems as well [52]. In<br />
fact, the protein 'which is going to be purified<br />
does not require a pre-concentration step if the<br />
conditions under 'which the <strong>chromatography</strong> is<br />
performed allow its adsorption.<br />
The application of HIC to the purification of<br />
nucleic acids using matrices of varied hydrophobicities<br />
according to the type of ligand attached<br />
or to its degree of subslitution awaits for further<br />
studies which may greatly improve the application<br />
of this technique. This aspect is especially<br />
interesting when the molecules are not stable or<br />
soluble at either too high or too low salt concentrations.<br />
The separation of particles, like sub-cellular<br />
fractions or entire cells [110, 121] has been reported,<br />
and it seems to be a very promising application<br />
of HIC, since membranes can be considered<br />
as aggregates of molecules of varied hydrophobicities.<br />
A similar application in a quite different<br />
field of biochemistry is preparation of enzymatic<br />
reactors through the immobilization of enzymes<br />
on hydrophobic supports [32, 101, 122, 126]. The<br />
basic idea is the possibility to have a reactor<br />
which can be periodically recycled, when the<br />
adsorbed material loses its activity by elution<br />
and adsorption of freshly active substances<br />
(which may be of the same or of another biological<br />
activity, but similarly adsorbed on the<br />
hydrophobic support). Thus, the hydrophobic<br />
matrix may function as an universal support of<br />
easier handling than those systems in which the<br />
immobilization necessitates a covalent linkage<br />
between the biomolecule and the adsorbent, with<br />
their corresponding limitations in function and<br />
utility,<br />
It should be reminded that some hydrophobic<br />
ligands denature proteins through a > action [96]. This effect can be reduced<br />
by employing hydrophobic ligands of milder<br />
influence, or by introducing additional polar or<br />
ionic groups in the hydrocarbon chain [9]. Finally,<br />
the problem of >, which<br />
constitutes another important drawback in protein<br />
separation by <strong>chromatography</strong> on hydrophobic<br />
columns, must be considered. If inhomogeneity<br />
occurs in the adsorbent, that is if the interacting<br />
sites on a given member of the suhstituted-agarose<br />
series are different in their strength of <strong>interaction</strong><br />
with a particular protein (fig. 2), elution by<br />
decreasing salt concentration or increasing concentration<br />
of > agents, will<br />
never result in a narrow peak but rather in flat<br />
curves, which increase contamination of the sample<br />
by other proteins. Some suggestions to avoid<br />
this phenomenon have already been mentioned<br />
taking advantage of the hydrophobicity of the<br />
matrix.<br />
In conclusion, the correct application of hydrophobic<br />
<strong>chromatography</strong> implies the consideration<br />
of three main factors :<br />
-- the nature and hydrophobicity of the adsorbent<br />
and of the protein,<br />
-- the conditions of the adsorption,<br />
-- the conditions of the elution of the adsorbed<br />
material.<br />
It may be expected that as the mellmd will be<br />
more and more aplied wi'.h various biomolecules,<br />
in different conditions, our understanding will be<br />
enriched so about many biological events in<br />
which a hydrophobic <strong>interaction</strong> is involved.<br />
Acknowledgments.<br />
The author expresses his gratitude to Dr. Jean-Marc<br />
Egly for his encouragement in the preparation of this<br />
article. Special thanks are due to Professor Shmuel<br />
Shaltiel and Professor Stellan Hjertdn for their criticism<br />
and suggestions, and to Professor Barbarin<br />
Arreguin for his continuous interest on my ,work. My<br />
appreciation to Dr. Dana Fawlkes for the linguistic<br />
reoision and to Mrs. Inga Johansson and Mr. GSsta<br />
ForMing for typing the manuscrip and drawing the<br />
figures, respectioely.<br />
To the National University of Mexico and the National<br />
Council of Sciences and Technology of Mexico<br />
many thanks for their financial support.<br />
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