PHYTOCHEMlCAL ANALYSIS, VOL.

2, 191-198 (1991)

RE VIEW PAPER
The Applications of Hydrophobic Interaction
Chromatography to the Purification of Plant
Proteins

E. J. M. Pennings*, A. H. Meijer, L. H. Stevens and R. Verpoorte

Center for Bio-Pharmaceutical Sciences, Division of Pharmacognosy, Leiden University, P.O. BOX9502, 2300 RA
Leiden, The Netherlands

Protein separation by hydrophobic interaction chromatography (HIC) depends on the differences in surface
hydrophobicity of the constituent proteins and is hence complementary to chromatographic techniques based on
the charge or the size of the macromolecule. This review summarises some of the recent developments in HIC
supports and discusses the parameters that must be considered in designing an efficient chromatographicsystem.
The application of HIC to the purification of enzymes of secondary compound biosynthesis that cannot be
obtained in pure form by size-exclusion and ion-exchange techniques alone is exemplified by reference to recent
studies on tryptophan decarboxylase, strictosidine synthase and geraniol-10-hydroxylase.

Keywords: Hydrophobic interaction chromatography; salt-promoted adsorption chromatography; stationary

phases; plant enzymes; protein purification.

INTRODUCTION HISTORY

Hydrophobic interaction chromatography (HIC) is a
purification technique based on the hydrophobic inter-
action between a macromolecule, usually of biological
origin, and a stationary phase. It is a powerful tech-
nique for the purification of proteins and usually results
in a high recovery of the biological activity of the
protein. The stationary phase consists of a weakly
hydrophobic ligand attached to a matrix. Proteins are
retained on the column in the presence of high salt
concentrations in an aqueous buffer and are then selec-
tively eluted by a decreasing salt gradient. For this
reason, the technique is also called salt-promoted
adsorption chromatography.
Hydrophobic interactions occur between non-polar
patches on the protein surface and the hydrophobic
groups on the column matrix. Non-polar patches on the
protein are formed by hyrophobic amino acid residues
such as isoleucine, valine and phenylalanine. The
retention of proteins is greatly affected by the type and
length of the hydrocarbon chains attached to the
matrix. In addition, the number, size, shape and hydro-
phobicity of the non-polar patches on the protein’s
surface determine the relative affinity of a protein for a
specific stationary phase. In other words, elution con-
ditions for a given protein are determined by the
hydrophobic character both of the protein and of the
stationary phase.
Author to whom correspondence should be addressed.
O958-O344/9 1/050191-08 $05.00
01991 by John Wiley & Sons, Ltd
In the early seventies, several reports showed that
proteins could bind to alkylamine derivatized supports.
Weiss and Biicher (1970) purified mitochondria1 mem-
brane proteins on a polyacrylic acid resin to which they
had attached aliphatic amines. Yon (1972) described
the preparation of aminodecylagarose and also a nega-
tively charged support produced by succinylating the
free amino groups. These derivatized agaroses were
used to purify aspartate transcarbamoylase and the
author also discussed alternative modes of operation
for supports containing both hydrophobic and ionic
groups. Hofstee (1973) studied the binding of various
proteins to agarose substituted with n-alkylamines of
varying carbon chain length. It was concluded that
binding to these stationary phases depended on the
cooperation of hydrophobic and electrostatic forces. At
the same time, attempts to purify enzymes involved in
glycogen metabolism led to the preparation of a series
of glycogen-coated aminoalkyl agaroses (Er-El et a l . ,
1972; Shaltiel and Er-El, 1973). Two glycogen-coated
agaroses, differing only in their hydrocarbon chain
length (Sepharose-C,-glycogen and Sepharose-C,-
glycogen), behaved differently towards glycogen phos-
phorylase b. The stationary phase with the C8-alkyl
bridge completely retained the enzyme, whereas the
corresponding phase with the C,-alkyl bridge did not
even retard it. Surprisingly, the corresponding station-
ary phases without glycogen behaved similarly and it
was concluded that the w-aminoalkyl chain was respon-
sible for binding the enzyme and that the length of the
Receiued 26 February 1991
Accepted 21 May 1991
192 E. J . M. PENNINGS E T A L

Table 1. Characteristics of some commercially available columns for high performance HIC: agarose-based and polymer-based
columns
Particle size Pore size
Column Support matrix (yml (A) Functional group Manufacture
HEMA-HIC 1000 hydrophilic polymer 1oa 300-400 underivatized Alltech
MacrospherelR HIC hydrophilic polymer 1oa 300-400 underjvatized Alltech
HRLC MP7 HIC hydrophilic polymer 7 900 methyl Bio-Rad
Polypore Phenyl HIC hydrophilic polymer 10 < 100 phenyl Brownlee
Hydrophase HP-Butyl hydrophilic polymer 10” 500 butyl Interaction
Alkyl Superose cross-linked agarose 10” 2 x l o 6 (MWIb neopentyl Pharmacia-LKB
Phenyl Superose cross-linked agarose 1oa 2 x lo6 (MWIb phenyl Pharmacia-LKB
Shodex PH-814 hydrophilic polymer 10 1000 phenyl Showa Denko
Ether-5PW hydrophilic polymer 10” 1000 oligoethylene glycol TosoHaas
Phenyl-5PW hydrophilic polymer 10” 1000 phenyl TosoHaas
Bakerbond WP HI-Propyl polyamide-coated spherical silica 5, 15a 300 ProPYl J. T. Baker
HIC Spherogel CAA spherical silica 5 300 polyether Beckman
Aquapore HIC spherical silica 7 300 ProPYl Brownlee
Euramid HIC polyamide-coated spherical silica 7 a 300, 1000,4000 proprionyl Knauer
PolyMethyl A, PolyEthyl A,
PolyPropyl A PAA-coated spherical silica“ 5, 12“ 300, 1000, 4000 short alkyl chain PolyLC
Hydropore-HIC spherical silica 12 300 polyethylene glycol Rainin
Butyl = 5300, Butyl = Si500 irregular silica 5, 10” 300, 500 butyl Serva
Shim-Pack HPC-C2, HPC-C3 spherical silica 5 300 ethyl, n-propyl Shimadzu
Supelcosil LC-HINT spherical silica 5 100 diol Supelco
SynChropak Methyl,
Hydroxypropyl, Propyl, Pentyl polyamide-coated spherical silica 6.5a 300 short alkyl chain SynChrom
a Manufacturer has additional materials available with larger particle size for preparative HIC.
The base material for these columns is Superose 12. Figures represent exclusion limits for globular proteins (MW: molecular
weight). The useful MW range is from 1000 to 300000.
PAA= poly(alky1 aspartamide).

alkyl chain was a determinant of the strength of the
binding. The phenomenon was further studied follow-
ing the synthesis of a homologous series of alkyl-
agaroses. Whereas Sepharose-C, and Sepharose-C, did
not bind phosphorylase b, Sepharose-C, slightly
retarded the enzyme and higher alkylagaroses retained
the enzyme. Interestingly, different proteins displayed
different adsorption characteristics and it was con-
cluded that purification of proteins was possible on the
basis of a new criterion, i.e. the hydrophobic nature of
the protein.
Since this initial work, HIC has developed into an
important separation mode with hydrophobic ligands
attached to cross-linked agaroses (Porath et al., 1973;
HjertCn et al., 1974; Jennissen and Heilmeyer, 1975;
Hofstee, 1979; Halperin et al., 1981; Maisano et al.,
198.5). Initially, this yielded soft gels with relatively
large bead size and low efficiency. More recently, Kato
et al. (1983) synthesized rigid HIC supports based on
silica and high performance HIC became available as a
purification tool. Since then, a number of packing
materials for high performance HIC have been deve-
loped (Fausnaugh et al., 1984; Gooding et al., 1984;
Miller et al., 198.5; Chang et al., 1985; Alpert, 1986;
HjertCn et al., 1986) bonded with weakly hydrophobic
ligands. A wide variety of high performance columns is
now commercially available (Table 1). Some of them
are silica-based sorbents, some are prepared from a
synthetic polymer and others are made from highly
cross-linked agarose. Uniform materials with small
particle size and large pore size are provided by all
these materials. The pH range for working with silica-
based matrices is limited to pH 2-8. This is the major
drawback in the use of silica-based packings. The
polymer-based packings and the highly cross-linked
agarose packings have a high stability over a wide pH
range. They may be used over the pH range 2-12 for
continuous operation and over the pH range 1-14 for
cleaning procedures. For this reason these latter sup-
ports have gained high popularity, particularly in the
pharmaceutical industry.
SEPARATION PARAMETERS
Hydrophobicity of the ligand
In true HIC, retention results from the partitioning of
the protein from the polar mobile phase into the hydro-
phobic stationary phase. The stationary phase should
be free of any charged groups as the latter can give
unwanted electrostatic interactions especially at low
salt concentrations. In fact, the supports used in the
early seventies did not meet this condition and proba-
bly all showed electrostatic effects superimposed on
hydrophobic interactions. In some cases such a mixed-
type stationary phase can afford an efficient ligand for a
protein. An example is the affinity of P-450 proteins for
the w-aminooctyl-type stationary phase (Janig and
Pfeil, 1984). In this case, affinity chromatography is a
more appropriate term to describe the mechanism of
the purification. Usually, however, the presence of
electrostatic effects render it difficult to elute the pro-
teins quantitatively and uncharged ligands are there-
fore preferred. Nowadays, the strategy is first to pre-
pare highly hydrophilic, yet uncharged, supports and
then to couple weakly hydrophobic ligands to the
matrix.
The hydrophobic character of the sorbent can be
controlled by the type of the ligand during the synthesis
 HYDROPHOBIC INTERACTION CHROMATOGRAPHY
of the stationary phase (Gooding et al., 1984; Miller et
al., 1985; Chang et al., 1985; Alpert,. 1986; Hjerten et
at., 1986). With the octyl chain as the ligand a station-
ary phase is obtained with a relatively high hydrophobic
character for many proteins. The hydrophobicity of the
phenyl ligand is less, between that of pentyl and tert
butyl, but proteins usually show more retention on the
phenyl packings than on the pentyl packings probably
because of stacking with aromatic amino acid residues.
Next in decreasing order of hydrophobicity come the
short alkyl chains: pentyl > butyl > propyl> methyl >
hydroxypropyl. The polyether bonded phases and the
polyethyleneglycol bonded phases have a low hydro-
phobic character, comparable to that of the shorter
alkyl chains.
Hydrophobicity of the protein
The strength of the hydrophobic interaction depends
on the number and size of the protein’s hydrophobic
contact areas. Membrane proteins may contain relati-
vely large spans of exposed hydrophobic amino acids
which easily interact with a hydrophobic stationary
phase. Cytoplasmic proteins carry ionic and polar resi-
dues o n their surface, whereas hydrophobic residues
are usually buried inside the protein. Each protein thus
carries a number of hydrophobic patches on its surface
and it is the difference in surface hydrophibicity of
proteins on which separation is based.
Clearly the interactions on which HIC retention is
based are similar to the interactions which maintain the
protein’s tertiary structure and those involved in the
hydrophilic associations of the protein with its environ-
ment. Therefore, the risk of partial or complete unfold-
ing of the protein with concomitant loss of biological
activity increases with the number of attachment points
of the protein to the stationary phase, that is with
tightness of the binding. For this reason, only weakly
hydrophibic stationary phases are used in most HIC
applications. Depending on the surface hydrophobicity
of the protein, a stronger or a weaker phase should be
used. For example, the less hydrophobic cytoplasmic
proteins are usually successfully chromatographed on
an octyl or phenyl bonded stationary phase. On the
other hand, for a very hydrophobic membrane protein,
a short alkyl chain phase should preferably be chosen.
Again, excessive binding may lead to mass loss or to
loss of biological activity.
Mobile phase composition
Hydrophobic interactions are promoted by high salt
concentrations. Elution of the bound proteins is usually
achieved by reducing the salt concentration, upon
which the proteins will elute in order of increasing
hydrophobic character. Salts can be arranged according
to the lyotropic series-the Hofmeister series (Hof-
meister, 1888; Von Hippel and Schleich, 1969)-that is
according to their abilities to precipitate proteins from
aqueous solutions. This series is very similar to the
arrangement of salts according to their abilities to
promote binding of a protein to a hydrophobic station-
ary phase. Salts at the beginning of this series are
water-structuring or antichaotropic; salts at the end o f
the series are considered chaotropic or randomizing the
structure of water. Whilst antichaotropic salts promote
hydrophobic interaction and protein adsorption, chao-
193
tropic salts either lessen such associations or may not
promote them at all. In these terms, HIC is described
as an entropically driven adsorption/desorption process
and the entropy gain for adsorption originates from the
dehydration of the hydrophobic patches on the pro-
tein’s surface (see also Norde et d . ,1986).
In order of their ability to promote hydrophobic
interactions, the series of anions is:
PO:- >SO,”- > CH3COO- > C1- > Br- > NO3-
> C104-> I - > SCN-
The lyotropic series for cations is:
NH4+> Rb’ > K+ > Na’ > Cs’ > Li+ > Mg2+
> Ca” > Ba2+
If under low salt conditions a protein is still bound, an
alcohol may be added to the eluent to achieve elution.
Alcohols change the polarity of the solvent and favour
the solvation of hydrophobic molecules. Alternatively,
the process can be considered as a competition between
the alcohol and the protein for the hydrophobic binding
sites of the stationary phase. Ethylene glycol is often
used as it is mild and non-denaturing to many proteins.
If retention of biological activity is not of any concern,
stronger alcoholic eluents (e.g. ethanol or (iso)propa-
nol) may be used. In fact, efficient cleaning of HIC
phases can be performed by washing successively with
water, ethanol and butanol. Polyethylene glycol 4000
added at low concentrations to the eluent (0.2%, w/w)
has been used to reduce the retentivity of the stationary
phase (El Rassi and Horvath, 1986). Similarly, chaotro-
pic agents such as urea and guanidine hydrochloride
(Kato et al., 1984) and surfactants (Buckley and
Wetlaufer, 1990) promote desorption, but they also
promote unfolding of proteins and should therefore be
used with caution.
Elution can sometimes be accomplished by changing
the protein’s conformation, whether this is done by
changing the pH or by using a “deforming” buffer (Er-
El et al., 1972). Obviously, when retention of biological
activity is a prerequisite, care should be taken not to
induce irreversible changes in the protein. The effect of
pH on the retention is different for different proteins.
Apart from inducing conformational changes, the
eluent pH can have an effect on local charge distribu-
tions and can therefore be an important separation
parameter. One might argue that hydrophobic interac-
tions are the strongest at the PI value of a protein,
where the net charge of the protein is zero and where
protein solubility in water is minimal, but this has not
generally been observed (Kato et al., 1984; Miller et al.,
1985; Engelhardt and Schon, 1986; Hjerten et al.,
1986). Apparently, it is not the net charge of the
protein, but the local charge distributions that are
important, and these affect the hydrophobic interac-
tions. Moreover, the pH determines the charge of the
ionizable groups, if present, of the matrix and it may
introduce electrostatic attraction or repulsion. The
effect of p H on the strength of the hydrophobic interac-
tion must therefore be called secondary and this effect
may be unpredictable. Nevertheless, the pH certainly
has an effect and this may be used to adjust the
retention and selectivity (Engelhardt and Schon, 1986;
194 E. J. M. PENNINGS E T A L .

Gooding et al., 1986; HjertCn et al., 1986; Buckley and
Wetlaufer, 1990).
Elution order is not necessarily correlated with the
hydrophobicity of a protein as determined by the
number and hydrophobicity of each contributing amino
acid. As mentioned before, hydrophobic groups may
be buried inside a protein and so may not contribute to
the protein’s surface hydrophobicity. On the other
hand, proteins with a higher degree of accessible non-
polar surface area are retained to a greater extent
(Katti et al., 1987) and the elution order appears to be
correlated with the surface hydrophobicity of a protein.
HIC has therefore been used to map the surface hydro-
phobicity of proteins (Melander and Horvath, 1977;
Kato et al., 1984; Katti et a f . , 1987).
Temperature
Lowering the temperature is another way of reducing
hydrophobic interactions. Thus, it has been found that
after binding phycoerythrin to Pentyl Sepharose 4B,
25-30% of the bound phycoerythrin can be desorbed
by transferring the suspension to 0°C (Hjerten et al.,
1974). Clearly, the degree of desorption is dependent
on the strength of the hydrophobic interaction,
although often the effect is too small to be suitable for
practical purposes. On the other hand, one must always
bear in mind that retention times may slightly change
when a method developed in the cold room is
attempted at ambient temperature and that the sepa-
ration may be affected (Miller et a f . , 1985).
CONFORMATIONAL ASPECTS
In the adsorbed state, hydrophobic patches on the
protein interact with the hydrophobic sorbent surface
under exclusion of water, and intramolecular hydro-
phobic bonding is less important as a factor stabilizing
the protein structure. Therefore, upon adsorption, con-
formational changes may be expected especially for
those proteins that have intramolecular hydrophobic
~
bonding as the main structure-stabilizing force. As
intramolecular hydrophobic bonds stabilize secondary
stuctures, such as a-helices, a reduction of the a-helix
content may be expected upon adsorption (Norde et
al., 1986). This reduction may partially remain after
desorption as has indeed been found for human plasma
albumin after desorption from several stationary phases
differing in hydrophilicity and hydrophobicity (Norde
et al., 1986). In addition to the dehydration of hydro-
phobic patches, the structural rearrangements provide
the entropy gain for the human plasma albumin mole-
cule during the adsorption process. This indicates that
protein adsorption can be accompanied by structural
changes in the protein molecule and that these changes
need not be fully reversible. This applies to adsorption
both onto hydrophobic surfaces and onto hydrophilic
surfaces.
SELECTED APPLICATIONS
Some recent examples of the use of HIC for the
purification of plant enzymes involved in the formation
of secondary metabolites are listed in Table 2. This list
is not exhaustive and is provided to indicate the import-
ance of HIC for the purification of enzymes that usually
occur in very low amounts in the plant tissue. It is often
a formidable task to purify such enzymes from crude
protein extracts, and in such cases it is profitable to use
as many different techniques as possible. It is in this
context that high performance HIC is becoming
increasingly popular.
Since 1985 we have used HIC successfully in our
research programme concerned with the production of
pharmaceutically interesting secondary products by
plant cell cultures. The main areas that we have studied
are the production of quinoline alkaloids (quinine and
quinidine) by Cinchona species, the production of
indole alkaloids (ajmalicine, serpentine and bisindole
alkaloids) by Catharanthus roseus, and the production
of related indole alkaloids and their dimerization to

Table 2. Selected recent applications of HIC in the purification of enzymes involved in plant secondary metabolism
Enzyme Plant source Type of HIC columnlmateriala Reference
Monoterpene cyclase Rauwolfia serpentina Phenyl Superose Uesato et a/. (1987)
L-Tyrosine decarboxylase Eschscholtzia californica Phenyl Superose Marques and Brodelius (1988)
6P-Hydroxyhyoscyamine epoxidase Hyoscyarnus niger Butyl Toyopearl 650M Hashimoto etal. (1989)
Cytochrome P450, 3,9-dihydroxypterocarpan
6a-hydroxylase Glycine rnax hexylagarose Kochs and Grisebach (1989)
6a-Hydroxymaackiain 3-0-methyltransferase Pisurn sativurn Phenyl-5PW Preisig e t a l . (1989)
17-0-Deacetylvindoline 17-0-acetyltransferase Catharanthus roseus hydroxyphenoxypropyl- Fahn and Stockigt (1990)
bonded
TSK HW-65 (Sib
3’-Hydroxy-N-methyl-(S)-coclaurine-4’-
0-methyltransferase Berberis koetineana Phenyl Sepharose Frenzel and Zenk (1990a)
( R , S)-Tetrahydrobenzylisoquinoline-
N-methyltransferase Berberis koetineana Phenyl Superose Frenzel and Zenk (1990b)
Diamine oxidase Hyoscyarnus niger Phenyl Superose Hashimoto e t a / . (1990)
(Sf-Tetrahydroprotoberberine-cis-
N-methyltransferase Eschscholtzia californica Phenyl Sepharose Rueffer et a/. (1990)
Hyoscyamine 6P-hydroxylase Hyoscyarnus niger Butyl-Toyopearl,
Phenyl Superose Yamada et a/. (1990)
a The high performance columns Phenyl Superose and Phenyl-5PW (with 10 p particle sizes) are primarily used at the end of
purification procedures. The other column materials have particle sizes greater than 20 p and are mainly used in initial purification
steps.
Prepared by authors from epoxide-activated Fractogel TSK HW-65 (S).
 HYDROPHOBIC INTERACTION CHROMATOGRAPHY 195

bisindole alkaloids by Tabernaemontana species. The

early steps of the biosynthetic pathways to these alka-
loids are identical in these plants. In order to increase
alkaloid production using the techniques of molecular
biology we have been interested to identify the limiting
steps in their biosynthesis. Our strategy has involved
the characterization of the metabolic intermediates and
the enzymes involved in their synthesis, and this will be
followed by a study of the regulation of the expression
of the corresponding genes. In order to identify these
genes, cDNA libraries producing fusion proteins have
been screened. An essential step in this process is the
raising of antibodies against the different purified
enzymes.
As mentioned above, the first steps in the biosynthe-
sis of the indole and the quinoline alkaloids are identi-
cal. The pathway starts with the decarboxylation of L-
tryptophan to tryptamine by the enzyme tryptophan
decarboxylase (TDC; EC 4.1.1.28), followed by the
elution volume i m l 1
stereospecific condensation of the monoterpenoid seco-
loganin and tryptamine to form strictosidine. This reac- Figure 1. Purification of tryptophan decarboxylase (TDCJfrom
tion is catalysed by the enzyme strictosidine synthase Catharanthus roseus by HIC on a Phenyl Sepharose CL-4B
(SSS; EC 4.3.3.2) which is a cytoplasmic glycoprotein. column (45 x 2.6 cm i.d.1. Elution was carried out using a linear
gradient of ammonium sulphate (0.2-0 M) (NH4)2S04in 0.1 M
The biosynthesis of secologanin is only partially Tris-HCI buffer (pH 7.5) containing 1 mM dithiothreitol and
resolved, but commences with the hydroxylation of 0.02 mM pyridoxaC5’-phosphate.
geraniol to 10-hydroxygeraniol by the enzyme geraniol-
10-hydroxylase (G10H) which is a membrane-bound
P-450 monooxygenase. We have isolated, purified and (1 M ammonium sulphate) although neither fraction
characterized these three enzymes using HIC as part of contained protein. The fraction corresponding with the
the purification protocol. first peak contained pyridoxal-5’-phosphate. After elu-
In our hands HIC appears to be an indispensable tion of these early peaks, the gradient was commenced
technique since other methods of protein purification and TDC was eluted as a sharp peak. From the elution
(e.g. size exclusion, ion exchange and chromatofocuss- pattern, and also from the SDS-PAGE pattern (not
ing) did not yield a sufficiently pure protein. Outlines of shown), we estimated that the purity of TDC before
the methods that we have employed are given below injection was already higher than 95%. Unbound
and these serve to demonstrate the usefulness and
versatility of high performance HIC in cases where a
highly purified protein sample is required for further
experimentation. 0.2

Tryptophan decarboxylase from Catharanthus roseus

-
-I
TDC from 2 kg (fresh weight) of C. roseus cells was I

concentrated by ammonium sulphate precipitation (42- I

I
55% saturation). The enzyme was resuspended in 0
m
N I
I
buffer with 0.2 M ammonium sulphate and further puri- 6 I
I
fied on a Phenyl Sepharose C L 4 B column
0.1 I
(45 x 2.6 cm) (Pennings et al., 1989a). TDC activity was
well separated from the main protein peak (Fig. 1).
After this step, the increase in specific activity was 14-
fold. The recovery of enzyme activity was high, i.e.
9470, which is particularly important as TDC is a labile
enzyme. In comparison with a previous procedure
(Pennings et al., 1988), the advantage of the present
method is that the final enzyme preparation has a
higher purity and that the desalting step following L J0
ammonium sulphate precipitation can be omitted. I I - 1 I

After the last purification step, the preparation still OO 10 20 30 LO

appeared to contain some minor impurities. High per-
formance HIC was therefore chosen as an additional
step and carried out on a TSK Phenyl-5PW column
(75 X 7.5 mm i.d.) (Pennings et al., 1989b); the result is
shown in Fig. 2. TDC was concentrated on the column
by eight consecutive 1 mL injections. Each injection
results in two peaks eluting under high salt conditions
Figure 2. HIC of tryptophan decarboxylase (TDC) from
Catharanthus roseus on a TSK Phenyl-5PW column
(75 x 7.5 mm i.d.1. The sample was applied by eight-repetitive
injections of 1 mL each. The flow rate was 0.5 mllmin: the
linear gradient was from 1 M ammonium sulphate in 50 mM
sodium phosphate buffer (pH 7.4) to water (without any salt)
during 15 min.
196 E. J. M. PENNINGS E T A L .
.too
75
.50
25
0 represents the pooled fractions.
volume I m l l
Figure 3. Purification of strictosidine synthase (SSS) from
Cinchona robusta on a Phenyl Sepharose C L 4 B column
(13x 1.6 cm). The ammonium sulphate gradient was 1.0-0 M in
20 mM sodium phosphate buffer (pH 6.8): the ethylene glycol
gradient was 0-50% (v/v) in the same buffer.
pyridoxal-5’-phosphate was separated from TDC in this
step, but bound cofactor was not removed (Pennings et
ul., 1989b).
Strictosidine synthase from Cinchona robusta
SSS from C. robusta consists of at least two isoforms
and these have a relatively small M , of about 35,000
(Pennings et al., 1990). Since most of the proteins in the
crude extract have higher M , vlaues, size-exclusion
chromatography appeared to be an efficient step and
was carried out first with HIC as the second chromato-
graphic step. After ammonium sulphate precipitation
(30-55% saturation) of the enzyme from the crude cell-
free extract (prepared from 750 g cells) and following
size-exclusion chromatography, SSS was applied to a
Phenyl Sepharose CL-4B column (13 X 1.6 cm).
Decreasing the ammonium sulphate concentration by a
linear gradient of 1.0-OM eluted most of the applied
protein but gave only a small amount of SSS activity.
An ethylene glycol gradient from 0 to 50% ( v h ) was
needed to recover the remainder of SSS from the
column (Fig. 3 ) . The purification factor was 14, but the
recovery was rather low (55%). When size-exclusion
chromatography was omitted and HIC was carried out
as the first column step, SSS eluted at low salt concen-
trations (50 mM sodium phosphate buffer, pH 6.8) with
a recovery of about 60% and ethylene glycol was not
required to elute the protein. This behaviour is an
example of how elution conditions may depend on the
degree of purity of the sample. It appears that contami-
nating proteins deactivate the most hydrophobic sites
of the stationary phase thereby making the column
apparently less hydrophobic. The low recovery for SSS
may indicate that this protein is relatively hydrophobic,
and it would therefore be interesting to study the
elution behaviour and recovery of SSS on a more
weakly hydrophobic stationary phase, for instance on
Butyl Sepharose.
Q I
I
06
2 8 0
\ 0
ID
5 6
0.4 x
-x . 4
Y
a 01
C
P,
0 02 Lz
-
6 2 s
0
2 4 0 00 I
0 20 GO 60
W
C
0)
- Elution volume (ml)
x
f
Figure 4. Purification of geraniol-10-hydroxylase (GlOH) from
I Catharanthus roseus by HIC on a TSK Phenyl-5PW column
I (75 x 7.5 m m i.d.). The column was pre-equilibrated with 20 mM
I potassium phosphate buffer (pH 7.7) containing 15% glycerol
and 0.1 % Renex 690. The enzyme was eluted by a linear gradient
of 0.1-0.6% Renex 690 in the same buffer. The shaded area
Geraniol-10-hydroxylase from Cutharunthus roseus
Cytochrome P-450-dependent G 10H from C. roseus
was isolated from membranes sedimenting in the range
20 min 1000 x g and 60 min 20,000 X g. The solubilized
monooxygenase was purified in a four-step procedure
comprising chromatography on DEAE Sephacel,
Hydroxyapatite-Ultrogel, o-aminooctylagarose and
TSK Phenyl-5PW (Meijer et al., 1990), the latter two
purification steps involving hydrophobic interactions.
o-Aminooctylagarose is often used for the purification
of cytochrome P-450 proteins since the aminooctyl
chain is an efficient ligand for the ferric form of this
type of heme-protein. The binding of proteins to this
stationary phase is based on both ionic and non-ionic
interactions. The binding of GlOH is probably mainly
of a hydrophobic nature since the enzyme can be eluted
with non-ionic detergent. The final step of the purifica-
tion procedure involved HIC on a uncharged high
performance TSK Phenyl-SPW column (75 x 7.5 mm
i.d.), and this step can be performed without applying a
high salt concentration to bind the GlOH. This enzyme
appeared to have such a high hydrophobicity that it was
even bound when the column was presaturated with a
buffer containing 15% glycerol and 0.1% of the non-
ionic polyoxyethylene-type detergent Renex 690.
Presaturation of the matrix with glycerol and detergent
created a condition that prevented the binding of most
of the contaminating proteins in the sample, thus mak-
ing HIC on TSK Phenyl-5PW an efficient method of
separation. The GlOH was eluted by increasing the
detergent concentration with a linear gradient of 0.1-
0.6% Renex 690 (Fig. 4). The peak fractions of GlOH
activity consisted of a near-homogeneous protein that
migrated with an M , of 56,000 on SDS-PAGE (Meijer
et al., 1990).
CONCLUSIONS
HIC has become a powerful technique for the purifica-
tion of proteins. As separation is based on the differ-
ences in surface hydrophobicity of proteins, HIC is
complementary to chromatographic techniques based
on the charge or the size of the protein and it may be
used advantageously for the purification of those pro-
 HYDROPHOBIC INTERACTION CHROMATOGRAPHY
teins that cannot be obtained in pure form by size-
exclusion and ion-exchange techniques alone.
Purifications by HIC are frequently carried out early
in a procedure usually after salt-mediated precipitation
of the crude protein: in such cases the desalting step can
be omitted. For these initial steps in a purification
procedure, many packing materials are available with
particle sizes up to approximately 165 p. The high
performance columns are preferentially used in the
final steps of a purification procedure. Many high
performance HIC columns are now available with high
efficiency and high stability. As the adsorption of pro-
teins is based on weak interactions and the elution
conditions are mild, recoveries of protein mass and
biological activity are usually high. However, excep-
tions exist, for example, the low recovery of SSS from
Phenyl Sepharose CL-4B. It is a good idea to use a
stationary phase with the least hydrophobic ligand
attached to the matrix, which is still able to bind the
protein of interest under high-salt conditions.
Alternatively, a stationary phase can be chosen to
which most contaminating proteins bind, but not the
protein of interest which is then bound on a second
REFERENCES
Alpert, A. J. (1986). High-performance hydrophobic interaction
chromatography of proteins on a series of
poly(alky1aspartamide)-silicas. J. Chromatogr. 359, 85-97.
Buckley, J. J. and Wetlaufer, D. B. (1990). Surfactant-mediated
hydrophobic interaction chromatography of proteins: gradi-
ent elution. J. Chromatogr. 518, 99-110.
Chang, J. P., El Rassi, 2. and Horvath, Cs. (1985). Silica-bound
polyethylene glycol as stationary phase for separation of
proteins by high performance liquid chromatography. J.
Chromatogr. 319, 396-399.
El Rassi, Z. and Horvath, Cs. (1986). Hydrophobic interaction
chromatography of t-RNAs and proteins. J. Liq.
Chromatogr. 9, 3245-3268.
Engelhardt, H. and Schon, U. (1986). Separation of proteins on
polar bonded phases by hydrophobic interaction chromato-
graphy. J. Liq. Chromatogr. 9, 3225-3244.
Er-El, Z., Zaidenzaig, Y. and Shaltiel, S. (1972).
Hydrocarbon-coated sepharoses. Use in the purification of
glycogen phosphorylase. Biochem. Biophys. Res. Commun.
49, 383-390.
Fahn, W. and Stockigt, J. (1990). Purification of acetyl-CoA:
17-0-deacetylvindoline 17-0-acetyltransferase from
Catharanthus roseus leaves. Plant Cell Rep. 8, 61 3-61 6.
Fausnaugh, J. L., Pfannkoch, E., Gupta, S. and Regnier, F. E.
(1984). High-performance hydrophobic interaction chroma-
tography of proteins. Anal. Biochem. 137, 464-472.
Frenzel, T. and Zenk, M. H. (1990a). S-Adenosyl-c-
methionine: 3’-hydroxy-N-methyl-(S)-coclaurine-4’-0-
methyltransferase, a regio- and stero-selective enzyme of
the (S)-reticuline pathway. fhytochemistry 29, 3505-351 1.
Frenzel, T. and Zenk, M. H. (1990b). Purification and characteri-
zation of three isoforms of S-adenosyl-L-methionine: (R,S)-
tetrahyrobenzylisoquinoline-N-methyltransferase from
Berberis koetineana cell cultures. fhytochemistry 29,
3491-3497.
Gooding, D. L., Schmuck, M. N. and Gooding, K. M. (1984).
Analysis of proteins with new, mildly hydrophobic high
performance liquid chromatography packing materials. J.
Chromatogr. 296, 107-1 14.
Gooding, D. L., Schmuck. M. N., Nowlan, M. P. and Gooding,
K. M. (1986). Optimization of preparative hydrophobic inter-
action chromatographic purification methods. J.
Chromatogr. 359, 331-337.
Halperin, G., Breitenbach, M., Tauber-Finkelstein, M. and
Shaltiel, S. (1981). Hydrophobic chromatography on
homologous series of alkylagaroses. A comparison of
197
column containing a more hydrophobic ligand. This
tandem chromatography is most suitable for a prelimi-
nary clean-up of the sample.
The type and concentration of the salt and of the
(po1y)alcoholic additive are important parameters in
controlling protein retention and selectivity. Gradient
design, pH and temperature can additionally be used to
optimize a separation. It can therefore be anticipated
that the popularity of the technique will become com-
parable to that of ion-exchange techniques. Also, new
ligands may be expected to be used for the synthesis of
new stationary phases. One such example is the
Immobilized Artifical Membrane support (Regis
Chemical Company, 1990) containing phosphatidylcho-
line covalently bound to propylamine-derivatized silica
and which can be used for the purification of membrane
proteins.
Acknowledgements
The authors thank Mr. A.G.M. Goosen for his technical assitance.
The ownership of all registered tradenames used in this review i s
acknowledged.
charged and electrically neutral column materials. J.
Chromatogr. 215, 21 1-228.
Hashimoto, T., Kohno, J. and Yamada, Y. (1989). 6p-
Hydroxyhyoscyamine epoxidase from cultured roots of
Hyoscyamus niger. fhytochemistry 28, 1077-1 082.
Hashimoto, T., Mitani, A. and Yamada, Y. (1990). Diamine
oxidase from cultured roots of Hyoscyamus niger. Its func-
tion in tropane alkaloid biosynthesis. Plant fhysiol. 93, 216-
221.
Hjerten, S., Rosengren, J. and PAhlman, S. (1974). Hydrophobic
interaction chromatography. The synthesis and the use of
some alkyl and aryl derivatives of agarose. J. Chromatogr.
101, 281-288.
Hjerten, S., Yao, K., Eriksson, K.-0. and Johansson, B. (1986).
Gradient and isocratic high-performance hydrophobic inter-
action chromatography of proteins on agarose columns. J.
Chromatogr. 359,99-109.
Hofmeister, F. (1888). Zur Lehre von der Wirkung der Salze.
Arch. Exp. Path. fharmacol. 24, 247-260.
Hofstee, B. H. J. (1973). Protein binding by agarose carrying
hydrophobic groups in conjunction with charges. Biochem.
Biophys. Res. Commun. 50, 751-757.
Hofstee, B. H. J. (1979). Non-ionic adsorption chromatography
and adsorptive immobilization of proteins. Pure Appl.
Chem. 51, 1537-1548.
Janig, G.-R. and Pfeil, D. (1984). Structure-function relation-
ships of the essential components of the liver microsomal
monooxygenase system. In Cytochrorne P-450 (Ruckpaul, K.
and Rein, H., eds.), pp. 58-1 10. Akademie-Verlag, Berlin.
Jennissen, H. P. and Heilmeyer Jr., L. M. G. (1975). General
aspects of hydrophobic chromatography. Adsorption and
elution characteristics of some skeletal muscle enzymes.
Biochemistry 14, 754-760.
Kato, Y., Kitamura, T. and Hashimoto, T. (1983).
High-performance hydrophobic interaction chromatogra-
phy of proteins. J. Chromatogr. 266, 49-54.
Kato, Y., Kitamura, T. and Hashimoto, T. (1984). Operational
variables i n high-performance hydrophobic interaction
chromatography of proteins on TSKgel phenyl-5PW. J.
Chromatogr. 298,407-418.
Katti, A., Maa, Y.-F. and Horvath, Cs. (1987). Protein surface and
retention in hydrophobic interaction chromatography.
Chromatographia 24, 646-650.
Kochs, G. and Grisebach, H. (1989). Phytoalexin synthesis in
soybean: purification and reconstitution of cytochrome
P450 3.9-dihydroxypterocarpan 6a-hydroxylase and sepa-
198 E. J. M.PENNINGS E T A L .
ration from cytochrome P450 cinnamate 4-hydroxylase.
Arch. Biochem. Biophys. 273, 543-553.
Maisano, F., Belew, M. and Porath, J. (1985). Synthesis of new
hydrophobic adsorbents based on homologous series of
uncharged alkyl sulphide agarose derivatives. J.
Chromatogr. 321,305-317.
Marques, I. A. and Brodelius, P. E. (1988). Elicitor-induced L-
tyrosine decarboxylase from plant cell suspension cultures.
I. Induction and purification. Plant Physiol. 88, 46-51.
Meijer, A. H., Pennings, E. J. M., De Waal, A. and Verpoorte, R.
(1990). Purification of cytochrome P-450-dependent
geraniol-10-hydroxylase from a cell suspension culture of
Catharanthus roseus. In Progress in Plant Cellular and
Molecular Biology (Nijkamp, H. J. J., Van Der Plas, L. H. W.
and Van Aartrijk, J., eds.), pp. 769-774. Kluwer Academic
Publishers, Dordrecht.
Melander, W. and Horvath, Cs. (1977). Salt effects on hydropho-
bic interactions in precipitation and chromatography of
proteins: an interpretation of the lyotropic series. Arch.
Biochem. Biophys. 183, 200-215.
Miller, N. T., Feibush, B. and Karger, B. L. (1985). Wide-pore
silica-based ether-bonded phases for separation of proteins
by high-performance hydrophobic interaction and size-
exclusion chromatography. J. Chromatogr. 316, 519-536.
Norde, W., MacRitchie, F., Nowicka, G. and Lyklema, J. (1986).
Protein adsorption at solid-liquid interfaces: reversibility and
conformation aspects. J. Colloid lnterface Sci. 112,447-456.
Pennings, E. J. M., Van Den Bosch, R., Van Der Heijden, R., Van
Der Leer, T. and Verpoorte, R. (1988). Rapid assay of
tryptophan decarboxylase from plant cell cultures. In
Manipulating Secondary Metabolism in Culture, (Robins, R.
J. and Rhodes, M. J. C., eds.), pp. 79-82. Cambridge
University Press, Cambridge.
Pennings, E. J. M., Verpoorte, R., Goddijn, 0.J. M. and Hoge,
J. H. C. (1989a). Purification of tryptophan decarboxylase
from a Catharanthus roseus cell suspension culture.
J. Chromatogr. 483, 311-318.
Pennings, E. J. M., Groen, B. W., Duine, J. A. and Verpoorte, R.
(1989b). Tryptophan decarboxylase from Catharanthus
roseus is a pyridoxoquinoprotein. FEBS Lett. 255, 97-100.
Pennings, E. J. M.. Giroud. C.. Stevens, L. H. and Verpoorte, R.
(1990). Purification of strictosidine synthase from a
Cinchona robusta cell suspension culture. Planta Med. 56,
599.
Porath, J., Sundberg, L., Fornstedt, N. and Olsson, I. (1973).
Salting-out in amphiphilic gels as a new appraoch to hydro-
phobic adsorption. Nature (London) 245, 465-466.
Preisig, C. L., Matthews, D. E. and VanEtten, H. D. (1989).
Purification and characterization of S-adenosyl-L-
methionine: 6a-hydroxymaackiain 3-0-methyltransferase
from Pisum sativum. Plant Physiol. 91, 559-566.
Regis Chemical Company (1990) Immobilized Artificial
Membrane-Data Sheet. Regis News.
Rueffer, M., Zumstein, G. and Zenk, M. H. (1990).Partial purifica-
tion and properties of S-adenosyl-L-methionine: 6)-
tetrahydroprotoberberine-cis-N-methyltransferase from
suspension-cultured cells of €schscholtzia and Corydalis.
Phytochemistry 29, 3727-3733.
Shaltiel, S. and Er-El, 2. (1973). Hydrophobic chromatography:
use for purification of glycogen synthetase. Proc. Natl. Acad.
Sci. 70, 778-781.
Uesato, S., Ikeda, H., Fujita, T., Inouye, H. and Zenk, M. H. (1987).
Elucidation of iridodial formation mechanism. Partial purifi-
cation and characterization of the novel monoterpene cyc-
lase from Rauwolfia serpentina cell suspension culture.
Tetrahedron Lett. 28, 4431-4434.
Von Hippel, P. H. and Schleich, T. (1969). The effects of neutral
salts on the structure and conformational stability of macro-
molecules in solution. In Structure and Stability of
Biological Macromolecules (Timasheff, S. N. and Fasman,
G. D., eds.), pp. 417-547. Marcel Dekker, New York.
Weiss, H. and Bucher, T. (1970). Chromatographic separation of
membrane proteins on lipophilic ion exchange resins. Eur.
J. Biochem. 17, 561-567.
Yamada, Y., Okabe, S. and Hashimoto, T. (1990). Homogeneous
hyoscyamine 6P-hydroxylase from cultured roots of
Hyoscyamus niger. Proc. Japan. Acad. 66, 73-76.
Yon, R. J. (1972). Chromatography of lipophilic proteins on
adsorbents containing mixed hydrophobic and ionic
groups. Biochem. J. 126,765-767.