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Colloids and Surfaces B: Biointerfaces 64 (2008) 145–149

Short communication

Anomalous adsorptive properties of HIV protease: Indication of

two-dimensional crystallization?
Petr Cı́gler a,b , Vladimı́r Král a , Milan Kožı́šek b , Jan Konvalinka b , Vladimir M. Mirsky c,∗
a Institute of Chemical Technology, Department of Analytical Chemistry, Technická 5, 166 28 Prague 6, Czech Republic
b Gilead Sciences and IOCB Research Center, Institute of Organic Chemistry and Biochemistry, AS CR,

Flemingovo n. 2, 166 10 Prague 6, Czech Republic

c University of Regensburg, Institute of Analytical Chemistry, Chemo- and Biosensors, D-93040 Regensburg, Germany

Received 31 October 2007; accepted 15 January 2008

Available online 19 January 2008

Abstract
Adsorption of HIV protease onto surfaces that are usually considered to be protein-resistant was studied quantitatively using surface plas-
mon resonance. Adsorption onto gold surfaces functionalized by OH-terminated alkyl chains was much stronger than onto oligo(ethylene
glycol)-terminated surfaces. Equilibrium and kinetic adsorption constants were determined. An anomalous mutual attraction between adsor-
bate molecules was observed, indicating the possibility of two-dimensional crystallization of HIV protease. These results are applicable for the
design of sensors/biosensors for HIV protease resistance detection and for proper manipulation of this enzyme in laboratory devices.
© 2008 Elsevier B.V. All rights reserved.

Keywords: HIV protease; Protein adsorption; Protein-resistant surfaces; Self-assembled monolayer; Surface plasmon resonance (SPR)

1. Introduction The virus-encoded aspartic protease from HIV (HIV PR) is

Self-assembled monolayers (SAMs) of thiols on a gold
surface have great potential for the development of medical
techniques and biosensors [1–3]. Most of these applications
demand protein-resistant surfaces that prevent the nonspecific
adsorption of biomolecules onto the solid–liquid interface.
Gold surfaces coated by linear alkylthiols ␻-substituted by
poly(ethylene glycol) chains or by shorter oligomers of
ethylene glycol (EGn , n = 3–6) have been found to demon-
strate very low nonspecific adsorption of fibrinogen [4–13],
ribonuclease A [4–6,8], pyruvate kinase [4–6], lysozyme
[4–6,8,10,11,13], chymotrypsinogen [4], carbonic anhydrase
[8], human immunoglobulin G and serum albumin [14], hep-
arinized plasma [9] and serum [9,15]. However, the large
differences between these proteins and their physico-chemical
properties do not allow extrapolation of these results to other
proteins.
∗ Corresponding author. Tel.: +49 9419434011; fax: +49 9419434064.
E-mail address: vladimir@mirsky.de (V.M. Mirsky).
a C2 symmetric homodimer [16]. HIV PR is essential for the
completion of the viral life-cycle, and if PR is afflicted or its
activity is inhibited, the virion cannot replicate and remains
non-infectious. Inhibition of PR by small molecule drugs is a
widely used approach for the treatment of HIV-positive patients.
However, the effectiveness of this treatment is compromised by
the rapid development of viral resistance [17]. Effective antivi-
ral therapy therefore requires a quantitative assessment of drug
resistance development throughout the course of the treatment.
Both methods currently in use (genotyping and phenotyping) are
expensive, labour-intensive, and time-consuming [18]. There-
fore, development of sensors and biosensors that would enable
direct quantitative detection of the binding of patient-derived
PR mutants to a specific PR inhibitor would be highly useful.
Several approaches to develop such a device have already been
suggested [19–28].
As a first step toward the development of such a biosensor,
we focused primarily on the reversible nonspecific adsorption of
HIV PR onto different model surfaces formed from ␻-terminated
SAMs. As a result of this investigation, we identified optimal
surfaces that prevent the nonspecific adsorption of this widely
investigated enzyme. Most interestingly, we observed unex-

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doi:10.1016/j.colsurfb.2008.01.011
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146 P. Cı́gler et al. / Colloids and Surfaces B: Biointerfaces 64 (2008) 145–149

Table 1
Thermodynamic and kinetic parameters of HIV PR adsorption on different surfaces
No. of thiol Structure of thiol K (M−1 ) koff (s−1 ) kon (s−1 M−1 ) Frumkin attraction parameter (a) Hill constant (n)

1 HS–(CH2 )11 –OH 8.1 × 105 a 80 6.5 × 107 c 1.20 4.8

2.2 × 106 d
2 HS–(CH2 )11 –(O–CH2 –CH2 )4 –OH 1.3 × 105 b 30 3.9 × 106 c – –
3.2 × 105 d
3 HS–(CH2 )16 –(O–CH2 –CH2 )2 –OH 1.5 × 105 b 28 4.2 × 106 c – –
3.4 × 105 d
a Frumkin isotherm (Eq. (2)).
b Henry isotherm θ = Kc.
c kon = koff K.
d From the initial slope of concentration dependence θ = Kc under the assumption that the saturated value corresponds to θ = 1 (see Fig. 2).

pected, strong cooperative effects of the absorptive behaviour
of HIV PR on the properly organized solid-liquid interface, and
we characterized these effects on a thermodynamic basis.
2. Materials and methods
2.1. Chemicals
P.a. quality organic solvents were obtained from J.T. Baker
and used without further purification. Millipore Milli-Q grade
water was used for the preparation of sample solutions. Thiol 1
was purchased from Sigma–Aldrich and thiol 2 from ProChimia
(Sopot, Poland). Thiol 3 was prepared by the procedure
described in the literature [29]. Expression, refolding, and purifi-
cation of wild type HIV PR were performed as previously
described, and the protein concentration was determined spec-
trophotometrically by titration using the tight-binding inhibitor
QF34 [30].
310 (www.biosuplar.com) SPR-reflectometer was used. The
measurements were performed in slope mode, i.e. as measure-
ments of the intensity of reflected light at a fixed angle. The angle
used for measurements was selected to be between the resonance
and total reflection angles so that 50% reflection was observed
in the buffer solution. All measurements were performed at
28.0 ◦ C.
The SPR measurement cell used had a thickness of about
0.3 mm. Estimations of the number of molecules present in the
cell indicated that the adsorption of an adsorbate monolayer in
such a thin bulk volume leads to an essential decrease in the bulk
(submicromolar) concentration. Continuous addition of protein
was impossible because of high protein consumption. To con-
serve protein, HIV PR injection was performed by several 5-s
cycles of turning the pump on and off. Injections were stopped
when no increase in the SPR-signal was observed. For deter-
mination of desorption kinetics, the cell was washed using the
working buffer.

2.2. Preparation of self-assembly monolayers

3. Results and discussion
The experiments were performed on a gold surface cov-
3.1. Reversibility of adsorption
ered by SAMs of thiols 1–3 (see Table 1). Gold-coated
glass slides for surface plasmon resonance (SPR) measure-
Adsorption of HIV PR1 onto all surfaces investigated was fast
ments were obtained from Analytical ␮-Systems/Mivitec GmbH
and reversible (Fig. 1). Depending on the protein concentration,
(www.biosuplar.com). Prior to SAM adsorption, the gold sur-
the typical adsorption kinetics fell into the second or tenths-of-
faces were ultrasonicated 1 min in chloroform, then ethanol,
a-second time scale. Because of the slow discharge of buffer into
and dried under nitrogen. The solutions for SAM deposition
the measurement cell, it was not possible to directly determine
were prepared by injection of stock solutions of thiols in ethanol
the kon (see below). Washing with buffer led to complete protein
into a 1 M aqueous solution of KCl (the final concentration of
desorption. Adsorption of most proteins is irreversible, which
ethanol was 3%), providing 50 ␮M solutions of the respective
excludes the possible analysis of adsorption isotherms, leav-
thiol. Deposition of thiols was performed at an electrode poten-
ing one to compare either the kinetic adsorption constants [15]
tial of +300 mV relative to Ag/AgCl(sat) for 3 h at 22 ◦ C. After
or some phenomenological adsorption characteristics [4–14].
SAM deposition, the electrodes were rinsed with 100 mM KCl
In this sense, the reversibility of the HIV PR adsorption is an
in 80% ethanol/water, ultrasonicated for 1 min in ethanol, and
unusual property, and it allowed us to make quasi-equilibrium
dried under nitrogen.
measurements of its binding isotherms.

2.3. Surface plasmon resonance (SPR) measurements

1 HIV PR is active as a homodimer. In this study we worked in a concentration
SPR measurements were performed with HIV PR solu- range between 50 and 800 nM, at which nearly all of the protein is dimerized
tions in 0.1 M acetate buffer pH 4.7, containing 0.3 M NaCl, [31]. Throughout the paper, when we refer to adsorption of “protease molecules”,
50 mM Na2 SO4 , and 2% dimethylsulfoxide. A BIOSUPLAR- we intend adsorption of the protease dimers.
 Author's personal copy
P. Cı́gler et al. / Colloids and Surfaces B: Biointerfaces 64 (2008) 145–149
Fig. 1. SPR response of self-assembly monolayer of thiol 1 to increasing con-
centrations of wild type HIV PR. The arrows indicate injections of HIV PR
solutions. Concentrations of HIV PR are indicated in nM. Protein injection was
performed as several 5-s cycles of turning the pump on and off. The injection was
stopped if no further increase in SPR-signal was observed. For determination of
desorption kinetics, the cell was washed using the working buffer.
3.2. Protein-resistant coatings
Adsorption of HIV PR onto a gold surface covered by classi-
cal protein-resistant coatings with different numbers of terminal
ethylene glycol (EG) groups is linearly dependent on the protein
concentration (Fig. 2). This is typical for Henry-type adsorp-
tion isotherms. Linear dependencies with the same slope were
observed for both EG-terminated alkylthiols 2 and 3, indicat-
ing that two EG units are sufficient to form the well-protected
surface suitable for further work with biosensors.
3.3. Cooperative adsorption on the OH-terminated
monolayer
Adsorption of HIV PR onto the gold surface coated by
12-hydroxydodecanethiol (thiol 1) exhibited linear dependence
Fig. 2. Adsorption isotherms of HIV PR on self-assembled monolayers of thiols
1 (circles), 2 (squares), and 3 (triangles). The particular structures of adsorbate
layer on the thiol 1 are indicated. Adsorption data of 1 were fitted using the
Frumkin isotherm (solid curve) (Eq. (2)) and the Hill isotherm (dashed curve)
(Eq. (1)).
147
on the protein concentration only at concentrations below
300 nM. The initial slope of this dependence, which charac-
terizes the binding constant, was about 5–6 times higher than
those obtained for thiols 2 and 3 (Table 1). At higher concen-
trations the dependence became superlinear. The tendency to
saturation was observed at a concentration of about 500 nM
(Fig. 2, circles). Such behaviour was reproducible and was
observed not only for the wild type HIV PR but also for
inhibitor-resistant PR species derived from patients [30] (data
not shown).
Superlinear behaviour of an adsorption isotherm is an indi-
cation of mutual attraction between adsorbed molecules. Two
models – the Hill and Frumkin isotherms – were considered in
order to describe the cooperative adsorption. Hill’s model pos-
tulates the formation of complexes of adsorbed molecules with
a fixed stoichiometry. This model can be described by the Hill
equation (Fig. 2, dashed curve)
θ
Kcn = (1)
1−θ
where K, c, n, and θ are the adsorption constant, the protease
concentration, the stoichiometry of adsorption, and the surface
coverage, respectively.
Fitting of the experimental data according to Eq. (1) gives a
stoichiometry of n = 4.8, i.e. complexes of adsorbed molecules
consisting of five HIV PR molecules. However, this model can-
not describe the initial part of the adsorption isotherm; the Hill
isotherm gives zero slope at zero concentration, while the exper-
imental data obeys linear dependence. A much better description
of the experimental data was obtained by fitting the data accord-
ing to the Frumkin isotherm (Fig. 2, solid curve)
θ
Kc = exp(−2aθ) (2)
1−θ
which yields a linear slope at low concentrations (θ = Kc). This
model provides an excellent fit of the experimental data with a
positive value of interaction parameter a = 1.2. A positive inter-
action parameter indicates a strong mutual attraction between
the adsorbed molecules. The superlinear behaviour of the graph
begins when about one third of the surface is occupied by HIV
PR.
The slope of the linear (Henry) adsorption isotherms and the
initial slope of the Frumkin isotherms allows us to determine
adsorption constants of HIV PR onto different surfaces. The des-
orption kinetics was measured directly, as the signal decreases
after changing the HIV PR solution for the solution without
PR (Table 1). Notably, the binding of HIV PR to EG-terminated
surface has about six times lower affinity than binding to the OH-
terminated surface. No effect of the number of EG-groups (for
the oligomers consisting of 2 and 4 EG-groups) was observed.
Desorption kinetic constants were estimated from monoex-
ponential fitting of the initial part of the desorption curves, in
which possible readsorption effects are ignorable. The values are
shown in Table 1. The definition of binding constant as the ratio
of adsorption and desorption kinetic constants K = kon /koff allows
calculation of adsorption kinetic constants. In the case of EG-
terminated surface, the initial part of the adsorption isotherm was
 Author's personal copy
148 P. Cı́gler et al. / Colloids and Surfaces B: Biointerfaces 64 (2008) 145–149
used for this analysis. The results are summarized in Table 1. The
protein-resistant EG-terminated surface displays a slightly lower
kinetic desorption constant and a much lower kinetic adsorption
constant than the OH-terminated surface.
3.4. Mean thickness of the adsorbed protein
The mean thickness of the adsorbed protein, d, can be esti-
mated using the equation [32]
 
ld n
d = − ln 1 − (3)
2 nmax
where ld is the delay length of the evanescent wave, nmax is
the difference between the refractive indices of buffer and pro-
tein, and n is the measured value of the refractive index due
to protein adsorption. Since ld = 230 nm for λ = 650 nm and
nmax = 0.234 RIU, we obtain a value for refractive index of
4.7 × 10−3 RIU, which corresponds to a thickness of the satu-
rated protein layer of about 2.3 nm. This value corresponds well
to the minimum size of one HIV PR molecule, which is 2.5 nm
(PDB entry: 1NH0) [33]. Therefore, we can suggest formation
of a monomolecular HIV PR layer.
Because all of the cooperative adsorption/desorption pro-
cesses were fast and fully reversible throughout the whole
concentration range, we assume that the saturated protein
layer on the OH-terminated surface is formed by native, non-
denaturated HIV PR organized similarly as in two-dimensional
crystals. Taking into account the quasi-ellipsoidic shape of
HIV PR (dimensions: 2.5 nm × 3.4 nm × 5.5 nm) [33], one can
speculate that the axis including the shortest dimension of the
molecule (and the substrate cavity of the enzyme) is oriented
perpendicular to the surface.
Our assumption regarding the formation of a monomolecu-
lar layer allows us to recalculate the observed SPR signals into
surface coverages. A deviation from linear adsorption caused
by attraction of adsorbate molecules was observed at a sur-
face coverage of about 0.3; the mean distance between adsorbed
molecules at this surface coverage is very close to the size of the
molecule. In the case of adsorption to the OH-terminated alkyl
chains, this attraction occurs at a protease concentration of about
300 nM. In the case of EG-terminated surface, such a coverage
was not reached. From linear extrapolation of the experimen-
tal dependencies, one can expect to obtain this coverage value
and the attraction of adsorbate molecules only at concentrations
higher than 2 ␮M.
4. Conclusions
The present work shows that the reversible adsorption of
HIV protease onto various surfaces that are usually consid-
ered to be protein-resistant differs substantially. Quantitative
SPR measurements revealed linear adsorption isotherms for
oligo(ethylene glycol)-terminated surfaces, which contrasted
with strongly cooperative adsorption on OH-terminated alkyl
chains. An anomalous mutual attraction between adsorbate
molecules indicating the possibility of two-dimensional crys-
tallization of HIV protease was observed.
The fact that deviation from a linear absorption pattern due
to intermolecular interactions can occur at surface coverage of
30% is useful for the design of surfaces for different applications.
Applications such as the development of biosensors for the deter-
mination of HIV PR drug resistance require minimal nonspecific
adsorption; therefore, the EG-terminated surfaces are preferable.
The observed high and cooperative adsorption of HIV PR onto
relatively protein-resistant surfaces should also be taken into
account during laboratory work with this enzyme, since protein
loss could occur due to adsorption onto the walls of laboratory
devices. EG-functionalized surfaces may minimize unwanted
adsorption during the manipulation with HIV PR solutions. One
can also speculate that the inter-protein interactions are relevant
for interpretation of protease function. The observed interaction
of adsorbate molecules indicates two-dimensional condensation
of HIV PR, which probably also occurs on biological membranes
with subsequent modulation of enzyme activity or influence on
the other membrane proteins.
Acknowledgements
The authors are grateful to ProChimia (Sopot, Poland) and
to Dr. M. Schaeferling for generously providing thiols 2 and 3.
The authors thank Professor O.S. Wolfbeis, Dr. A. Zybin, Dr.
D. Boecker, and Dr. Jiri Brynda for helpful discussions. The
research was supported by a grant from the Ministry of Educa-
tion (MSMT) of the Czech Republic within Program 1M0508
“Research Centre for New Antivirals and Antineoplastics” to
P.C., M.K., and J.K., by EC grant SP5A-CT-2006-044515 RASP
to V.M., and by the Ministry of Education, Youth and Sports of
the Czech Republic (MSM 6046137307 and LC512) to V.K. and
P.C.
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