You are on page 1of 5

Author's personal copy

Available online at www.sciencedirect.com

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


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

0927-7765/$ – see front matter © 2008 Elsevier B.V. All rights reserved.
doi:10.1016/j.colsurfb.2008.01.011
Author's personal copy

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 310 (www.biosuplar.com) SPR-reflectometer was used. The
of HIV PR on the properly organized solid-liquid interface, and measurements were performed in slope mode, i.e. as measure-
we characterized these effects on a thermodynamic basis. ments of the intensity of reflected light at a fixed angle. The angle
used for measurements was selected to be between the resonance
2. Materials and methods and total reflection angles so that 50% reflection was observed
in the buffer solution. All measurements were performed at
2.1. Chemicals 28.0 ◦ C.
The SPR measurement cell used had a thickness of about
P.a. quality organic solvents were obtained from J.T. Baker 0.3 mm. Estimations of the number of molecules present in the
and used without further purification. Millipore Milli-Q grade cell indicated that the adsorption of an adsorbate monolayer in
water was used for the preparation of sample solutions. Thiol 1 such a thin bulk volume leads to an essential decrease in the bulk
was purchased from Sigma–Aldrich and thiol 2 from ProChimia (submicromolar) concentration. Continuous addition of protein
(Sopot, Poland). Thiol 3 was prepared by the procedure was impossible because of high protein consumption. To con-
described in the literature [29]. Expression, refolding, and purifi- serve protein, HIV PR injection was performed by several 5-s
cation of wild type HIV PR were performed as previously cycles of turning the pump on and off. Injections were stopped
described, and the protein concentration was determined spec- when no increase in the SPR-signal was observed. For deter-
trophotometrically by titration using the tight-binding inhibitor mination of desorption kinetics, the cell was washed using the
QF34 [30]. 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) 145–149 147

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
Fig. 1. SPR response of self-assembly monolayer of thiol 1 to increasing con- a fixed stoichiometry. This model can be described by the Hill
centrations of wild type HIV PR. The arrows indicate injections of HIV PR equation (Fig. 2, dashed curve)
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 θ
stopped if no further increase in SPR-signal was observed. For determination of Kcn = (1)
1−θ
desorption kinetics, the cell was washed using the working buffer.
where K, c, n, and θ are the adsorption constant, the protease
3.2. Protein-resistant coatings concentration, the stoichiometry of adsorption, and the surface
coverage, respectively.
Adsorption of HIV PR onto a gold surface covered by classi- Fitting of the experimental data according to Eq. (1) gives a
cal protein-resistant coatings with different numbers of terminal stoichiometry of n = 4.8, i.e. complexes of adsorbed molecules
ethylene glycol (EG) groups is linearly dependent on the protein consisting of five HIV PR molecules. However, this model can-
concentration (Fig. 2). This is typical for Henry-type adsorp- not describe the initial part of the adsorption isotherm; the Hill
tion isotherms. Linear dependencies with the same slope were isotherm gives zero slope at zero concentration, while the exper-
observed for both EG-terminated alkylthiols 2 and 3, indicat- imental data obeys linear dependence. A much better description
ing that two EG units are sufficient to form the well-protected of the experimental data was obtained by fitting the data accord-
surface suitable for further work with biosensors. ing to the Frumkin isotherm (Fig. 2, solid curve)
θ
Kc = exp(−2aθ) (2)
3.3. Cooperative adsorption on the OH-terminated 1−θ
monolayer which yields a linear slope at low concentrations (θ = Kc). This
model provides an excellent fit of the experimental data with a
Adsorption of HIV PR onto the gold surface coated by positive value of interaction parameter a = 1.2. A positive inter-
12-hydroxydodecanethiol (thiol 1) exhibited linear dependence 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
Fig. 2. Adsorption isotherms of HIV PR on self-assembled monolayers of thiols
shown in Table 1. The definition of binding constant as the ratio
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 of adsorption and desorption kinetic constants K = kon /koff allows
Frumkin isotherm (solid curve) (Eq. (2)) and the Hill isotherm (dashed curve) calculation of adsorption kinetic constants. In the case of EG-
(Eq. (1)). 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 The fact that deviation from a linear absorption pattern due
protein-resistant EG-terminated surface displays a slightly lower to intermolecular interactions can occur at surface coverage of
kinetic desorption constant and a much lower kinetic adsorption 30% is useful for the design of surfaces for different applications.
constant than the OH-terminated surface. Applications such as the development of biosensors for the deter-
mination of HIV PR drug resistance require minimal nonspecific
3.4. Mean thickness of the adsorbed protein adsorption; therefore, the EG-terminated surfaces are preferable.
The observed high and cooperative adsorption of HIV PR onto
The mean thickness of the adsorbed protein, d, can be esti- relatively protein-resistant surfaces should also be taken into
mated using the equation [32] account during laboratory work with this enzyme, since protein
  loss could occur due to adsorption onto the walls of laboratory
ld n
d = − ln 1 − (3) devices. EG-functionalized surfaces may minimize unwanted
2 nmax adsorption during the manipulation with HIV PR solutions. One
where ld is the delay length of the evanescent wave, nmax is can also speculate that the inter-protein interactions are relevant
the difference between the refractive indices of buffer and pro- for interpretation of protease function. The observed interaction
tein, and n is the measured value of the refractive index due of adsorbate molecules indicates two-dimensional condensation
to protein adsorption. Since ld = 230 nm for λ = 650 nm and of HIV PR, which probably also occurs on biological membranes
nmax = 0.234 RIU, we obtain a value for refractive index of with subsequent modulation of enzyme activity or influence on
4.7 × 10−3 RIU, which corresponds to a thickness of the satu- the other membrane proteins.
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 Acknowledgements
(PDB entry: 1NH0) [33]. Therefore, we can suggest formation
of a monomolecular HIV PR layer. The authors are grateful to ProChimia (Sopot, Poland) and
Because all of the cooperative adsorption/desorption pro- to Dr. M. Schaeferling for generously providing thiols 2 and 3.
cesses were fast and fully reversible throughout the whole The authors thank Professor O.S. Wolfbeis, Dr. A. Zybin, Dr.
concentration range, we assume that the saturated protein D. Boecker, and Dr. Jiri Brynda for helpful discussions. The
layer on the OH-terminated surface is formed by native, non- research was supported by a grant from the Ministry of Educa-
denaturated HIV PR organized similarly as in two-dimensional tion (MSMT) of the Czech Republic within Program 1M0508
crystals. Taking into account the quasi-ellipsoidic shape of “Research Centre for New Antivirals and Antineoplastics” to
HIV PR (dimensions: 2.5 nm × 3.4 nm × 5.5 nm) [33], one can P.C., M.K., and J.K., by EC grant SP5A-CT-2006-044515 RASP
speculate that the axis including the shortest dimension of the to V.M., and by the Ministry of Education, Youth and Sports of
molecule (and the substrate cavity of the enzyme) is oriented the Czech Republic (MSM 6046137307 and LC512) to V.K. and
perpendicular to the surface. P.C.
Our assumption regarding the formation of a monomolecu-
lar layer allows us to recalculate the observed SPR signals into References
surface coverages. A deviation from linear adsorption caused
by attraction of adsorbate molecules was observed at a sur- [1] J.C. Love, L.A. Estroff, J.K. Kriebel, R.G. Nuzzo, G.M. Whitesides, Chem.
face coverage of about 0.3; the mean distance between adsorbed Rev. 105 (2005) 1103–1169.
molecules at this surface coverage is very close to the size of the [2] V.M. Mirsky, Trends Anal. Chem. 21 (2002) 439–450.
[3] T. Wink, S.J. van Zuilen, A. Bult, W.P. van Bennkom, Analyst 122 (1997)
molecule. In the case of adsorption to the OH-terminated alkyl
43R–50R.
chains, this attraction occurs at a protease concentration of about [4] K.L. Prime, G.M. Whitesides, Science 252 (1991) 1164–1167.
300 nM. In the case of EG-terminated surface, such a coverage [5] K.L. Prime, G.M. Whitesides, J. Am. Chem. Soc. 115 (1993) 10714–10721.
was not reached. From linear extrapolation of the experimen- [6] G.B. Mrksich, G.M. Sigal, Whitesides, Langmuir 11 (1995) 4383–4385.
tal dependencies, one can expect to obtain this coverage value [7] K. Feldman, G. Haehner, N.D. Spencer, P. Harder, M. Grunze, J. Am. Chem.
Soc. 121 (1999) 10134–10141.
and the attraction of adsorbate molecules only at concentrations
[8] R.G. Chapman, E. Ostuni, L. Yan, G.M. Whitesides, Langmuir 16 (2000)
higher than 2 ␮M. 6927–6936.
[9] J. Benesch, S. Svedhem, S.C.T. Svensson, R. Valiokas, B. Liedberg, P.
4. Conclusions Tengvall, J. Biomater. Sci. Polym. Ed. 12 (2001) 581–597.
[10] E. Ostuni, R.G. Chapman, M.N. Liang, G. Meluleni, G. Pier, D.E. Ingber,
The present work shows that the reversible adsorption of G.M. Whitesides, Langmuir 17 (2001) 6336–6343.
[11] E. Ostuni, R.G. Chapman, R.E. Holmlin, S. Takayama, G.M. Whitesides,
HIV protease onto various surfaces that are usually consid- Langmuir 17 (2001) 5605–5620.
ered to be protein-resistant differs substantially. Quantitative [12] S. Herrwerth, W. Eck, S. Reinhardt, M. Grunze, J. Am. Chem. Soc. 125
SPR measurements revealed linear adsorption isotherms for (2003) 9359–9366.
oligo(ethylene glycol)-terminated surfaces, which contrasted [13] L. Li, S. Chen, J. Zheng, B.D. Ratner, S. Jiang, J. Phys. Chem. B 109 (2005)
with strongly cooperative adsorption on OH-terminated alkyl 2934–2941.
[14] V. Silin, H. Weetall, D.J. Vanderah, J. Colloid Interface Sci. 185 (1997)
chains. An anomalous mutual attraction between adsorbate 94–103.
molecules indicating the possibility of two-dimensional crys- [15] B. Menz, R. Knerr, A. Goepferich, C. Steinem, Biomaterials 26 (2005)
tallization of HIV protease was observed. 4237–4243.
Author's personal copy

P. Cı́gler et al. / Colloids and Surfaces B: Biointerfaces 64 (2008) 145–149 149

[16] A. Wlodawer, M. Miller, M. Jaskolski, B.K. Sathyanarayana, E. Baldwin, [26] M.D. Hamalainen, P.O. Markgren, W. Schaal, A. Karlen, B. Classon, L.
I.T. Weber, L.M. Selk, L. Clawson, J. Schneider, S.B. Kent, Science 245 Vrang, B. Samuelsson, A. Hallberg, U.H. Danielson, J. Biomol. Screen. 5
(1989) 616–621. (2000) 353–359.
[17] S. Gulnik, J.W. Erickson, D. Xie, Vitam. Horm. 58 (2000) 213–256. [27] P.O. Markgren, M. Hamalainen, U.H. Danielson, Anal. Biochem. 279
[18] J. Prejdova, M. Soucek, J. Konvalinka, Curr. Drug Targets Infect. Disord. (2000) 71–78.
4 (2004) 137–152. [28] P.O. Markgren, M. Hamalainen, U.H. Danielson, Anal. Biochem. 265
[19] C.F. Shuman, L. Vrang, U.H. Danielson, J. Med. Chem. 47 (2004) (1998) 340–350.
5953–5961. [29] M. Riepl, K. Enander, B. Liedberg, M. Schaeferling, M. Kruschina, F.
[20] C.F. Shuman, M.D. Haemaelaeinen, U.H. Danielson, J. Mol. Recognit. 17 Ortigao, Langmuir 18 (2002) 7016–7023.
(2004) 106–119. [30] J. Weber, J.R. Mesters, M. Lepsik, J. Prejdova, M. Svec, J. Sponarova,
[21] T. Gossas, U.H. Danielson, J. Mol. Recognit. 16 (2003) 203–212. P. Mlcochova, K. Skalicka, K. Strisovsky, T. Uhlikova, M. Soucek, L.
[22] C.F. Shuman, P.O. Markgren, M. Hamalainen, U.H. Danielson, Antiviral Machala, M. Stankova, J. Vondrasek, T. Klimkait, H.G. Kraeusslich, R.
Res. 58 (2003) 235–242. Hilgenfeld, J. Konvalinka, J. Mol. Biol. 324 (2002) 739–754.
[23] P.O. Markgren, W. Schaal, M. Haemaelaeinen, A. Karlen, A. Hallberg, B. [31] M. Ingr, T. Uhlikova, K. Strisovsky, E. Majerova, J. Konvalinka, Protein
Samuelsson, U.H. Danielson, J. Med. Chem. 45 (2002) 5430–5439. Sci. 12 (2003) 2173–2182.
[24] M. Alterman, H. Sjobom, P. Safsten, P.O. Markgren, U.H. Danielson, M. [32] L.S. Jung, C.T. Campbell, T.M. Chinowsky, M.N. Mar, S.S. Yee, Langmuir
Hamalainen, S. Lofas, J. Hulten, B. Classon, B. Samuelsson, A. Hallberg, 14 (1998) 5636–5648.
Eur. J. Pharm. Sci. 13 (2001) 203–212. [33] J. Brynda, P. Rezacova, M. Fabry, M. Horejsi, R. Stouracova, M. Soucek, M.
[25] P.O. Markgren, M.T. Lindgren, K. Gertow, R. Karlsson, M. Hamalainen, Hradilek, J. Konvalinka, J. Sedlacek, Acta Crystallogr. D Biol. Crystallogr.
U.H. Danielson, Anal. Biochem. 291 (2001) 207–218. 60 (11) (2004) 1943–1948.