Self-assembled monolayer of a pepstatin fragment as a sensing element for aspartyl proteases

Article abstract of DOI:10.1021/ac0488558

A novel disulfide, which carried two pepstatin fragments at both ends, was prepared by the coupling of 11,11′-dithiobisundecanoic acid (DTUA) with a fragment (Val-Val-Sta) carrying a n-hexyl end (Pepsta(h)). The compound obtained (DTUA-Pepsta(h)) formed a

Full text of DOI:10.1021/ac0488558

Anal. Chem. 2005, 77, 1588-1595
Self-Assembled Monolayer of a Pepstatin Fragment
as a Sensing Element for Aspartyl Proteases
Hiromi Kitano,* Yoshinobu Makino, Hideaki Kawasaki, and Yusuke Sumi
Department of Chemical and Biochemical Engineering, Toyama University, Toyama 930-8555, Japan
composed of natural lipids or synthetic lipid analogues have very
often been used. We have been investigating molecular recogni-
tion processes of proteins and sugar derivatives on the surface of
model cell membranes. For example, amphiphiles with many
pendent sugar groups were prepared, and recognition of the sugar
residues in the amphiphiles incorporated in liposomes by lectins
and enzymes was examined.7-13
Alkyl or aromatic disulfides and thiols form a close-packed
ordered monolayer, “self-assembled monolayer” (SAM), on gold
or silver surfaces via chemisorptive S-Au or S-Ag bonds.14-21
Di-n-alkyl sulfides form SAM on metal surfaces, too, though the
stability of the SAM is not sufficient.22,23 Because of their structural
analogy to biomembranes, ease of preparation, and apparent
stability, SAMs have extensively been studied as novel cell
membrane mimetics in recent years.24-31 For example, SAMs of
A novel disulfide, which carried two pepstatin fragments
at both ends, was prepared by the coupling of 11,11′-
dithiobisundecanoic acid (DTUA) with a fragment (Val-
Val-Sta) carrying a n-hexyl end (Pepsta(h)). The com-
pound obtained (DTUA-Pepsta(h)) formed a self-assembled
monolayer (SAM) on a gold electrode and vacuum-
evaporated gold thin film as proven by cyclic voltammetry
and reflection absorption infrared spectroscopy, respec-
tively. When the SAM-modified gold electrode was incu-
bated with a solution of aspartyl protease, pepsin, a
decrease in both anodic and cathodic peak currents and
an increase in potential difference were observed in the
cyclic voltamogram of hydroquinone as a probe, whereas
a coexistence of free pepstatin fragment inhibited these
phenomena, indicating the specific binding of pepsin to
the fragment at the exterior of the SAM. The binding rate
of the enzyme to the SAM was largely dependent on the
surface density of the fragment moiety in the SAM.
Furthermore, when the SAM of DTUA-Pepsta(h) on a gold
colloid array deposited on an amino group-modified glass
plate was immersed in a pepsin solution, absorption of
the glass plate at 550 nm corresponding to a localized
surface plasmon resonance of the gold colloid abruptly
increased and slightly red-shifted, and a further addition
of pepstatin A gradually decreased the absorbance. From
the increasing and decreasing profiles of absorbance, the
association constant (Kassoc) for pepsin with the fragment
on the SAM was determined. Similar phenomena were
observed upon immersion of the fragment-modified SAM
in a solution of HIV-1 protease, suggesting a usability of
the pepstatin fragment SAM for the detection and removal
of the enzyme from biological fluids.
(4) Ringsdorf, H.; Schlarb, B.; Venzmer, J. Angew. Chem., Int. Ed. Engl. 1988,
27, 114-158.
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1995, 170, 18-24.
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361-366.
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Interface Sci. 1999, 210, 290-295.
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T. Bioconjugate Chem. 1999, 10, 354-360.
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Many kinds of proteins and peptides participate in many
biological processes including specific recognition and response
to external signals (i.e., functional mediator of information) on
membrane surfaces.1,2 To investigate diverse interactions on
membrane surfaces, simple biomembrane mimetic systems such
as monolayers,3 liposomes,4-6 and Langmuir-Blodgett films
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* To
whom
correspondence
should
be
addressed.
E-mail:
kitano@eng.toyama-u.ac.jp.
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S. J. Biol. Chem. 1989, 264, 9476-9484.
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165-170.
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1588 Analytical Chemistry, Vol. 77, No. 6, March 15, 2005
10.1021/ac0488558 CCC: $30.25 © 2005 American Chemical Society
Published on Web 02/09/2005

ω-mercaptoalkanoic acid32 and phenylboronic acid,33 and that of
a polymer chain with many pendent glucose residues,34,35 were
constructed on silver colloids or gold electrode to analyze the
interaction between proteins and biomembranes. Furthermore,
thiolated cyclodextrin (CD) derivatives were fixed as a sensing
device due to the molecular recognition properties of CD.36-41
Studies on SAM of substrates for enzymes have also been
reported. However, it is very difficult for an enzyme to pursue
catalysis to a substrate at an exterior surface of the SAM. To
realize the specific adsorption of various substances including
enzymes to the SAM-modified surface, it is important to construct
the SAM with optimum performances; that is, it resists against
nonspecific adsorption of the target molecule and affords a suitable
environment for functional fragments (surface density, distance
from the solid surface, etc).42,43 In contrast with the substrate-
carrying SAM, only a few reports concerning SAM of enzyme in-
hibitors have been made despite their importance in basic and
practical research fields.44,45 The enzyme inhibition has very often
been examined in solution phase for determining concentrations
of the inhibitors themselves, whereas it has not been adopted for
determining concentrations of the corresponding enzymes so
often.
Meanwhile, cyclic voltammetry (CV) is an electrochemical
technique that monitors processes occurring at the interface of
the electrode. The CV method involves measurement of current
flow as a function of applied potential and gives a great deal of
information about the redox activity of a compound and the
stability and accessibility of its reduced and oxidized forms.46
Using this technique, furthermore, it is possible to obtain valuable
information about a microenvironment near the electrode surface.
For that purpose, an electrochemically active compound such as
ferricyanide ion33-35,38 and hydroquinone40,41 can be used as a
probe. The fixation of an azurin (blue copper protein from
Pseudomonas aeruginosa) to the gold electrode, for example,
resulted in a strongly asymmetric CV profile of ferricyanide ion
with a markedly smaller cathodic current and a large peak
separation due to effective blocking of the faradaic process.
Recently a label-free localized surface plasmon resonance
absorption spectroscopy (abbreviated as LSPR-AS) has drawn
attention.47-51 When a monolayer of gold colloid deposited on a
glass plate was incubated with thiolated compound, absorption
around 525 nm corresponding to a localized surface plasmon
resonance of the gold colloid increased and red-shifted. An
attachment of macromolecules to the SAM induced further
increase in absorbance and red-shift. Using this technique, a
binding process of streptavidin to a biotin-carrying SAM could
easily be followed in situ.47
Aspartyl proteases including pepsin, chymosin, and HIV-1
protease are characterized by the presence of two active site
aspartate residues whose carboxylate side chains are involved in
catalysis. This family of enzymes is characterized by their
inhibition with a low level of pepstatin (Pepstatin A, Iva-Val-Val-
Sta-Ala-Sta) from Streptomyces sp.52 The unit of statine (4-amino-
3-hydroxy-6-methylheptanoic acid) in the pepstatin molecule
resembles the tetrahedral intermediate in the hydrolysis of
peptides, which results in the effective inhibition of catalyses by
aspartyl proteases.53
In this report, a SAM carrying a fragment of pepstatin was
constructed on a gold surface, and recognition of the fragment at
the SAM surface by proteolytic enzymes, pepsin and HIV-1
protease, was studied using both the LSPR-AS technique and the
CV method with hydroquinone as a probe. The effect of the sur-
face density of the inhibitor moiety in the SAM on the recognition
by the enzyme was investigated. In addition, the effect of chain
length at the end of SAM on the formation of enzyme-inhibitor
complex was also examined. Furthermore, using the LSPR-AS
technique, we could construct a sensor chip to follow association
and dissociation processes of aspartyl proteases. This chip would
be useful for a highly sensitive detection of the aspartyl proteases.
EXPERIMENTAL SECTION
Material. Pepsin (porcine pancreas, three times recrystallized,
3000 units/mg), trypsin (porcine pancreas, three times recrystal-
lized, 5600 units/mg), peroxidase (horseradish (HRP), 100 units/
mg), and 2-hydroxyethyl disulfide (2-HEDS) were purchased from
Wako Pure Chemicals (Osaka, Japan). Pepstatin A was purchased
from Peptide Institute, Inc. (Osaka, Japan). HIV-1 protease
(recombinant, expressed in Escherichia coli) was from Sigma (St.
Louis, MO). 11,11′-Dithiobisundecanoic acid (DTUA) was pre-
pared from 11-bromoundecanoic acid as previously reported.54
Synthetic procedures of a conjugate of DTUA with a pepstatin
fragment (Val-Val-Sta) carrying a n-hexyl end (DTUA-Pepsta(h),
Scheme 1) and that carrying an ethyl end (DTUA-Pepsta(e))55-57
are described in Supporting Information. Other reagents were
commercially available. A Milli-Q grade water was used for
preparation of sample solutions.
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Analytical Chemistry, Vol. 77, No. 6, March 15, 2005 1589

Scheme 1. Synthesis of DTUA-Pepsta(h)
Scheme 2. Construction of the Sensor Chip
Japan) and function generator (HA-104, Hokuto-Denko). Outputs
of the potentiostat were converted by an A/D converter and
collected by a microcomputer (PC-486 SE, Epson, Suwa, Japan).
Data analyses were performed with an in-house program. Gold
and Pt electrodes and a KCl-saturated calomel electrode were used
as working, counter, and reference electrodes, respectively. An
electrochemical cell was thermostated at 25 °C by a circulating
water bath (RM6, Lauda, Postfach, Germany).
The gold electrode (area, 0.023 cm2) was incubated with
various compositions of DTUA-Pepsta(h) and 2-hydroxyethyl
disulfide mixture (total concentration, 1 mM; solvent, chloroform)
for 24 h at room temperature and rinsed with chloroform,
methanol, and Milli-Q grade water several times before the
voltammetric measurement. The electroactive probe used was 1
mM hydroquinone in a 0.1 M Na2SO4-10 mM phosphate buffer
(pH 7.0).40,41 The scan rate was 10 mV/s.
The electrochemical reductive desorption of the SAM from the
electrode was performed in a 0.5 M KOH solution by scanning
from 0 to -1.2 V at a scan rate of 100 mV/s. The corresponding
process is represented by the following equation:
Absorption Measurements. (a) Enzyme Reaction.55,56,58,59
A procedure of kinetic measurements is described in Supporting
Information in detail.
(b) Localized Surface Plasmon Resonance Absorption
Spectroscopy.47 Gold colloid was prepared by the conventional
procedure using HAuCl4 and citric acid.33 The hydrodynamic
diameter of the colloid was determined to be 19 nm on average
using a dynamic light scattering technique (He-Ne laser at 632.8
nm, DLS-7000, Ohtsuka Electronics, Hirakata, Japan).
Glass plate (20 × 60 × 0.15 mm, Matsunami, Osaka, Japan)
was rinsed with nitric acid and water. The glass plate was
incubated with (3-aminopropyl)triethoxysilane (10 (v/v)% in etha-
nol) for 1 h at room temperature. The glass plate modified with
aminopropyl groups was further incubated with the gold colloid,
washed several times with water, and finally dried at 65 °C. The
pink-colored glass plate was incubated with various compositions
of DTUA-Pepsta(h) and 2-hydroxyethyl disulfide mixture (total
concentration, 1 mM; solvent, chloroform) for 24 h at room
temperature and washed with chloroform, methanol, and water
several times (Scheme 2).
R-S-Au + e- ) Au + R-S-
(1)
Contact Angle Measurements. A method for contact angle
measurements by sessile drop method was described in Support-
ing Information.
RESULTS AND DISCUSSION
Inhibitory Effect of Pepstatin Fragment. At first, an inhibi-
tory effect of the pepstatin fragment on the catalysis by pepsin
was examined. The addition of pepstatin fragment inhibited the
pepsin-catalyzed hydrolysis of peroxidase, and consequently, the
rate of absorbance change at 492 nm corresponding to the product
of peroxidase-catalyzed reaction, quinoneimine dye, increased
(indicated in Supporting Information).
Table 1 shows the concentration of 50% inhibition (IC50) and
inhibition constant (Ki) for two kinds of pepstatin fragments. The
Ki value for Boc-Pepsta(h) was in good agreement with the
literature value, showing the validity of experimental procedures
adopted in this work. In addition, the side chain of the C-terminus
represents a very effective part of the inhibitors, as described by
Kratzel et al.55,59 In general, it was said that the hydrophobic
A solution of pepsin was incubated with the fragment-modified
glass plate in a quartz cell, and the absorbance change around
550 nm was observed using a spectrophotometer (Lambda 19 UV/
VIS/NIR spectrometer, Perkin-Elmer).
Electrochemical Measurements. CV measurements were
performed with a potentiostat (HA-301, Hokuto-Denko, Tokyo,
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1590 Analytical Chemistry, Vol. 77, No. 6, March 15, 2005

Table 1. IC50 and Ki Values of Pepstatin Fragments for
Pepsin
Table 2. Γ Values for Various SAMs
disulfide
Γ (nm2)
inhibitor
IC50 (µM)
Ki (µM)
DTUA-Pepsta(h)
DTUA-Pepsta(h):2-HEDS ) 9:1
2-HEDS
0.90
0.4 ( 0.3a
1.7 ( 0.5
0.82
Boc-Pepsta(h)
Boc-Pepsta(e)
2.6 ( 0.3
3.8 ( 0.5
0.56 (0.59)a
1.50
DTUA
a Literature value, (7.6 ( 3.6) × 10-7 M.56
a Reference 66.
environment surrounding the statine unit does a potent role for
inhibitors of aspartyl proteases, which was consistent with the
results obtained in this work.
Cyclic Voltammetry of DTUA-Pepsta(h). Next, a character-
ization of SAM formed on a gold electrode was carried out using
cyclic voltammetry with hydroquinone (HQ) as a probe. The
oxidation of HQ gives p-benzoquinone via a two-step mechanism
shown below, and HQ is used in electrochemical analysis in the
vicinity of the electrode surface.
2-HEDS (9:1) and that only with 2-HEDS were 411 and 347 mV,
respectively. The resistance of the mixed SAM for HQ to give an
electrode reaction was slightly larger than that of pure 2-HEDS
SAM.
The gold electrode, which had been incubated with a solution
of DTUA-Pepsta(h) beforehand, showed a peak at ∼ -0.9 V by
scanning from 0 to -1.2 V in a 0.5 M KOH solution (see
Supporting Information Figure S-2). This peak has been attributed
to the reductive desorption of thiolated compounds, which are
chemisorbed to Au (eq 1),60,61 which supports the fact that DTUA-
Pepsta(h) was sorbed onto the gold electrode with the cleavage
of the S-S bond and following Au-S bond formation. Using the
peak area of reductive desorption curve, the molecular occupation
area (abbreviated as “Γ” hereafter) for various SAMs formed on
the electrode surface was determined (Table 2).
The table shows that the addition of 2-HEDS reduced the Γ
value for the SAM (DTUA-Pepsta(h):2-HEDS ) 9:1) in comparison
with that for the pure DTUA-Pepsta(h) SAM, indicating that, by
the coexistence of a disulfide of shorter molecular length, the
density of SAM on the electrode surface was increased. The
previous studies on alkanethiolates chemisorbed to Au (111)
surfaces showed that the symmetry of sulfur atoms is hexagonal
with an S-S spacing of 0.50 nm, and the calculated area per
molecule is 0.20 nm2,62-64 which is much smaller than the Γ value
for DTUA-Pepsta(h)/2-HEDS mixed SAM (9:1) in the table (0.82
nm2).
HQH2 T HQ2- + 2H+
HQ2- T HQ + 2e-
(2)
(3)
The deceleration of the diffusion at solution-electrode inter-
faces due to the adsorption of substrate brings about inhibition
of the electron transfer on a working electrode. The potential
difference (∆Ep) between the oxidation (Epa) and reduction peaks
(Epc) of the bare electrode was 98 mV. After incubation of the
electrode with DTUA-Pepsta(h) and DTUA-Pepsta(e), the ∆Ep
value between Epa and Epc values increased to 524 mV and 417
mV, respectively (Figure 1). Correspondingly, the anodic peak
When 2-HEDS was incubated solely with the gold electrode,
the packing density of the SAM was the largest, probably due to
a smaller volume of 2-HEDS moiety than that of DTUA-Pepsta-
(h) moiety. For the same reason, the Γ value for DTUA-Pepsta-
(h) SAM was larger than those for 2-HEDS SAM and DTUA-
Pepsta(h)/2-HEDS mixed SAM.
Effect of Pepsin on the Cyclic Voltamograms. Upon im-
mersion in a pepsin solution, the voltamogram of the electrode
modified with DTUA-Pepsta SAM changed somewhat (Figure
2A,B). Compared with quartz crystal microbalance measurements,
which have been used for the observation of various interfacial
reactions,65 it was previously shown that both the initial rate of
decrease in anodic peak current (d∆Ia/dt) and that of increase in
∆Ep (d∆Ep/dt) were linearly correlated with the initial rate of
increase in weight of the materials attached to the surface of
Figure 1. Cyclic voltammograms of various SAM-modified Au
electrodes: bare Au (solid line), DTUA-Pepsta(h) (dashed line), and
DTUA-Pepsta(e) (dotted line). Scan rate, 10 m/s. [HQ] ) 1 mM, in a
0.1 M Na2SO4-10 mM phosphate buffer (pH 7.0) solution at 25 °C.
current (Ia) decreased from 3.97 to 2.58 and 2.78 µA, respectively.
A quite similar tendency was observed in the decrease in cathodic
peak current (-∆Ic), and therefore, we will discuss the -∆Ia and
∆Ep values hereafter. The CV measurements at different scan rates
showed that the anodic peak current was proportional to the
square root of the scan rate (data not shown). Therefore, both
the decrease in anodic peak current (-∆Ia) and the increase in
∆Ep value indicated that the SAM formed on the electrode surface
decelerated the diffusion of the probe. Similarly, the ∆Ep values
of the electrode incubated with a mixture of DTUA-Pepsta(h) and
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Analytical Chemistry, Vol. 77, No. 6, March 15, 2005 1591

Figure 3. Effect of the mixing ratio of DTUA-Pepsta and 2-HEDS
(total concentration, 1 mM) on the saturated values of the change in
potential difference (d∆Ep sat) after incubation with pepsin (1 µM)
dissolved in 0.05 N HCl solution for 2.5 h. DTUA-Pepsta: b, n-hexyl.
O, ethyl; 2, absolute value of the decrease in anodic peak current
(∆Ia) of DTUA-Pepsta(h)/2-HEDS mixed SAM-modified Au electrode.
Scheme 3. Susceptible Structure of the Mixed
SAM of DTUA-Pepsta(h) and 2-HEDS on the Au
Surface
specific. It should be mentioned here that, by the UV-visible
absorption measurements, it was confirmed that 1 mM HQ did
not affect the catalytic activity of pepsin in the hydrolysis of HRP.
Varying the mixing ratio of DTUA-Pepsta and 2-HEDS fed at
the construction of SAM, the changes in ∆Ep and ∆Ia values 2.5
h after the incubation with pepsin solution were followed (Figure
3). The increase in potential difference (d∆Ep) and the absolute
value of the decrease in anodic peak current (∆Ia) were the largest
at the ratio of DTUA-Pepsta(h):2-HEDS ) 9:1, which might reflect
the optimum conditions both in the distance between the fragment
moieties and the flexibility of undecanoyl Pepsta(h) chain within
the SAM. Below 80% in the mixing ratio of DTUA-Pepsta(h), the
surface density of Pepsta(h) groups in the SAM might be
insufficient, resulting in the decrease in both ∆Ia and d∆Ep values.
The same tendency was observed for DTUA-Pepsta(e) SAM.
However, the d∆Ep values for DTUA-Pepsta(h) and DTUA-Pepsta-
(e) were different from each other, especially at the mixing ratio
of 9:1, probably due to the difference in the amount of pepsin
adsorbed to the exterior of SAM. As previously mentioned, the
hydrophobic environment surrounding the statine unit has a
potent role for the binding stability between pepstatin and aspartyl
proteases.
Figure 2. (A) Cyclic voltammograms of DTUA-Pepsta(h)-modified
electrode after onset of the incubation with the pepsin solution (1 µM).
(B) Effect of the concentration of pepsin on the potential difference
of DTUA-Pepsta(h)-modified Au electrode. O, [pepsin] ) 1 µM. b, 2
µM pepsin was dissolved in 0.05 N HCl. Scan rate, 10 mV/s. (C) Time
dependences of the value of the increase in potential difference of
DTUA-Pepsta(h)/2-HEDS (9:1)-modified electrode. [pepsin] ) 1 µM,
[pepstatin A] ) 15 µM dissolved in 0.05 N HCl/methanol (3:1). O,
pepsin only. b, pepsin + pepstatin A.
SAM.66 Therefore, both the d∆Ia/dt and d∆Ep/dt values can be
used as an index of the binding process at the solution-electrode
interfaces. The figure indicated that the binding of pepsin to the
SAM decelerated the diffusion of the probe at the electrode
surface.
Aspartyl proteases have a specific affinity for pepstatin.52
Therefore, the increase in ∆Ia and d∆Ep values upon the incuba-
tion with pepsin could be attributed to the binding of enzyme to
the pepstatin analogous groups in the SAM and concomitant
increase in the average thickness of the adsorbed substance,
which decelerated the diffusion of the probe, and consequently,
increased the ∆Ia and d∆Ep values (Scheme 3). The inhibitory
effect of pepstatin A on the initial increase in ∆Ep value for the
DTUA-Pepsta(h)/2-HEDS (9:1)-modified electrode (Figure 2C)
suggested that the binding of pepsin to Pepsta(h) moieties was
Comparing the spectrum obtained by using IR-reflection
absorption spectroscopy for the pure DTUA-Pepsta(h) SAM with
that for the mixed SAM of DTUA-Pepsta(h) and 2-HEDS (9:1)
(data not shown), it could be estimated that the occupation of
DTUA-Pepsta(h) in the mixed SAM was 33%. This result indicated
that it is relatively much easier for 2-HEDS to form a Au-S bond
(66) Kitano, H.; Saito, T.; Kanayama, N. J. Colloid Interface Sci. 2002, 250, 134-
141.
1592 Analytical Chemistry, Vol. 77, No. 6, March 15, 2005

Figure 5. Absorption spectra of amino group-modified glass plates.
(a) Amino group-modified glass plate. (b) After incubation with gold
colloids. (c) After incubation with DTUA-Pepsta(h).
Figure 4. Time dependence of the change in potential difference
of the DTUA-Pepsta/2-HEDS (9:1)-modified electrode after the
incubation with HIV-1 protease solution (1 µM). b, DTUA-Pepsta(h).
O, DTUA-Pepsta(e). [HQ] ) 1 mM, in a 0.1 M Na2SO4-10 mM
phosphate buffer (pH 7.0) solution at 25 °C. HIV-1 protease was
dissolved in 0.05 N HCl. Scan rate, 10 mV/s.
similar to pepsin. But the saturation occurred at much shorter
incubation time than in the case of pepsin, probably due to the
difference in molecular size of these enzymes (Mw of pepsin and
HIV-1 protease, 34 and 11 kDa, respectively). These results
definitely show that the Pepsta(h) group-carrying SAM on the gold
electrode could be recognized and bound by aspartyl proteases
such as pepsin and HIV-1 protease. Using the pepstatin fragment
SAM system, therefore, it will be possible to effectively remove
HIV-1 protease from biological fluids.
Localized Surface Plasmon Resonance Absorption Spec-
troscopy. When a dispersion of gold colloid was incubated with
amino group-modified glass plate, the glass plate changed to pink
(Figure 5b), showing that the surface of the glass was covered
with the gold colloid. The gold colloid was so strongly bound to
the glass plate that the colloid could not be desorbed even by an
incubation with 3 M NaCl solution.70 When a mixed solution of
DTUA-Pepsta(h) and 2-HEDS (solvent, chloroform) was incubated
with the gold colloid-modified glass plate, the absorption of the
glass plate further changed, indicating that the SAM of the
fragment was constructed on the gold colloid (Figure 5c). When
a pepsin solution was incubated with the SAM-modified glass plate,
moreover, the absorption around 550 nm further increased and
the position of the absorbance peak (λmax) made a red shift (Figure
6).
(in other words, SAM) than for DTUA-Pepsta(h). After incubation
of the mixed SAM with pepsin solution, a large absorption increase
around 1600-1700 cm-1, which was ascribable to amide bonds
in polypeptides, was clearly observed, supporting the fixation of
pepsin to the SAM.
The contact angle of a water droplet for the SAM of various
mixing ratios showed that wettability of the SAM was increased
with a decrease in the mixing ratio of DTUA-Pepsta(h) (Supporting
Information Table S-1). However, one should note that, since one
drop of water has a much larger size than those of the disulfide
molecules examined, the contact angle does not sensitively reflect
the hydrophilicity of disulfides, but simply reflects the average
wettability of the monolayer.
Furthermore, when the same CV experiments as Figure 3 were
carried out for comparison using trypsin, which has no affinity
for pepstatin,67,68 only a very small increase in ∆EP (50 mV in 2.5
h at DTUA-Pepsta(h):2-HEDS ) 9:1) due to a nonspecific adsorp-
tion of trypsin on the SAM was observed. Various molecules
including enzymes often adsorb to a hydrophobic interface (such
as a bare Au surface, hydrocarbon surface, etc,) irreversibly
(thermodynamically stable). However, each d∆EP value for DTUA-
Pepsta(h and e)/2-HEDS mixed (9:1) SAM-modified electrode
incubated in pepsin solution was larger than that of pure DTUA-
Pepsta(h and e) SAM-modified electrode despite the smaller
hydrophobicity of DTUA-Pepsta/2-HEDS mixed (9:1) SAM-modi-
fied surface than that of DTUA-Pepsta SAM-modified surface (by
contact angle measurements, Supporting Information Table S-1).
These results suggested that the binding of pepsin to the electrode
could be attributed to the specific binding of the enzyme to the
pepstatin fragment at the exterior of the SAM. This point will be
discussed further in the following section using SPR absorption
spectroscopy.
Figure 6 shows the gradual increase in absorbance of the glass
chip at 550 nm upon immersion in pepsin solution. By the
incubation of pepstatin A with the glass plate, the absorbance
gradually decreased indicating the desorption of pepsin. From the
relaxation times for the increase and decrease in absorbance, the
binding and desorption rate constants (kassoc and kdiss) were
evaluated using equations shown below.71
∆Absassoc ) Abst - Abso )
[Pepsin]kassoc∆Absmax {1 - exp [-([Pepsin]kassoc + kdiss)t]}
[Pepsin] kassoc + kdiss
(4)
To examine whether other kinds of aspartyl proteases could
be accumulated on the surface of SAM, HIV-1 protease was
incubated with the SAM-modified electrode. Previously, a strong
binding of pepstatin A to HIV protease was reported by Sarubbi
et al.69 As shown in Figure 4, the d∆Ep value increased in a manner
∆Absdiss ) Abst - Abstf∞ ) (Abso - Abstf∞
)
exp(-kdiss t) + Abstf∞ (5)
where Abso, Abst, and Abstf∞ are the absorbance at time 0, t, and
infinity, respectively.
(67) Kunimoto, S.; Aoyagi, T.; Morishima, H.; Takeuchi, T.; Umezawa, H. J.
Antibiot. 1972, 25, 251-255.
(68) McConnell, R. M.; Frizzell, D.; Camp, A.; Evans, A.; Jones, W.; Cagle, C. J.
(70) Morokoshi, S.; Ohhori, K.; Mizukami, K.; Kitano, H. Langmuir 2004, 20,
8897-8902.
Med. Chem. 1991, 34, 2298-2300.
(69) Sarubbi, E.; Seneci, P. F.; Angelastro, M. R.; Peet, N. P.; Denaro, M.; Islam,
K. FEBS Lett. 1993, 319, 253-256.
(71) Lookene, A.; Chevreuile, O.; Østergaard, P.; Olivecrona, G. Biochemistry
1996, 35, 12155-12163.
Analytical Chemistry, Vol. 77, No. 6, March 15, 2005 1593

Table 3. Kassoc Values of Pepsin with Various Mixed
SAMs
DTUA-Pepsta(h)
(%)a
Kassoc
b
(×106 M-1
)
R2 c
100
90
2.8
5.8
4.7
2.8
0.90
0.94
0.94
0.94
80
70
a Mol % at the immersion with a gold colloid-modified cover glass.
b 1/Ki value for Boc-Pepsta(h) fragment, 2.5 × 106 M-1
.
c Coefficient
of determination.
noticeably observed upon the same treatment (data not shown).
At first we considered that the incomplete desorption in Figure
6B would be attributable to the physical adsorption of enzyme to
the alkyl end group of the SAM. To clarify this point, we evaluated
the inadequate desorption using conventional surface plasmon
resonance absorption spectroscopy (SPR-AS, continuous flow
system; see Supporting Information). The complete desorption
of adsorbed pepsin (Figure S-3) seemed to support a specific
binding of pepsin on the SAM-modified surface.42 We consider
that HIV-1 protease, another kind of aspartyl protease, also has a
similar tendency. Because of both high affinity of the pepstatin
fragment for aspartyl protease and the nonflow assay system of
CV and LSPR-AS, the complete desorption was not observed by
these methods.
Figure 6. (A) Absorbance spectra of SAM-modified gold colloid on
glass plate. (a) DTUA-Pepsta(h)-modified gold colloids. (b) after
Incubation with a solution of pepsin. (c) After incubation with a solution
of pepstatin A. (B) Time dependence of absorbance of gold colloids
on a glass plate at 550 nm. (a) Incubation of DTUA-Pepsta(h)-
modified gold colloids on a glass plate with pepsin. (b) After incubation
with pepstatin A. [pepsin] ) 1 µM dissolved in a 0.05 N HCl, [pepstatin
A] ) 3 µM in 0.05 N HCl and methanol (3:1).
The association constants (Kassoc ) kassoc/kdiss) for pepsin with
the pepstatin fragment above the SAM of various compositions
(Table 3) were comparable with the value for the free fragment
pepstatin-pepsin system and had a maximum at the mixing ratio
of 9:1 (DTUA-Pepsta(h):2-HEDS) at the construction of SAM,
which was quite similar to the ∆Ep value at the CV measurements
(Figure 3).
As indicated in Figure 6, the immersion of the sensor chip,
which had been in contact with the pepsin solution beforehand,
in the pepstatin A solution decreased the absorbance. In the case
of bare chips, on the contrary, no absorbance decrease was
The addition of trypsin did not induce such absorption changes,
whereas the incubation of HIV-1 protease induced the similar
absorption increase and decrease due to the specific binding giving
the Kassoc value of 1.8 × 105 M-1
.
Finally, the detectability of the sensor chip modified with
DTUA-Pepsta(h)/2-HEDS (9:1) SAM was estimated using various
concentrations of pepsin. Figure 7 shows that the maximum
amount of pepsin bound to the surface in the steady state is
directly related to the solution concentration, and an absorbance
change at 550 nm could only be observed when the solution
concentration was above 8 nM.
Figure 7. Time dependence of absorbance of DTUA-Pepsta(h)/2-HEDS (9:1) SAM-modified sensor chip at 550 nm after immersion in aspartyl
protease solution, and absorbance change in 120 min after the immersion as a function of the protease concentration. Upper (a-g) [pepsin] )
1.0 × 10-6, 1.0 × 10-7, 1.0 × 10-8, 8.0 × 10-9, 6.0 × 10-9, 3.0 × 10-9, and 1.0 × 10-9 M. Lower, (a-f) [HIV-1 protease] ) 1.0 × 10-6, 1.0
× 10-7, 5.0 × 10-8, 3.0 × 10-8, 1.0 × 10-8, and 5.0 × 10-9 M.
1594 Analytical Chemistry, Vol. 77, No. 6, March 15, 2005

In the case of HIV-1 protease, the detection limit was 50 nM,
probably due to the much larger Ki value of pepstatin A for the
HIV-1 protease-catalyzed reaction (5-35 nM)72 than that for the
pepsin-catalyzed one (0.046 nM) in the solution phase.68 The very
small detection limit of pepsin using the DTUA-Pepsta(h) SAM-
carrying sensor chip indicates that this sensor chip has a very
high relative sensitivity.
changed with the surface density of the fragment moiety in the
SAM. HIV-1 protease, another kind of aspartyl protease, strongly
bound to the fragment-modified electrode, too. The present results
will be quite useful to further enlarge the usability of SAMs
carrying enzyme inhibitor moieties as molecular sensing devices
for enzymes.
There have been some reports concerning biological reactions
at the surface of SAM.66,73,74 However, only a few kinetic studies
have yet been made.42 The results obtained in this work will be
highly useful to develop electrochemical and spectrophotometric
biosensors to detect enzymes in solution using a rate assay.
ACKNOWLEDGMENT
This work was supported by the Grant-in-Aid for Scientific
Research (16205015) from the Japan Society for the Promotion
of Science. Presented at the 52nd Annual Meeting of the Society
of Polymer Science, Japan, in Nagoya in May 2003.
CONCLUSION
SUPPORTING INFORMATION AVAILABLE
Syntheses of pepstatin fragment-DTUA conjugates (DTUA-
Pepsta(h) and DTUA-Pepsta(e)). Data and additional descriptions
for inhibitory effect of pepstatin fragment, voltammograms of the
reductive desorption of various SAMs, contact angle measurement,
and surface plasmon resonance absorption spectroscopy. This
material is available free of charge via the Internet at http://
pubs.acs.org.
For the construction of a sensing device for aspartyl proteases,
the pepstatin fragment-carrying SAM could easily be prepared on
the metal surfaces, and the density of fragment in the SAM was
adjusted by coexistence of a disulfide of short chain length. The
cyclic voltammetry and localized surface plasmon resonance
absorption spectroscopy showed that the fragment-carrying SAM
could specifically be bound by pepsin to form a very stable
complex. The binding rate of pepsin to the fragment moiety
(72) Richards, A. D.; Roberts, R.; Dunn, B. M.; Graves, M. C.; Kay, J. FEBS Lett.
1989, 247, 113-117.
(73) Yoshida, T.; Sato, M.; Ozawa, T.; Umezawa, Y. Anal. Chem. 2000, 72, 6-11.
(74) Ratner, D. M.; Adams, E. W.; Su, J.; O’Keefe, B. R.; Mrksich, M.; Seeberger,
P. H. Chembiochem 2004, 5, 379-383.
Received for review August 4, 2004. Accepted December
16, 2004.
AC0488558
Analytical Chemistry, Vol. 77, No. 6, March 15, 2005 1595