# Monitoring of Enzymatic Proteolysis Using Self-Assembled Quantum Dot-Protein Substrate Sensors

Authors: Aaron R. Clapp; Ellen R. Goldman; H. Tetsuo Uyeda; Eddie L. Chang; Jessica L. Whitley; Igor L. Medintz
Journal: Journal of Sensors
 (2008)

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## Abstract

We have previously utilized hybrid semiconductor quantum dot- (QD-) peptide substrates for monitoring of enzymatic proteolysis. In this report, we expand on this sensing strategy to further monitor protein-protease interactions. We utilize QDs self-assembled with multiple copies of dye-labeled proteins as substrates for the sensing of protease activity. Detection of proteolysis is based on changes in the rate of fluorescence resonance energy transfer (FRET) between the QDs and the proximal dye-labeled proteins following protein digestion by added enzyme. Our study focused on two representative proteolytic enzymes: the cysteine protease papain and the serine protease endoproteinase K. Analysis of the enzymatic digestion allowed us to estimate minimal values for the enzymatic activities of each enzyme used. Mechanisms of enzymatic inhibition were also inferred from the FRET data collected in the presence of inhibitors. Potential applications of this technology include drug discovery assays and in vivo cellular monitoring of enzymatic activity.


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## Body

## 1. Introduction

Proteases have critical functions in many normal and aberrant biochemical processes that occur at the cellular and
organismal levels [1]. This includes
roles in the development of tissues and organs, regulation of wound healing,
necrosis, and apoptosis [1–4]. Proteases
also play active roles in diseases, such as stroke, cancer, and over 50 genetic
disorders [2, 5–8]. Furthermore, many
infectious microorganisms, including viruses and bacteria, use proteases as
essential virulence factors [2, 9–12]. In
combination, these properties indicate that proteases are important research
and pharmaceutical targets that require the development of improved sensitive
techniques to monitor their activity both in vivo and in vitro.Most assays designed to monitor
protease activity rely on detecting the cleavage or digestion of an appropriate
substrate, such as a peptide or a protein, recognized by the target protease [13, 14]. For signal transduction, these assays
often use changes in fluorescence resonance energy transfer (FRET) between two distinct
fluorophores attached to the opposite ends of the substrate, following
interactions with the protease [14–16]. The
fluorophores used in these assays are usually organic dyes or fluorescent
proteins. Alternatively, when the assay requires a complete intact native
protein substrate rather than a minimal peptide sequence, a combination of
other analytical techniques such as radioactive labeling, gel electrophoresis, or
mass spectral analysis are used [17, 18]. Each of these techniques has limitations, however. For example, fluorescent proteins and organic
dyes used in FRET-based detection have absorption and emission spectra that are
closely spaced (small Stoke’s shifts) which invariably leads to donor-acceptor signal
cross-talk [19]. Successful implementation of this detection scheme often requires
sophisticated instrumentation and complex data analysis.  Conventional organic fluorophores can also be
pH sensitive and photolabile.
Radioactive isotopes, on the other hand, are increasingly difficult to
work with due to safety and disposal problems, while mass spectrometry requires
complex instrumentation and time-consuming sample preparation and analysis.It has recently been demonstrated
that luminescent QDs are effective fluorophores for use in a variety of
biosensing configurations (e.g., immunoassay labels or cellular probes) to
detect specific analytes [20, 21].  Along
with high quantum yield and pronounced photostability as compared to
conventional dyes or fluorescent proteins, QDs also offer several unique
advantages as FRET donors [22, 23]. These include the ability to tune the spectral overlap with a particular
acceptor through choice of QD donor emission along with the option of exciting the
system at a wavelength far removed from the acceptor absorption peak, thus
reducing direct excitation of the acceptor. A central QD can interact with multiple acceptors within a single QD-bioconjugate,
which increases the overall FRET efficiency due to multiple energy transfer interactions
per conjugate [23, 24].  Additionally,
one excitation source can be used with almost any QD which, along with a wide choice
in emission colors, allows compatibility with almost any instrumental
configuration.  We and other groups have
applied QD-based FRET to the detection of soluble molecules, such as the
nutrient sugar maltose, the  explosive trinitrotoluene, and specific DNA
segments [25–29]. Cumulatively, these demonstrations show the potential of
QD-protein bioconjugates for improved FRET-based biosensing including
in proteolytic monitoring [19, 30].We have previously
reported that the
use of designer peptide
substrates conjugated to QDs, where control over the average number of peptides
attached per QD along with their sequence, allowed us to specifically detect the
activity of various proteases including caspase-1, thrombin, chymotrypsin, and
collagenase [31].  Proteolytic assays
were carried out under both excess enzyme and excess substrate conditions
allowing quantitative monitoring of protease activity and providing insight
into mechanisms of enzymatic inhibition. To demonstrate the potential for these bioconjugates, a number of
inhibitory compounds were tested against the QD-thrombin-specific peptide
substrate in a pharmaceutical screening assay. However, as substrates, peptides are limited due to their small size which
in turn limits their potential conformations and full protein-on-protein
interactions are in many cases more reflective of biochemical interactions and
true cellular context. In
this report, we expand this QD-based sensing strategy to include full proteins
as substrates. We
demonstrate FRET-based monitoring of protein digestion by proteolytic enzymes,
namely, proteinase K and papain, using QD-protein conjugates as substrates (see
Figure 1). The enzymatic digestion data
were analyzed in a manner that allowed us to estimate the minimal values for enzymatic
activity or velocity for each enzyme used within that assay. Mechanisms of enzymatic inhibition were also
inferred from assays performed in the presence of specific inhibitors.(a) Schematic
depiction of the QD-protein bioconjugates and the change in PL signal following
interactions with target protease.
Self-assembly of Cy3-labeled proteins on the QDs induces efficient FRET
between the QD and dye. Only one protein is shown for brevity, not to
scale.  Added protease cleaves/digests
the protein and changes the FRET signature. (b) Normalized absorption and emission spectra for Cy3-labeled MyG
(Cy3 QY = 0.20,ε≅150,000M−1cm−1, λexc=555nm, λem=570nm) and
530 nm
emitting QDs (QY≅0.20, QD-Cy3  R0≅52.5Å).
Note: the QD absorbance is not normalized.
(a)(b)


## 2. Experimental

## 2.1. QD Synthesis

CdSe-ZnS core-shell QDs employed in this study were synthesized step-wise
by reacting organometallic precursors at high temperature in coordinating solvent
mixtures of trioctyl phosphine/trioctyphophine oxide (TOP/TOPO) and amines. This synthesis scheme is detailed in previous
reports [32–36] and consistently provides nanocrystals with low polydispersity
and high quantum yields (QYs).  Water-soluble
QDs were prepared by exchanging the native TOP/TOPO surface capping ligands
with dihydrolipoic acid (DHLA) [35]. Two
QD populations with emission maxima centered at 520 nm and 530 nm were used;they both have favorable spectral overlap
with the Cy3 dye absorption (see Figure 1).


## 2.2. Protein Purification and Labeling with Dye Acceptor

Proteins used in this study include maltose
binding protein and apomyoglobin.  Maltose
binding protein (MBP, MW ~ 44 kDa) appended with a C-terminal pentahistidine
(His5)
sequence and expressing a unique cysteine at residue 95C
as well as apomyoglobin (MyG, MW ~ 17 kDa) expressing a unique cysteine at residue
116C and appended with a C-terminal His6 were prepared as described previously [37].  Both MBP95C and MyG116C were labeled with
maleimide-activated Cy3 dye (Amersham Biosciences, Piscataway, NJ); following
purification, this yielded average dye-to-protein (D/P) ratios of ~1 as
expected [25, 38].


## 2.3. Assembly and Characterization of the Dye-Labeled QD-Protein Nanosensors

We first characterize the FRET properties of
the QD-protein substrates used in this study.
The QD-MyG-Cy3 assemblies used as substrates for papain assays were
prepared by mixing 20 pmol of DHLA-capped QDs with increasing concentration of
MyG-Cy3 (increasing Cy3-to-QD ratio,n)
in 100 μL
10 mM sodium tetraborate buffer pH 8.5 and letting the solution incubate for
20–30 minutes; this corresponds to a concentration of 0.2 μM
QDs. Similarly, the QD-MBP-Cy3 nanoassemblies targeting proteinase K were
prepared in 10 mM HEPES buffer pH 8.0 (also at 0.2 μM QDs). Aliquots of these solutions were then loaded
in a microtiter plate wells and the fluorescence spectra were collected at each
dye-to-QD ratio for the two sets of assemblies.
Data on the FRET efficiency (derived from donor quenching) versus
average number of dye-to-QD ratio, n,
were used to construct standard/calibration curves, establishing a one-to-one
functional relationship between the measured FRET efficiency and the number of
labeled-proteins per QD-conjugate for each system. These standard curves were subsequently used
to convert changes in the FRET efficiency measured for conjugates interacting
with the targeted enzyme into enzymatic activity (see below).The data for FRET efficiency versusn were also analyzed within the Förster formalism to determine values
for center-to-center (QD-to-dye) separation distance r and to gain insights into the conjugate structure using the following
expression (developed for a centro-symmetric QD-protein-dye conjugate) [23]: (1)r=(n(1−En)En)1/6R0.En is the FRET efficiency and R0 designates the Förster radius corresponding to En=1=0.5 [19]. R0 is expressed as (2)R0=9.78×103[κ2nDQDI]1/6,where nD is the refractive index of the medium, QD is the
PL quantum yield of the donor in the absence of acceptor, I is the integral of the spectral overlap, and
κ2 is the dipole orientation factor. A 2/3 value
for κ2 corresponding to a
random dipole orientation was shown to be appropriate for our self-assembled
QD-protein-dye and QD-peptide-dye conjugates, as detailed in  [23]. Experimentally,
the FRET efficiency E was determined using (3)En=(FD−FDA)FD,where FD and FDA designate the fluorescence intensities of the donor
alone and the donor in the presence of acceptor(s), respectively [19].The QD-MyG-Cy3 substrates used for assaying papain activity were
prepared by mixing 20 pmol of DHLA-capped QDs (emission at 520 nm) with 60 pmol of MyG-Cy3 (ratio QD:MyG of 1:3) in sodium tetraborate buffer pH 8.5 and
let incubate for 20–30 minutes; for this initial stock solutionEn=3≈0.84.
Aliquots of these solutions were then loaded in a microtiter plate wells. Carica
papaya (Sigma, MW ~ 23 kDa, EC 3.4.22.2, with specific activity of 3.3
units/mg powder) dissolved in borate buffer at the desired concentrations was
added to each well (to a total of 100 μL
of reaction solution) and allowed to react with the QD conjugates for 7–10 minutes
at room temperature.  The final substrate
concentration was 0.2 μM
QDs (and 0.6 μM
MyG-Cy3).  The PL emission spectra were then
collected at each enzyme concentration.
For the papain inhibition assay, three types of inhibitors were added to
the appropriate wells at the indicated concentration before fluorescence data
collection: α-iodoacetamide (AIA, Sigma-Aldrich, St.
Louis, Miss), a known noncompetitive inhibitor of proteases, was added at 10 mM
concentration and functioned as a positive control; Pepstatin A (Sigma), a
peptidyl inhibitor of acid proteases and known to have no specificity for
cysteine proteases such as papain, was used at the recommended concentration of
1 μM
(negative control); and leupeptin (Sigma),
a peptidyl inhibitor of cysteine protease known to function as a competitive inhibitor
was tested at 100 μM and 300 μM concentrations.For substrates used in assaying the activity of proteinase K,
a stock solution of QD-MBP-Cy3 conjugates was prepared at a ration=5 corresponding to an initial FRET
efficiency En=5=0.6;
aliquots of this solution were loaded into the wells of a microtiter
plate.  Proteinase K (Pro-K, MW ~ 27 kDa,
EC 3.4.21.64, specific activity of 30 units/mg protein, New England Biolabs,
Beverly, Mass) diluted in 10 mM HEPES buffer at pH 8.0 was added to the wells at
several concentrations (to a total of 100 μL
of reaction solution) and allowed to
react for 7–10 minutes at room temperature before fluorescence spectra collection
and analysis. Final QD-MBP (QD-substrate) concentration
in each well was 0.2 μM
of QDs. For the Pro-K inhibition assay, 426 μM
kanamycin (Sigma, St Louis, Miss) was added to the appropriate
wells along with the enzyme followed by PL data collection, at the same QD-MBP
(QD-substrate) concentration. All assays were performed at least in
triplicate with standard deviations shown where appropriate.Fluorescence spectra were collected on a Tecan Safire Dual
Monochromator Multifunction Microtiter Plate Reader (Tecan US, Research Triangle Park, NC). Samples were excited at 300 nm and either
full PL emission spectra or partial integration from spectral windows (width ~2.5
nm) at the peak emission of QD and
dye was carried out.


## 2.4. Enzymatic Data Analysis

Changes in the FRET efficiencies collected for the series of enzyme concentrations were
then compared to the calibration curves to derive an estimate for the ratio of
intact proteins per QD-conjugate left in the solution following the reaction.  By comparing to the fluorescence data of the
initial stock solution, a measure for the amount (concentration) of digested substrate
is determined.  Enzymatic activity rates
or “velocities” were then deduced by converting the amount of digested proteins
[P] to a concentration of digested substrate per unit time (d[P]/dt).  In this study, we define proteolytic (or enzymatic)
velocity in units of molar protein cleaved per minute. The proteases utilized
in this study recognize and cleave many sequences in each of the protein
substrates, however, it is only the digestion of the specific dye-labeled
segment/region of a protein substrate that is the key transduction event.
Although we cannot observe many of the “silent” cleavage events, by treating
the observed FRET changes from corresponding digestion as a single event and
correlating it to molar protein units, we are able to quantitatively estimate a
minimal value for enzymatic activity in these assay formats. In essence, we are
estimating the lower boundary of enzymatic velocity as actual velocity will
always be at least this value or higher. Further, we use the terms maximum
velocity orVmax and
apparent maximum velocity or Vmaxapp to
describe the highest measured activity of an enzyme in the absence or presence
of an inhibitor in an assay. Within a particular assay, fitting of the activity
data curve allows us to derive the asymptote, providing an estimate of the
enzymatic turnover rate or velocity. Again these values represent a minimal or lower
boundary. A broad range of enzyme concentrations was explored.  Starting from a concentration where a near
complete substrate digestion is expected to occur within an allotted reaction
time of 20 minutes (stock solution) [39], a serial dilution over several orders
was prepared and subsequently used in the digestion experiments.  By limiting the digestion experiments to
concentrations far enough below that of the stock solution and using a reaction
time of ~7–10 minutes, we ensured that initial rates were always measured
within the experimental conditions.


## 3. Results and Discussion

As with the previous QD-peptide substrates, the QD-protein
conjugation method used for this study is based on metal-affinity-driven
self-assembly.  This process is initiated
by interactions between the terminal polyhistidine tract appended at the C- or
N-terminus of the protein and the metallic surface of DHLA-capped CdSe-ZnS QDs.  In a recent study, we have demonstrated that
such conjugation is not necessarily driven by electrostatic attractions, since
it can be implemented using QDs capped with polyethylene glycol- (PEG-) terminated
DHLA ligands (neutral DHLA-PEG ligands), provided that the polyhistidine sequence
itself is well exposed and available; this permits the His-tract to interdigitate
with the DHLA-PEG ligands and directly access the ZnS surface of the
nanocrystals for strong binding [40].  Further,
our study showed that these interactions are stable with an anticipated dissociation
constant of ~1–10 nM.  Metal-affinity-driven
self-assembly provides stable and functional conjugates within 10–15 minutes
of reagent mixing [31].  This conjugation
method also offers a unique advantage by allowing control over the average number
of attached protein/peptides per QD and potentially the orientation of proteins
or peptides within the QD-bioconjugates [23, 41–43].


## 3.1. QD-Protein Assemblies and FRET Efficiency

Figure2(a) shows the composite PL spectra collected
from solutions of QD-MyG-Cy3 (520 nm QDs) conjugates for increasing Cy3-to-QD
ratio, n.  Figure 2(b) shows a plot of the FRET efficiency (together
with the QD PL
loss) versus n; this constitutes the
standard curve introduced above.  The
efficiency values were calculated from the QD PL
loss (after deconvoluting the
composite spectra shown in Figure 2(a)).  Similar
data for the raw spectra, QD PL
loss and FRET efficiency were collected for the QD-MBP-Cy3 (530 nm QDs) assemblies
(data not shown).  The efficiency data were
further analyzed using (1) and
(2) to
extract estimates for the center-to-center separation distance, r, for the two sets of QD-protein assemblies.  Due to the high FRET efficiencies measured
for these systems, heterogeneity in the conjugate valence was accounted for when
fitting the data to the Förster formalism using Poisson distributions as
detailed in  [41].Center-to-center QD-dye separation distances determined from the
520 nm QD-MyG-Cy3 (R0≅51 Å) and 530 nm QD-MBP-Cy3 (R0≅52.5 Å)
assemblies were of 51 ± 2 Å and 65 ± 3 Å, respectively. The difference in
distances derived above is primarily due to differences in the protein
dimensions and labeling site, since 520 nm and 530 nm,
QDs have hard radii that
differ by only ~2–4 Å [22, 23]. These
values are consistent with the protein dimensions, as anticipated from their
respective molecular weights, the crystallographic data, and the specific dye-labeled
residues in the protein sequences [44, 45].
The value measured
for the QD-MBP-Cy3 assemblies is in agreement with previous distances measured for
dye-labeling of this particular residue (95C) [23, 43].  In contrast, MyG is a relatively small
protein (MW ~ 17 kDa compared to MW ~ 44 kDa for MBP), which results in a shorter
separation distance and produces higher FRET efficiencies even at small
dye-to-QD ratio; this was reflected by ~50% loss in QD PL at n=1 (r~R0 for this QD-dye pair).  We should emphasize that
calibration curves using either FRET efficiency versus n, or dye-to-QD PL ratios versus n, can be used to obtain information on
the enzyme kinetic parameters and inhibition.
Both forms provide similar information on the enzyme kinetics, as we
have recently shown for QD-peptide substrates [31].(a) Composite PL spectra showing 520 nm QDs
self-assembled with an increasing number of MyG116C-Cy3 per QD-conjugate. (b) FRET
efficiency versus MyG-Cy3-to-QD ration, derived from the deconvoluted integrated QD PL
intensity.  Inset shows the corresponding PL intensity at
520 nm versus
n. Similar data were collected for the 530 nm
QD-MBP-Cy3 assemblies.
(a)(b)


## 3.2. QD-Based Papain and Proteinase-K Sensing

In this study, we focused on two representative proteases, papain and proteinase-K.  Papain is a prototypical cysteine protease
that cleaves the peptide bonds of basic amino acids, leucine and glycine, as
well as hydrolyzes esters and amides.  It is commonly used for extracting fragments from whole antibodies, dissociating
cells from surfaces or tissues, and as a meat tenderizer [46].  Proteinase K (endopeptidase K or Pro-K)
is a
non-specific serine protease isolated from the mold Tritirachium album limber, with a high specific activity; it
is well suited for experiments requiring short digestion times.  Pro-K belongs to the superfamily of
subtilisin proteases and can hydrolyze both native and denatured proteins.  Further, Pro-K is stable across a wide range
of pHs and temperatures and its activity is not affected by the presence of chelating
agents such as EDTA.  It has found use in
an array of biotechnological applications, including the digestion of proteins and
inactivation of nucleases (e.g., isolation of mRNA and high molecular weight
DNA) and as a tool for the purification of target nucleic acid material from contaminating proteins.  Pro-K has also been used for the production
of characteristic protein fragments that can be used in protein
structure/function studies [47, 48].  In
addition, Pro-K can inactivate other enzymes and can thus be used as a
potential inhibitor.  Papain was tested
with 520 nm QD-MyG-Cy3 and Pro-K was tested with 530 nm QD-MBP-Cy3 substrates, respectively.Addition of the protease to a solution of
QD-protein conjugates digests/cleaves the protein substrate, allowing the dye
acceptor to diffuse away from the QD surface. This systematically alters the FRET efficiency.  Monitoring changes in the rate of FRET as a
function of enzyme concentration over a given reaction time forms the basis of
our assays.  Comparing those changes
against the standard curves can provide estimates of the amount of digested
substrate per unit reaction time (i.e., velocity reported in molar
concentration of MyG or MBP cleaved per min).
In choosing the starting QD-protein solution for mixing with the enzyme,
we refer to the FRET calibration curve where selection of dye-to-QD ratio satisfies
two criteria:  (1) high initial FRET
efficiency, followed
by (2) a large change in the measured FRET efficiency upon interaction with the
target enzyme, which would result in a broad dynamic range of accessible
velocities.  As such, we chose an
ensemble ratio of 3 MyG-Cy3 per QD for assays targeting papain and 5 MBP-Cy3 per QD for
assays targeting Pro-K; this corresponds toEn≈0.6 and 0.84 for the QD-MBP-Cy3 and QD-MyG-Cy3
assemblies, respectively (see Figure 2).
Such ratios should provide large changes in the FRET efficiency upon
cleavage of the QD-bound substrate. PL emission from solutions containing equivalent
amounts of free protein-dye (accounting for direct-acceptor excitation
contribution) were collected and subtracted from the composite spectra before
data analysis (data not shown).For the inhibition of papain, we tested the
protease activity in the presence of AIA at a strong inhibitory concentration, as
well as an inhibitor known not to affect papain activity, Pepstatin A.  AIA is an alkylating reagent specific to the cysteine/histidine
residues in proteins and is commonly used as an irreversible enzyme and
protease inhibitor [49]. Pepstatin A (sequence:
Ac-Val-Val-Sta-Ala-Sta, where Ac=acetyl and Sta=3S,4S-4-amino-3-hydroxy-6-methylheptanoic
acid) is a peptidyl inhibitor of acid proteases (aspartyl peptidases),
including pepsin, renin, bovine chymosin, and protease B. It is highly
selective and known not to inhibit serine or cysteine proteases such as papain
(i.e., negative control).  We also
explored papain inhibition by leupeptin at two different concentrations.  Leupeptin is a peptidyl
(Acetyl-Leu-Leu-Arg-aldehyde) inhibitor of serine and cysteine proteases.  To investigate the inhibition of Pro-K, we
used kanamycin at 426 μM. Kanamycin is generally used as a broad-spectrum
aminoglycoside-antibiotic as it inhibits ribosomal protein translocation among
other complex inhibitory functions [50]. We should stress that in
choosing the initial concentration of a particular inhibitor, we referred to the recommended
experimental protocols specific for each enzyme-inhibitor pair provided by the
manufacturer. Therefore, the inhibitor concentration
used in each case is a priori
expected to effectively alter the activity of the target enzyme.Figure3(a) shows plots of the estimated velocity (d[P]/dt) versus
increasing concentration of papain alone (blue squares, Vmax=9 nM/min), papain in the presence of 10 mM
of AIA (red circles, Vmaxapp=3.4 nM/min), and papain in the presence of 1 μM
of pepstatin (purple triangles, Vmaxapp~9 nM/min).  The observed data and
significant decrease in maximum velocity indicates that the AIA presence directly
affects the enzymatic activity.  This
finding is consistent with AIA’s function as a nonspecific alkylating agent
which would modify both the proteins reaction site and other allosteric
residues. In comparison, essentially no significant changes were measured in
the presence of pepstatin. Figure 3(b) shows plots
for the velocity (d[P]/dt) for
the assay using QD-MBP-Cy3 substrate reacted with Pro-K alone (blue curve, Vmax=5.6 nM) or with Pro-K mixed with kanamycin
inhibitor (red curve, Vmaxapp=2.3 nM).
The values extracted for Vmax are about an order of magnitude smaller than those measured for papain, a
property attributable to the much higher specific activity of Pro-K (Pro-K
activity is 30 units/mg compared to 3.3 units/mg for papain). Addition of
kanamycin, a broad ranging antibiotic agent derived from Streptomyces kanamyceticus, to the assay solution results in a
substantial decrease in the Vmax (by a factor of ~2.5)  similar to what was observed for papain assayed
in the presence of AIA.(a) Results from assaying an increasing concentration of papain
against a constant amount of QD-MyG-Cy3 in the absence
(blue squares) and in presence (red circles) of 10 mM AIA (red
squares) or 1μM pepstatin (purple triangles) inhibitors. Changes
in FRET efficiency were converted to activity (nM MyG substrate
cleaved/min) as described in the text. (b) Velocity
versus increasing Pro-K concentration assayed against a fixed
QD-MBP-Cy3 substrate concentration in the absence (blue squares)
and presence of 426 μM of kanamycin inhibitor (red circles).
Solid lines are best fits to the data to aid the eye.
(a)(b)


## 3.3. Kinetics of Enzymatic Inhibition

The
nature of the changes in the velocity versus enzyme concentration observed upon
addition of inhibitors (shown in Figure3) can reflect the mechanism of inhibition
involved and potentially its kinetics.   Lineweaver-Burk (L-B) analysis, which uses a
plot of the inverse velocity, 1/V,
against inverse enzyme concentration, 1/[E],
is commonly employed to identify the mechanism(s) of enzymatic inhibition from
both the changes in axis intercept values and the relative positions of the inhibited
plots [51].  The Lineweaver-Burk analysis
applied to the assay results shown in Figure 3 yields similar behaviors for the
QD-MyG reacting with papain-AIA and for QD-MBP reacting with Pro-K-kanamycin
(see plots in Figure 4). Within the Lineweaver-Burk plot, the y-axis intercept
is equal to 1/Vmax and the results confirm that the inhibitors are
altering the maximum velocity (Vmaxapp≠Vmax).  The appearance of these types of changes in an
L-B plot also
suggests that a mixed noncompetitive inhibition dominates the
interactions for these systems in our assay format [52].  This confirms what is anticipated based on
the structure and function of AIA, which would alter the proteases’ binding
site and other potentially allosteric sites. Due to the complexity of
its function, the exact mechanism of Kanamycin inhibition of Pro-K is, however,
not yet fully known.(a) Lineweaver-Burk (L-B) double reciprocal plots of the data shown in Figure3 for
the assay using QD-MyG to test the inhibition-activity of papain-AIA; papain
only (blue squares) and papain plus AIA inhibitor (red circles). (b) L-B plots of the data shown in
Figure 3(b) assaying for the Pro-K-kanamycin pair; shown are Pro-K only (blue
squares) and Pro-K plus kanamycin inhibitor (red circles). Data show that the Vmax are affected by the
presence of kanamycin inhibitor.
(a)(b)


## 3.4. Leupeptin Inhibition

Leupeptin
is a known competitive inhibitor of serine and cysteine proteases and its
recommended effective range is 10–100μM for bacterial cell lysates
(Sigma-Aldrich.com).  Initial assaying of
the QD-Myg-papain system at both 100 and 300 μM resulted in modest changes in Vmax while the overall
shapes of the curves essentially remained the same which is indicative of a
competitive inhibition process (see Figure 5(a)) [52].   We
opted to confirm the inhibitory effects in an alternate format by adding
increasing concentrations of leupeptin (mixed with papain) to solutions
containing QD-MyG substrates.  For this,
we fixed the substrate concentration at 0.2 μM QDs (with 3 MyG-Cy3
per conjugate),and maintained a high concentration of
papain (15 μM, near saturation based on the above
results derived in Figure 5(a)), but progressively increased the leupeptin
concentration.  Using such high papain
concentration ensured that for a reasonably small inhibitor concentration we
measured velocities near Vmax (appropriate for a non-competitive inhibitor). The velocity data collected from
these solutions show a progressive decrease with increasing inhibitor
concentration and allow us to estimate an inhibitor concentration that
corresponds to a significant decrease in activity (>50%) of >2 mM (Figure
5(b)).(a) velocity versus papain enzyme concentration for QD-MyG assayed alone and in the
presence of 100μM and 300 μM leupeptin. (b)
Plot of the velocity versus leupeptin concentration measured using a
concentration of papain near saturation. Solid lines are best fits
to the data to aid the eye.
(a)(b)Cumulatively, these results confirm a competitive inhibitory
process in the assay and indicate that the effective inhibitory concentration
of leupeptin on papain is ~20 times higher than that suggested for cysteine
proteases of bacterial origin (as recommended by the manufacturer). However, it should be noted that although
papain is a member of the same superfamily of cysteine proteases, it most
probably has a different structure and function because it originates from the fruit
of a plant. This result also highlights
the importance of pretesting any enzyme-inhibitor combination before selecting
an effective inhibitory concentration in a targeted application, such as
extracting nucleic acids from plants or fruits as opposed to extraction from
bacteria.


## 4. Conclusions

In this report, we expanded on our previous results with
QD-peptide substrates and demonstrated the use of luminescent QDs as
nanoscaffolds for self-assembling FRET-based protein substrates to semiquantitatively
detect proteolytic enzyme activity derived from protein-protease interactions.  Experiments testing QD-protein substrates in
assays with and without added inhibitors showed that enzymatic digestion along
with mechanism of inhibition can be inferred from assay results. The current sensing assemblies
also benefit from the ability to array multiple copies of dye-labeled proteins around
a single QD which serves to both improve FRET efficiency and allow simultaneously
interrogation of multiple protein substrates in the same FRET pair by the
enzyme (and inhibitor).A few specific aspects of these sensing assemblies should
be clarified.  In contrast to our previous studies [23],
we do not use additional unlabeled proteins to the QD assemblies.  These are usually added as they help
ameliorate conjugate heterogeneity caused by Poisson distribution kinetics when
self-assembling small ratios of proteins to QDs [31, 41, 42] and to improve the
QY of the QD in the conjugate through surface passivation effects [22, 53].  The error introduced by essentially
ignoring heterogeneity and the overall changes in the QY of the conjugates is
rather small in comparison with changes in measured FRET efficiencies.  Further, the nature of the proteases and the
assay formats used only allow us to derive the minimal enzymatic activity and
not other values such as the Michaelis constant or turnover number.Both of the enzymes utilized here
can function at a wide range of pH’s and with a variety of additives present,
and the choice of slightly basic pH is dictated by the solubility of the QDs
when capped with DHLA.  Further, it is
well known that even small changes in assay format such as relative
concentration, pH or salt concentration can dramatically alter activity
kinetics [51, 52], thus it is hard to compare the assay data and activity
collected here to that published in other reports. However, it appears that, in
general, the amount of substrate utilized and minimal amount of enzyme detected
along with assay time is far less (>1 order of magnitude for substrate
concentration) than for reports using the same enzymes [54].  This strongly suggests that the QD size and
self-assembled protein nanostructure do not interfere with proteolytic
activity, at least for the enzymes tested here.
The combination of the unique QD stability and the photophysical
properties mentioned earlier along with access to higher sensitivity are all
desirable attributes in any proteolytic assay format.Finally, such QD-protein substrates may be directly
applicable in assays where monitoring of nonspecific proteolysis is important
such as during DNA preparation [47, 48].
Other dye-labeled
biological molecules such as peptides, DNA, RNA, or aptamers that are
responsive to enzymes, drugs, or other chemicals can be easily adapted to this
format and indeed a few preliminary qualitative enzymatic studies using
QD-substrates have been reported, including a QD-sensor for monitoring
lactamase activity associated with antibiotic resistance [55–58].  With the large number of putative proteases
in the human genome (~560) as well as those utilized by pathogens, many will require
intact protein substrates for recognition and cleavage/digestion [1–4]. The QD-protein conjugates described here
provide a viable architecture to detect protein-digestion activity using FRET
for signal transduction [1, 2].



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*Source: 797436-2008-07-20.xml*