Fluorometric assay for tissue transglutaminase-mediated transamidation activity

Article abstract of DOI:10.1016/j.bmc.2009.07.031

Herein, we report the development of a direct discontinuous fluorometric transamidation assay for determining tissue transglutaminase (TG2) activity. In the assay reaction, TG2 catalyzes the formation of a biotin-fluorophore conjugate, using a fluorescent, high affinity γ-glutamyl donor substrate and a biotinylated amine as a γ-glutamyl acceptor substrate. After the reaction, the conjugate is fixed on streptavidin-coated beads and excess substrate is washed away, allowing the transamidation activity to be quantified by fluorescence measurement. This method was used to detect the activity of as little as 0.6 mU of purified TG2, and can be used for detection of activity from crude cellular lysates. Furthermore, this assay can be used for screening potential inhibitors and synthetic substrates, the latter of which was demonstrated herein.

Full text of DOI:10.1016/j.bmc.2009.07.031

Bioorganic & Medicinal Chemistry 17 (2009) 6354–6359
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Fluorometric assay for tissue transglutaminase-mediated
transamidation activity
*
Claudio Gnaccarini, Wajih Ben-Tahar, William D. Lubell, Joelle N. Pelletier, Jeffrey W. Keillor
Département de chimie, Université de Montréal, C.P. 6128, Succursale centre-ville, Montréal, Québec, Canada H3C 3J7
a r t i c l e i n f o
a b s t r a c t
Article history:
Herein, we report the development of a direct discontinuous fluorometric transamidation assay for deter-
mining tissue transglutaminase (TG2) activity. In the assay reaction, TG2 catalyzes the formation of a bio-
Received 26 April 2009
Revised 12 July 2009
Accepted 15 July 2009
Available online 18 July 2009
tin-fluorophore conjugate, using
a fluorescent, high affinity c-glutamyl donor substrate and a
biotinylated amine as a -glutamyl acceptor substrate. After the reaction, the conjugate is fixed on strep-
c
tavidin-coated beads and excess substrate is washed away, allowing the transamidation activity to be
quantified by fluorescence measurement. This method was used to detect the activity of as little as
0.6 mU of purified TG2, and can be used for detection of activity from crude cellular lysates. Furthermore,
this assay can be used for screening potential inhibitors and synthetic substrates, the latter of which was
demonstrated herein.
Keywords:
Transglutaminase
Biotin
Streptavidin
Activity assay
Screening
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
mechanism and the modification of its activity.20 Standard assays
for reporting TG2 specific activity are based on hydroxamate
Transglutaminases (TGases) are a class of Ca2+-dependent en-
zymes that catalyze transamidation from the -carboxamide of a
formation21 or incorporation of radiolabeled putrescine into N,N-
dimethyl casein.22 Another assay featuring enzymatic release of
p-nitrophenol has seen application in kinetic studies.23 Our group
has reported additional assays for measuring TG2 activity by mon-
itoring the enzymatic production of a chromophoric anilide,24 or
the enzymatic release of 7-hydroxycoumarin.25 Although sensitive,
these methods use chromogenic or fluorogenic substrate analogues
that may react differently than the native substrates. Assays that
detect released ammonia from glutamine acyl-donor substrates
may provide better mechanistic insight into the native enzyme
activity.26–28 However, these assays detect product formation lead-
ing to the acyl-enzyme intermediate that can then react with water
or a primary amine. As such, they do not specifically monitor the
formation of the transamidation product. Recently, three methods
have been reported for detection of TG2-mediated isopeptide bond
formation. Two detect increased fluorescence of a dansyl group on
the acyl-acceptor substrate. In the first case, the fluorescence in-
c
peptide- or protein-bound glutamine with a primary amine, a pep-
tide- or protein-bound lysine generally serving as the amine sub-
strate. The reaction yields a crosslink between two proteins to
form an isopeptide e-(c
-glutaminyl)lysinyl bond.1–3 The transami-
dation mechanism starts with the nucleophilic attack by the thiol
group of an active-site cysteine residue on the donor substrate
c-carboxamide group, leading to loss of an equivalent of ammonia
and formation of a covalent thiolester intermediate. The acyl group
of the transient thiolester is transferred to the acceptor amine sub-
strate in the second step.3 TGases can recognize a broad range of
primary amine acyl-acceptor substrates. However, recognition of
the acyl-donor substrate in vivo is restricted to the
ide of glutamine within an apparently relatively small subset of
sequence contexts.4–6 At
physiological level, tissue TGases
(TG2) are involved in normal cellular processes, including cell
adhesion,7 formation of the extracellular matrix,8 and apoptosis.9
Diminished regulation of TG2 activity is implicated in a number
of diseases.10–13 Exploiting its stringent acyl-donor and broad
acyl-acceptor specificities, TG2 has been employed in site-specific
protein labeling,14–16 glycosylation,17,18 and PEG-ylation.19
c-carboxyam-
a
crease is due to
p-stacking with a second dansyl group on the
acyl-donor substrate after ligation.29 In the second case, dansyl
fluorescence increases after ligation to the donor substrate case-
in.30,31 In the third method, the persistent fluorescence of a solu-
tion of casein labeled in this manner is measured after removal
of excess unreacted dansyl acceptor substrate.32 Alternative activ-
ity assays33–36 have been developed that use TGase-mediated bio-
tinylation of immobilized protein and subsequent detection with
streptavidin or antibody conjugates of horseradish peroxidase or
alkaline phosphatase, whose catalytic activities amplify the signal.
Methods for monitoring TG2 activity, in purified form and in
complex solution, are fundamental for the study of its catalytic
* Corresponding author. Tel.: +1 514 343 6219; fax: +1 514 343 7586.
E-mail address: jw.keillor@umontreal.ca (J.W. Keillor).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.07.031

C. Gnaccarini et al. / Bioorg. Med. Chem. 17 (2009) 6354–6359
6355
Streptavidin-based detection of biotinylated protein products has
(Boc)2O, NEt3
X
X
H2N
NHBoc
also been applied in gel electrophoresis and dot blot assays.37,38
Although these methods are sensitive, their reliance on a protein
substrate impedes structural modifications such that these meth-
ods are only useful for characterizing native activity and screening
inhibitors, instead of studying TG2 reactivity toward small mole-
cule substrates.
H2N
NH2
dioxane:water
(9:1)
7a-c
8a-c
X= a CH2
b O(CH2)2O
c S-S
O
D-biotin, TBTU,
DIEA, DMF
Herein, we present a discontinuous assay in which TG2 cata-
lyzes the reaction between an acyl-donor substrate bearing a fluo-
rescein derivative,39 and an amine acceptor substrate bearing a
biotin group. The resulting product bearing both biotin and fluoro-
phore is then immobilized on streptavidin beads. After washing to
remove excess substrate, fluorescence is measured to ascertain
TG2 activity. Convenient for use in 96-well microtiter plates, this
assay is sensitive, allowing detection of as little as 0.6 mU of TG2
activity from a sample of purified enzyme. TG2 activity was also
detected effectively in complex biological samples. Molecules that
interfere with reaction of the test substrates were also detected.
This assay is particularly designed for screening TG2 reactivity to-
ward synthetic substrates, because the tagged substrate structures
may be readily modified.
S
X
R
N
H
N
H
9a-c
R = Boc
10a-c
NH
HN
MeOH:HCl (1M)
R = H
O
Scheme 2. Synthesis of acyl-acceptor substrates 10a–c.
pling. Dansyl chloride and fluorescein isothiocyanate were tested
as fluorophores, because they were commercially available and
readily amenable to coupling chemistry. Donor substrates 6a–c
were found to have low KM values of 28.7 6.1, 13.1 1.8, and
72.5 6.0
tion 4).
l
M, respectively, using the DMPDA method24 (see Sec-
2. Results and discussion
Biotinylated amines 10a–c were synthesized by coupling biotin
to the corresponding diamines 7a–c:1,5-diaminopentane (7a),
tris(ethylene glycol)-1,8-diamine (7b) and 2-(2-aminoethyldisulfo-
nyl)-ethylamine (7c). These diamines were selected because of
their different lengths and polarities, as well as to have a cleavable
disulfide bond. They were then tested as acceptor substrates. In the
The fluorescent acyl-donor substrates 6a–c were synthesized
from the known40 acyl-donor substrate, tetrapeptide Gln-Leu-
Pro-Phe, which was conjugated to one of three fluorophores—To-
kyo Green,39 fluorescein isothiocyanate and dansyl—by way of an
aminocaproate spacer (Scheme 1). The biotinylated acyl-acceptor
substrates 10a–c were prepared from three diamines 7a–c
(Scheme 2).
Cbz-L
-Glu(O-p-nitrophenyl)Gly assay23 biotinylated amines 10a–c
served as acceptor substrates and were found to have KM values
of 2.3 0.4, 11.3 3.7, and 1.6 0.7 mM, respectively (see Section
4).
The influence of the acceptor substrate spacer on assay sensitiv-
ity was determined by incubating acyl-donor substrate 6a and
acyl-acceptor substrates 10a–c, respectively, at final concentra-
The fluorescent acyl-donor substrates 6a–c were prepared by
solid-phase synthesis (see Supplementary data). Tokyo Green
was chosen as a fluorophore because of its high quantum yield
(U
= 0.93)39 and carboxylate suitably disposed for peptide cou-
tions of 30 lM, in the presence or absence of TG2 (Fig. 1). Acceptor
substrates 10a–c gave different levels of fluorescence that have an
inverse correlation with their KM values. Thus, the lowest and high-
O
H
N
N
O
O
O
NH
O
70000
O
FmocHN
N
H
NH2
5
+TGase
5
60000
O
Blank
1) Fmoc deprotection
2) TG-OH, EDC-HCl,
HOBt, DIEA, DMF, 6 h
3) TFA: DCM (1:1)
1) Fmoc deprotection
2) Dansyl chloride,
DIEA, DMF, 6 h
1) Fmoc deprotection
2) FITC, DIEA,
DMF, 6 h
50000
40000
30000
20000
10000
0
3) TFA: DCM (1:1)
3) TFA: DCM (1:1)
TG-AC-Gln-Leu-Pro-Phe
Dansyl-AC-Gln-Leu-Pro-Phe
6a
FTU-AC-Gln-Leu-Pro-Phe
6c
TG:
6b
O
Dansyl:
O
FTU:
S
S
O
NH
OH
O
N
6a + 10a
6a + 10b
6a + 10c
HO
O
O
Substrates
Figure 1. Comparison of assay sensitivity using acyl-donor substrate 6a and acyl-
acceptor substrates 10a–c. Error bars represent the standard deviation from the
mean of triplicate measurements.
HO
O
O
Scheme 1. Synthesis of acyl-donor substrates 6a–c.

6356
C. Gnaccarini et al. / Bioorg. Med. Chem. 17 (2009) 6354–6359
est fluorescence measurements were exhibited by the substrate
harboring the polyethylene glycol (10b) and disulfide (10c) spac-
ers, respectively, consistent with their KM values.
70000
60000
50000
40000
30000
20000
10000
0
+TGase
Blank
Acceptor substrate 10c gave the highest signal and contains a
disulfide spacer for ‘reversible’ biotinylation by reductive cleavage
of the disulfide bond. In cleaving the disulfide after conjugation,
the influence of streptavidin bead conjugation on fluorescence
could be quantified. Thus, following enzymatic coupling of the do-
nor and acceptor substrates 6a and 10c, trapping on the streptavi-
din-coated beads and washing, reduction using D,L-dithiothreitol
(DTT) and separation by centrifugation allowed measurement of
the released fluorophore in the absence of the beads (Table 1). This
demonstrated comparable fluorescence intensity (+DTT, before or
after centrifugation, and ÀDTT, before centrifugation). A low level
of fluorescence was apparently released from the beads even in
the absence of DTT (ÀDTT, after centrifugation). Nonetheless, fluo-
rescence measurement directly on the beads was not significantly
hampered and the additional steps required to release the fluoro-
phore were not deemed to be justified. As compound 10c yielded
the highest fluorescence after coupling to the donor substrate 6a
(Fig. 1), it was retained as the most suitable acceptor substrate
for further measurements.
6a + 10c
6b + 10c
6c + 10c
Substrates
To evaluate the effect of the fluorophore on assay sensitivity,
acyl-acceptor substrate 10c was incubated with purified TG2 and
acyl-donor substrates 6a–c. Substrate 6c was deemed to be insuf-
ficient as a fluorophore and was not retained for further testing
(Fig. 2). Substrates 6a and 6b showed significant signal relative
to control. The fluorescence of the blank for 6b was slightly higher
than that of 6a.
Figure 2. Comparison of assay sensitivity using acyl-donor substrates 6a–c and
acyl-acceptor substrate 10c. Error bars represent the standard deviation from the
mean of triplicate measurements.
60000
The limit of detection of the assay with respect to TG2 concen-
tration was then determined using different concentrations of
purified TG2 with acyl-donor 6b and acyl-acceptor 10c (see Section
4). As little as 0.6 mU of purified TG2 provided a signal of
(9.0 0.4) Â 103 afu. This was easily detectable above the back-
ground fluorescence of the blank of (6.7 0.5) Â 103 afu (Fig. 3).
Although higher than that of methods incorporating enzyme-cata-
lyzed signal amplication,34,35 our limit of detection is comparable
with other fluorescence-based methods.25,29,32,38
50000
40000
35000
40000
30000
25000
30000
20000
15000
10000
20000
For practical applications, the enzymatic assay was evaluated in
biological samples. In particular, acyl-donor 6b and acyl-acceptor
10c were coupled using crude lysate of Escherichia coli expressing
recombinant TG2, and compared with lysate of E. coli void of the
expression plasmid as a negative control. When analyzing TGase
activity in complex biological samples, assays that detect the prod-
uct of the enzyme acylation process may overestimate transamida-
tion activity due to competing hydrolysis of the acyl-enzyme
intermediate. Using the present assay, authentic transamidation
5000
0
0
10
20
30
10000
0
mUTGase
0
50
100
150
200
250
300
mUTGase
Figure 3. Limit of detection of the reaction of substrates 6b and 10c with respect to
concentration of purified TG2. Inset shows the linearity of the low concentration
data. Error bars represent the standard deviation from the mean of triplicate
measurements.
product was detected directly in 5 lL of crude lysate of bacteria
expressing TG2 ((48.4 5.5) Â 103 afu), relative to void lysate
((7.0 1.2) Â 103 afu) (see Section 4). In this way, transpeptidation
activity exhibited by as little as 91 mU of TG2 was detected in the
biological sample.
ferent specificity. In an alternative application requiring no
further synthesis, the reaction of substrates 6b and 10c was used
to screen for synthetic substrates in competition experiments with
six primary amines. Addition of each amine to the assay incubation
mixture resulted in varying decreases of fluorescence after capture
on streptavidin beads. Correlating changes in fluorescence in the
presence and absence of added amine allowed their relative activ-
ities to be ascertained, as shown in Figure 4.
One of the distinctive advantages of this small molecule-based
assay is its flexibility with respect to the substrate structures.
Other acyl-donor and acyl-acceptor substrates could easily be cou-
pled to a fluorophore and to biotin, providing direct assays of dif-
Table 1
Effect of cleavage of disulfide spacer of 10c on assay signal
Fluorescence (afu)a
From Figure 4 it is apparent that at 1 mM added amine, the
known substrates N-Ac-
effectively compete with 30
strates being present at concentrations 10- to 50-fold below their
KM values. Notably, the methyl ester of -Ala was also found to
be an effective substrate. At 5 mM of each amine, the activity of
L
-Lys-OMe and Gly-Gly-NH2 were able to
+DTT
ÀDTT
lM of substrate 10c, all of these sub-
Before centrifugation
Supernatant after centrifugation
6.3 Â 104
4.6 Â 104
4.2 Â 104
1.0 Â 104
L
a
kexc = 485 nm; kem = 535 nm.

C. Gnaccarini et al. / Bioorg. Med. Chem. 17 (2009) 6354–6359
6357
Potential substrate
Ac-L-Lys-OMe Gly-Gly-NH2
PABA
D-Ala-OMe
L-Ala-OMe
Phe-NH2
100%
90%
80%
70%
60%
50%
40%
30%
20%
10%
0%
1 mM
5 mM
-10%
-20%
Figure 4. Screening of potential substrates of TG2. Activity was measured relative to the control reaction of 30
lM 6b and 30
l
M 10c. Error bars represent the standard
deviation from the mean of triplicate measurements.
each of these substrates increased as expected, and the activity of
D-Ala-OMe was also detected. No significant increase of these
activities was detected at 10 mM added amine (not shown). The
activity of both alanine esters as substrates (and not as inhibitors
of the reaction of 6b and 10c) was verified using an independent
nitrile/water) at the Centre Régional de Spectrométrie de Masse de
l’Université de Montréal.
The general procedure used for Fmoc deprotection is as follows:
Wang resin (0.81 mmol/g) was treated with a mixture of 20%
piperidine in DMF, shaken for 30 min, filtered and washed as de-
scribed above. Deprotection was verified by positive Kaiser test
on a sample of a few beads.41 The level of loading of the amino acid
on the resin after the first coupling step was used as the resin load-
ing capacity for all subsequent steps, as determined by spectro-
scopic measurement of the UV absorbance of the piperidine
dibenzofulvalene adduct.
assay method24 and the KM value of
L-Ala-OMe was determined
to be 7.4 1.9 mM. This demonstrates the speed, sensitivity and
concentration-dependent stringency of this assay, for screening
for substrates or inhibitors.32
3. Conclusions
Peptide sequence purity was performed on an aliquot of resin
using LCMS-MS on a Agilent LCMSD, column (C6 Phenyl, 3H,
50 Â 4.6 Gemini, 0.5 mL/ min) in two different solvent systems:
method A: 40–80% CH3CN in H2O with 0.1% formic acid,
k = 214 nm, or B: 60–90% MeOH in H2O with 0.1% formic acid,
k = 254 nm.
In summary, a sensitive assay for the detection of tissue TGase
activity has been developed. This method can be used to detect
TGase cross-linking activity samples prepared from crude bacterial
lysate as well as purified TGase. Because both acyl-donor and acyl-
acceptor substrates are synthetic, they can easily be modified for
application to high throughput screening of libraries of mutant
TGase having potentially different substrate specificity, libraries
of potential TGase inhibitors, and libraries of potential non-native
substrates, as demonstrated herein.
For detailed procedures and characterization data, see the Sup-
plementary data.
4.2. Kinetics
4. Experimental
Kinetic parameters for all donor substrates were determined
independently using the N,N-dimethyl-1,4-phenylenediamine
(DMPDA) method,24 using a range of donor substrate concentra-
tions of 0.5 mM to 20 mM and a constant acceptor substrate con-
centration of 20 mM. Kinetic parameters for acceptor substrates
4.1. General synthesis
All solvents used for reactions were purified with solvent filtra-
tion systems (Glass Contour, Irvine, CA). 4-Carboxy-Tokyo Green
was synthesized according to a published procedure.39 Wang resin
and amino acids were purchased from Nova Biochem except for
were obtained using the chromogenic donor substrate, Cbz-L-
Glu(O-p-nitrophenyl)Gly,23 using a range of acceptor substrate
concentrations of 0.04 mM to 30 mM and a constant donor sub-
strate concentration of 0.108 mM (fivefold apparent KM). Initial
rates were measured as a function of varied substrate concentra-
tion and kinetic parameters were determined from non-linear
regression to the hyperbolic Michaelis–Menten equation. The buf-
fer solution in all assays gave a final concentration of 100 mM
TrisÁHCl (pH 8), 0.6 mM EDTA, and 10 mM CaCl2. The washing buf-
fer solution was composed of 100 mM phosphate (pH 8), 100 mM
NaCl, and 0.1% SDS. Water was purified using a Millipore BioCell
water purification system. Streptavidin beads (Pierce) were sup-
Fmoc-Gln-OH, obtained from Advanced ChemTech, and Fmoc-e-
aminocaproic acid, obtained from Bachem Chemical. All other re-
agents were obtained from Sigma–Aldrich. 1H and 13C NMR spectra
were recorded on Bruker NMR spectrometers.
Reactor tubes for solid-phase peptide synthesis were obtained
from Alltec Associates Inc. and shaking was performed on a recip-
rocating shaker (SK-300 Jeio Tech). All resins were swelled in DMF
and washing steps were performed using CH2Cl2 and DMF (EMD
Chemicals) using five DMF washes followed by three CH2Cl2
washes. Purification of all peptides was performed using a prepara-
tive HPLC method. Mass spectral data (MS, LCMS) were all
obtained using two different solvent systems (MeOH/water; aceto-
plied as a suspension capable of binding 15–28 lg biotin/mL of set-
tled resin (a capacity corresponding to ꢀ1–3 mg biotinylated BSA/
mL of resin) on a support of 6% crosslinked beaded agarose.

6358
C. Gnaccarini et al. / Bioorg. Med. Chem. 17 (2009) 6354–6359
4.3. Enzyme preparation
4.6. Assay procedure using crude bacterial lysate
Recombinant guinea pig liver TGase was over-expressed and
purified from E. coli following our previously published proce-
dure.42 The purified TG2 solution used in all assays had a specific
activity of 14 U/mg in 25 mM Tris–acetate (pH 7) according to
All assays were performed in triplicate. Donor substrate 6a
(10
6 mM stock solution) 80
were combined in a final volume of 195
ated by addition of 5 L of crude bacterial lysate expressing TG2, or
l
L of 6 mM stock solution), acceptor substrate 10c (10
L of buffer solution, and 95 L of water
L. The reaction was initi-
lL of
l
l
l
the hydroxamate activity assay,20 in which Cbz-
L
-Gln-Gly and
l
hydroxylamine are used as acyl-donor and acyl-acceptor sub-
strates respectively.
bacterial lysate void of the TG2 expression plasmid as a blank. Fol-
lowing incubation for 60 min at 25 °C, a fraction of the solution
(10
l
L) was added to a mixture of 180
lL of the washing buffer
and 10
l
L of streptavidin bead suspension. The mixture was incu-
4.4. Preparation of crude bacterial lysate
bated for 30 min at 25 °C, and centrifuged for 1 min at 1400 RPM
(microcentrifuge). The pellets were washed three times as de-
scribed above, resuspended in washing buffer (100 lL) and trans-
ferred to a polystyrene 96-well plate for analysis of enzyme
activity by measuring fluorescence relative to the blank, as de-
scribed above.
4.4.1. First day
For expression of recombinant guinea pig liver TGase, 4 mL of
Terrific Broth (TB),43 containing 100
l
g/mL ampicillin (Amp) and
30
lg/mL chloramphenicol (Chl) was inoculated with E. coli XL1-
Blue harboring the plasmids pDnaKJ (Chl-resistant) and pQE-32
TG2 (Amp-resistant). E. coli XL1-Blue harboring pDnaKJ only, inoc-
ulated in TB + Chl, was used as a control cell line not expressing
TG2. All cultures were incubated for 17 h at 37 °C with shaking
at 240 RPM in a C25 Incubator Shaker (New Brunswick Scientific
Edison, NJ. USA).
4.7. Reduction of the disulfide-bridged acyl-acceptor substrate
Substrates 6a and 10c were incubated for 60 min at 25 °C
(30
EDTA, and 350 mU TG2 in a total volume of 200
10 L of streptavidin bead suspension was added. The mixture
l
M 6a, 30
lM 10c, 100 mM Tris–HCl, 10 mM CaCl2, 0.6 mM
lL), after which
l
4.4.2. Second day
was incubated for a further 60 min. The tubes were centrifuged
Aliquots of 1.5 lL of pre-culture were used to inoculate 1.5 mL
for 1 min at 1400 Â RPM (microcentrifuge) and the supernatant
of fresh TB medium containing the appropriate antibiotics in wells
of a 48-well plate (maximum volume: 6 mL/well) at a ratio of 1/20.
The bacteria were propagated at 37 °C with shaking at 240 RPM.
When the OD (600 nm) reached approximately 0.5, over-expres-
sion of TG2 was induced by addition of IPTG to a final concentra-
tion of 1 mM. The plate was then incubated at 28 °C with
shaking at 800 RPM for 20 h in a Titer Plate Shaker (Lab-Line
Instruments, Inc.).
discarded. The resin was washed twice by adding 200 lL of wash
buffer, centrifuging for 1 min at 1400 Â RPM (microcentrifuge)
and discarding the supernatant. Finally the beads were suspended
in 100
lL of wash buffer for treatment under one of the following
conditions:
1. 100 lL of 0.1 M DTT was added, followed by 15 min incubation,
centrifugation and removal of the supernatant for fluorescence
detection.
4.4.3. Third day
2. 100
and the entire mixture was used for fluorescence detection.
3. 100 L of water was added, followed by centrifugation and
removal of the supernatant for fluorescence detection.
4. 100 L of water was added, and the entire mixture was used for
lL of 0.1 M DTT was added, followed by 15 min incubation,
The plate was centrifuged for 30 min at 4 °C and 3000 RPM
(AllegraTM 6R Centrifuge). The supernatant was discarded. The bac-
terial cell pellets were stored at À80 °C for 10 min, thawed, resus-
l
pended in 200 l
L of CelLyticTM B cell lysis reagent (Sigma) and
l
incubated at 28 °C for 20 min. The crude bacterial lysates thus ob-
tained and used for subsequent tests were determined by hydroxa-
mate test to show an activity of ꢀ18 U/mL.
fluorescence detection.
4.8. Screening procedure for synthetic substrates using purified
enzyme
4.5. Assay procedure using purified enzyme
All assays were performed in triplicate. Donor substrate 6b
All assays were performed in triplicate. Donor substrate 6a, b,
(10
of 0.6 mM stock solution), potential synthetic substrate (20
of 100, or 50 or 10 mM stock solution) 70
and 80 L of water were combined in a final volume of 190
The reaction was initiated by addition of 350 mU (10 L) of puri-
l
L of 0.6 mM stock solution), acceptor substrate 10c (10
l
l
L
L
or c (10
or c (10
l
l
L of 0.6 mM stock solution), acceptor substrate 10a, b,
L of 0.6 mM stock solution), 80 L of buffer solution,
L of water were combined in a final volume of 190 L.
The reaction was initiated by addition of 350 mU (10 L) of puri-
l
l
L of buffer solution,
L.
and 90
l
l
l
l
l
l
fied TG2 or an equivalent volume of 25 mM Tris–acetate assay
fied TG2 or an equivalent volume of 25 mM Tris–acetate assay
buffer (pH 7.0) for the blank. Following incubation for 50 min
at 25 °C, 10 lL of the streptavidin bead suspension was added.
The mixture was incubated for 30 min at 25 °C. The tubes were
centrifuged for 1 min at 1400 RPM (micro centrifuge) and the
supernatant was discarded. The pellets were washed by resus-
buffer for the blank. Following incubation for 30 min at 25 °C,
10 lL of the streptavidin bead suspension was added. The
mixture was incubated for 30 min at 25 °C. The tubes were centri-
fuged for 1 min at 1400 RPM (micro centrifuge). The supernatant
was discarded. The pellets were washed by resuspension in the
washing buffer described above (200
above. The pellets were washed twice more, resuspended in
washing buffer (100 L) and transferred to a polystyrene 96-well
lL) and centrifuged as
pension in the washing buffer (200
scribed above. The pellets were washed twice more,
resuspended in washing buffer (100 L) and transferred to a
lL) and centrifuged as de-
l
l
plate. Enzyme activity was detected using a Perkin Elmer Bio
Assay Reader (HTS 7000) using kexc = 485 nm, kem = 535 nm for
substrates 6a and 6b, and kexc = 360 nm, kem = 535 nm for sub-
strate 6c, by measuring the increased fluorescence signal relative
to the blank.
polystyrene 96-well plate. Enzyme activity was detected using
a Perkin Elmer Bio Assay Reader (HTS 7000) using kexc = 485 nm,
kem = 535 nm by measuring the increased fluorescence signal rel-
ative to the blank.

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