Highly specific substrates of proteinase 3 containing 3-(2-Benzoxazol-5-yl) - l -alanine and their application for detection of this enzyme in human serum

Article abstract of DOI:10.1021/ac1004103

A set of benzoxazolyl-l-alanine derivates along with the MCA moiety (donors of fluorescence) were introduced into a proteinase 3 (PR3) substrate with a C-terminal ANB-NH₂ that serves as a fluorescence acceptor. Five substrates with general formula X-Tyr-Tyr-Abu-ANB-NH₂ were synthesized, and their kinetic parameters against proteinase 3 were determined. The highest k_cat/K_M value, 1.5 × 10⁶ M^-1 s^-1, was obtained for (Pyr)Box-Ala-Tyr-Tyr-Abu-NH ₂ where (Pyr)Box-Ala stands for N-methylpyrrole benzoxazole-l- alanine. Titration of this peptide with proteinase 3 resulted in measurable fluorescence at an enzyme amount equal to 29 pmol. This substrate was selected used to detect quantifiable levels of proteinase 3 in serum samples, including those of normal subjects. For all c-ANCA-positive samples (diagnosed Wegener granulomatosis), a significant increase of PR3 concentration was observed. Wegener granulomatosis is a severe autoimmune disease causing inflammation of the blood vessels. Our results clearly show that this substrate can be used for the construction of a very reliable, inexpensive, and easy to use diagnostic test for PR3 determination.

Full text of DOI:10.1021/ac1004103

Anal. Chem. 2010, 82, 3883–3889
Highly Specific Substrates of Proteinase 3
Containing 3-(2-Benzoxazol-5-yl)-L-alanine and
Their Application for Detection of This Enzyme in
Human Serum
Magdalena Wysocka,† Adam Lesner,*,† Katarzyna Guzow,† Julia Kulczycka,‡ Anna Łe¸ gowska,†
Wiesław Wiczk,† and Krzysztof Rolka†
Faculty of Chemistry, University of Gdan`ısk, 80-952 Gdan`ısk, Poland, and Laboratory of Clinical Immunology, Gdansk
Medical University, 80-952 Gdansk, Poland
A set of benzoxazolyl-
L
-alanine derivates along with the
activation,4 and antimicrobial activity.5 Current work reveals that
only PR3 is involved in the regulation of cell proliferation6 and
apoptosis.7
MCA moiety (donors of fluorescence) were introduced
into a proteinase 3 (PR3) substrate with a C-terminal
ANB-NH2 that serves as a fluorescence acceptor. Five
substrates with general formula X-Tyr-Tyr-Abu-ANB-
NH2 were synthesized, and their kinetic parameters
Moreover, PR3 is known as the principal target antigen of
antineutrophil cytoplasm antibodies (c-ANCA) that are formed in
patients with Wegener’s granulomatosis (WG).8,9 This severe
disease leads to destruction of small blood vessels within the
inflammatory tract caused by necrotizing neutrophils. The neu-
trophils appear to play a dual role in WG so that they are targets
of autoimmunity and are also involved in causing tissue injury.
PR3 becomes the pathogenic factor that boosts the progress of
the disease. Data provided by Uriarte and Brachemi10,11 indicate
that the presence of c-ANCA activates the neutrophil population,
leading to increased secretion of PR3. The secreted enzyme can
be bound to the leukocyte membrane or released directly into
the bloodstream.
against proteinase 3 were determined. The highest kcat
/
KM value, 1.5 × 106 M-1
s
-1, was obtained for (Pyr)-
Box-Ala-Tyr-Tyr-Abu-NH2 where (Pyr)Box-Ala stands
for N-methylpyrrole benzoxazole-L-alanine. Titration of
this peptide with proteinase 3 resulted in measurable
fluorescence at an enzyme amount equal to 29 pmol.
This substrate was selected used to detect quantifiable
levels of proteinase 3 in serum samples, including
those of normal subjects. For all c-ANCA-positive
samples (diagnosed Wegener granulomatosis), a sig-
nificant increase of PR3 concentration was observed.
Wegener granulomatosis is a severe autoimmune
disease causing inflammation of the blood vessels. Our
results clearly show that this substrate can be used for
the construction of a very reliable, inexpensive, and
easy to use diagnostic test for PR3 determination.
Recently, we reported a selective fluorogenic-chromogenic
PR3 substrate, ABZ-Tyr-Tyr-Abu-ANB-NH2,
12 containing a donor
(ABZ, 2-aminobenzoic acid) and acceptor (ANB-NH2, amide of
5-amino-2-nitrobenzoic acid) of fluorescence. Using this com-
pound as a lead structure, we designed more efficient and
specific substrates of PR3 which could be applied for determi-
nation of this enzyme in humans. Since there are no diagnostic
tests available for direct determination of PR3 in serum
(however, the procedure for c-ANCA determination is common
Proteinase 3 (PR3) is a neutrophil serine protease localized
within azurophilic cytoplasmic granules of polymorphonuclear
neutrophils (PMNs). Along with cathepsin G (CG) and neutrophil
elastase, proteinase 3 is released upon leukocyte activation in the
inflammatory site.1 Historically, all neutrophil serine proteases
(NSP) are described as elastin-degrading enzymes;2 however,
recent data reveal its contribution to several physiologically
important processes such as control of cytokine activity (e.g.,
tumor necrosis factor-a [TNF-a], interleukin 1b [IL-1b]),3 platelet
(4) Renesto, P.; Halbwasch-Mecarelli, L.; Nusbaum, P.; Lesavre, P.; Chignard,
M. J. Immunol. 1994, 152, 4612–4617
(5) Gabay, J. E.; Scott, R. W.; Campanelli, D.; Griffith, J.; Wilde, C.; Marra,
.
M. N.; Seeger, M.; Nathan, C. F. Proc. Natl. Acad. Sci. U.S.A. 1989, 86,
5610–5614
.
(6) Desmedt, C.; Ouriaghli, F. E.; Durbecq, V.; Soree, A.; Colozza, M. A.;
Azambuja, E.; Paesmans, M.; Larsimont, D.; Buyse, M.; Harris, A.; Piccart,
M.; Martiat, P.; Sotiriou, C. Int. J. Cancer 2006, 119, 2539–2545
(7) Yang, J. J.; Preston, G. A.; Pendergraft, W. F.; Segelmark, M.; Heeringa,
.
P.; Hogan, S. L.; Jennette, J. C.; Falk, R. J. Am. J. Pathol. 2001, 158, 581–
592
.
* Corresponding author. Tel: ++48585235363. Fax: ++4858523472. E-mail:
adas@chem.univ.gda.pl.
(8) Kantari, C.; Pederzoli-Ribeil, M.; Amir-Moazami, O.; Gausson-Dorey, V.;
Moura, I. C.; Lecomte, M. C.; Benhamou, M.; Witko-Sarsat, V. Blood 2007,
† University of Gdan`ısk.
110, 4086–4095
(9) Jennette, J. C.; Falk, R. J. Curr. Opin. Rheumatol. 2008, 20, 55–60
(10) Uriarte, S. M.; McLeish, K. R.; Ward, R. A. Nephrol. Dial. Transplant. 2009,
24, 1150–1157
(11) Brachemi, S.; Mambole, A.; Fakhouri, F.; Mouthon, L.; Guillevin, L.; Lesavre,
P.; Halbwachs-Mecarelli, L. J. Am. Soc. Nephrol. 2007, 18, 2330–2339
(12) Wysocka, M.; Lesner, A.; Guzow, K.; Mackiewicz, L.; Łe¸gowska, A.; Wiczk,
W.; Rolka, K. Anal. Biochem. 2008, 378, 208–215
.
‡ Gdan`ısk Medical University.
(1) Rao, N. V.; Wehner, N. G.; Marshall, B. C.; Gray, W. R.; Hoidal, J. R. J. Biol.
.
Chem. 1991, 266, 9540–9548
(2) Heck, L. W.; Blackburn, W. D.; Irwin, M. H.; Abrahamson, D. R. Am. J.
Pathol. 1990, 136, 1267–1274
(3) Bedard, M.; McClure, C. D.; Schiller, N. L.; Francoeur, C.; Cantin, A.; Denis,
M. Am. J. Respir. Cell Mol. Biol. 1993, 9, 455–462
.
.
.
.
.
.
10.1021/ac1004103  2010 American Chemical Society
Published on Web 04/02/2010
Analytical Chemistry, Vol. 82, No. 9, May 1, 2010 3883

Figure 1. Chemical structures of 3-(2-benzoxazol-5-yl)alanine derivatives, ABZ, and MCA that are introduced on the N-termini of peptides.
Table 1. Physicochemical and Biochemical Characteristics of Peptides with General Formula
X-Tyr-Tyr-Abu-ANB-NH2
MW calcd/
obtained
kcat/KM
sequence where X )
ABZ12
tR [min]a F1/F0 λext/λem [nm] ETE* [%] kcat [s-1
]
KM [µM]
[s-1 × M-1] × 10-4
689.4/690.3
808.3/809.3
20.45
25.08
20.42
12.49
19.94
22.12
7.4
5.2
11.8
14.1
10.2
8.6
325/400
315/410
340/400
310/360
314/360
315/390
86
80
91
93
90
88
5.9 0.3
31.4 3.2
18.8 0.2
1.4 0.1
6.2 0.5
44.1 0.4
154.2 17.1
4.0 0.1
MCA
1.1 0.1 737.3 94.5
1.2 0.1 201.4 18.2
7.2 0.3 163.2 9.7
[(2-OMe-4-NMe)2Ph]Box-Ala (1) 929.4/930.4
(2-im)Box-Ala (2)
(Pyr)Box-Ala (3)
(2-Q)Box-Ala (4)
846.3/847.3
859.3/860.2
907.3/908.2
18.1 2.1
3.5 0.2
8.5 1.3
875 69.8
a HPLC analysis indicated *ETE-energy transfer efficiency.
and widely used), we sought PR3 substrate(s) that could be
applied for the construction of a reliable, inexpensive test for
use in medical practice. In the synthesized peptide substrates,
the N-terminal ABZ moiety was replaced by a set of benzox-
azolyl-L-alanine (Box-Ala) derivates (shown in Figure 1). These
derivates proved to be efficient donors of fluorescence as
substrates of cysteine proteinases.13 Box-Ala and ANB-NH2 at N-
and C-termini of the synthesized peptides served as donor and
acceptor, respectively. These substituents allow fluorescence
resonance energy transfer (FRET) to occur within the peptides.
FRET peptides are commonly used as fluorogenic substrates
for biochemical characterization of proteases. Energy transfer
occurs between donor and acceptor groups located within the
peptide backbone. The key factor is that energy transfer
(quenching) depends on the distance between the donor and
acceptor groups. Upon enzymatic proteolysis, the ANB-NH2 is
released, the distance between the donor and acceptor in-
creases, and a boost in fluorescence is observed. Low molecular
weight chromogenic/fluorogenic substrates are commonly
used for determination of proteinases, even as a noncovalent
complex with its cognate inhibitor (i.e., PR3 R-PI).14 Bieth and
others proved that such complexes are disrupted in the
presence of a large excess of substrate.15
MATERIALS AND METHODS
Chemical Synthesis. Benzoxazolyl Alanine Derivates. N-Boc-
3-[2-[2-(1′-methyl)pyrrolo]benzoxazol-5-yl]-
Ala) and N-Boc-3-[2-(2-imidazolyl)benzoxazol-5-yl] -
(2-im)Box-Ala) were synthesized from N-Boc-protected 3-amino-
-tyrosine methyl ester whereas N-Boc-3-[2-(2-quinolinyl)benzoxazol-
5-yl]alanine (Boc-(2-Q)Box-Ala) and N-Boc-3-[2-(2′-methoxy-4′-
dimethylaminophenyl)benzoxazol-5-yl]- -alanine (Boc-[(2-OMe-4-
L-alanine (Boc-(Pyr)Box-
L-alanine (Boc-
L
L
NMe2)Ph]Box-Ala) were synthesized from N-Boc-protected
3-amino-L-tyrosine, via the intermediate Schiff base, which
(13) Szabelski, M.; Rogiewicz, M.; Wiczk, W. Anal. Biochem. 2005, 342, 20–
(14) Duranton, J.; Bieth, J. G. Am. J. Respir. Cell Mol. Biol. 2003, 29, 57–61
(15) Faller, B.; Bieth, J. G. Biochem. J. 1991, 280, 27–32
.
27
.
.
3884 Analytical Chemistry, Vol. 82, No. 9, May 1, 2010

Figure 2. Superposition of absorption spectrum of acceptor (ANB-NH2) and emission spectrum of donors (A-F).
underwent oxidative cyclization to the heterocyclic compound
in the presence of lead tetraacetate in DMSO, according to
Peptide Synthesis. All peptides were synthesized manually by
the solid-phase method using Fmoc chemistry, as described
previously.20 TentaGel S RAM (substitution 0.25 mequiv/g) (RAPP
Polymere, Germany) was used as a solid support. The R-amino
,17
the procedure published previously.16 The methyl ester was
removed by saponification using 1 N NaOH in methanol. 3-Nitro-
L
-tyrosine methyl ester, and N-Boc-3-nitro-
L-tyrosine methyl ester
(17) Guzow, K.; Szmigiel, D.; Wro´blewski, D.; Milewska, M.; Karolczak, J.; Wiczk,
and N-Boc-3-nitro- -tyrosine, were prepared according to literature
procedures published in refs 18 and 19, respectively.
L
W. J. Photochem. Photobiol., A 2007, 187, 87–96
(18) Wu¨nsch, E. Synthese von Peptiden, 15, Houben-Weyl, Mu¨ller, E., Ed.; Georg
.
Thieme Verlag: Stuttgart, 1974; p 317.
(16) Guzow, K.; Zielin´ska, J.; Mazurkiewicz, K.; Karolczak, J.; Wiczk, W. J.
(19) Stewart, J. M.; Young, J. D. Solid Phase Peptide Synthesis, 2nd ed.; Pierce
Chemical Company: Rockford, IL, 1984; p63.
Photochem. Photobiol., A 2005, 175, 57–68
.
Analytical Chemistry, Vol. 82, No. 9, May 1, 2010 3885

groups of amino acids were Fmoc-protected. 5-Amino-2-nitroben-
zoic acid (ANB) was attached to the resin by the TBTU/DMAP
method.21 Briefly, 2 mmol of ANB was dissolved in 5 mL of DMF,
and 2 mmol of TBTU was added, which was followed by the
addition of 1 mmol of DMAP. The resulting solution was added
to 1 g of the resin and after 30 s supplemented with 4 mmol of
DIPEA. The whole mixture was stirred for 3 h. The procedure
was repeated three times. The C-terminal amino acid residues
were incorporated using POCl3 as the coupling reagent.21 The
other amino acid derivatives were coupled by the DIPCI/HOBt
method. Briefly, a mixture of N-protected amino acid, DIPCI,
and HOBt (molar ratio, 1:1:1) was dissolved in DMF:NMP
solution (1:1, v/v) and added to the ANB-resin. A 3-fold excess
to the resin active sites was used. The tert-butyloxycarbonyl
derivates of benzoxazolyl-L-alanine (Boc-(X)Box-Ala) were
introduced into the peptide chain using prolonged coupling (4
h) under the same conditions. After completion of the synthe-
sis, the peptides were cleaved from the resin using a TFA/
phenol/triisopropylsilane/H2O mixture (88:5:2:5, v/v).22 Purity
of individual peptides was checked on an RP-HPLC Pro Star
system (Varian, Australia) equipped with a Kromasil 100 C8
column (8 × 250 mm) (Knauer, Germany) and a UV-vis
detector. A linear gradient from 20 to 80% B during 40 min
was applied (A: 0.1% TFA; B: 80% acetonitrile in A). The
analyzed peptides were monitored at 226 nm.
Enzymatic Studies. UV Studies. All UV measurements were
performed using a Cary 3E spectrophotometer (Varian, Australia).
Bovine ꢀ-trypsin and turkey ovomucoid third domain (OMTKY3)
come from Sigma-Aldrich (Germany). Proteinase 3 and human
neutrophil elastase (HNE) come from Elastin Products Co., Inc.
(Owensville, MO). The concentration of bovine ꢀ-trypsin stock
solution was determined by titration with NPGB. OMTKY3 was
used as a mutual inhibitor of bovine ꢀ-trypsin, proteinase 3, and
HNE for titration of the active form of proteinase 3 and elastase.
The stock solutions of the synthesized substrates were
prepared by dissolving about 3 mg of each peptide in 250 µL of
DMSO and were further diluted 2-100 times; the enzyme
concentration was 2.06 × 10-9 M for PR3 and 2.8 × 10-5 M for
HNE. Three to five measurements were carried out for each
compound (systematic error expressed as a standard deviation
never exceeded 20%). All details of kinetic studies and the
method of calculating kinetic parameters, Michaelis constants
(KM), catalytic constants (kcat), and specificity constants (kcat
/
,24
KM) have been described in our previous papers.23
Titration Curve. A constant amount (3.3 µM) of (Pyr)Box-Ala-
Tyr-Tyr-Abu-ANB-NH2 (3) was added into a buffered solution
of proteinase 3 in Tris-HCl, pH 7.5, supplemented with 500 mM
NaCl. The amount of assayed enzyme ranged from 2.2 × 10-9
M to 1.2 × 10-10 M. The absorbance increase at 405 nm versus
time was measured. All obtained values were measured against
substrate concentration with no enzyme added. The threshold
Figure 3. Proteolytic cleavage pattern of (Pyr)Box-Ala-Tyr-Tyr-Abu-
ANB-NH2 (3) in the presence of (A) proteinase 3, (B) cathepsin G,
and (C) human neutrophil elastase. The incubation mixture was
analyzed using the following time points: (A) 5 and 60 min; (B and
C) 60 min and 24 h. MALDI-TOF analysis indicates that peak 1
corresponds to intact substrate, peak 2 to (Pyr)Box-Ala-Tyr-Tyr-Abu,
and peak 3 to the amide of ANB.
limit for all measurements was 3:1, expressed as signal-to-noise
ratio.
(21) Hojo, K.; Maeda, M.; Iguchi, S.; Smith, T.; Okamoto, H.; Kawasami, K. Chem.
Pharm. Bull. 2000, 48, 1740–1745
(20) Wysocka, M.; Łe¸gowska, A.; Bulak, E.; Jas´kiewicz, A.; Miecznikowska, H.;
Lesner, A.; Rolka, K. Mol. Divers. 2007, 11, 1193–1199
(22) Sole, N. A.; Barany, G. J. Org. Chem. 1992, 57, 5399–5403
(23) Lesner, A.; Brzozowski, K.; Kupryszewski, G.; Rolka, K. Biochem. Biophys.
Res. Commun. 2000, 269, 81–85
(24) Lesner, A.; Kupryszewski, G.; Rolka, K. Biochem. Biophys. Res. Commun.
2001, 285, 1350–1354
.
Proteolytic Cleavage Pattern Determination. Selected substrates
were mixed with a 2-fold molar excess of the enzyme in a buffer
used for kinetic studies. HPLC analysis of this mixture was
performed after the following incubation times: 5, 60 min in the
case of proteinase 3, and 1 and 24 h in the case of cathepsin G
(CG) and neutrophil elastase.
.
.
.
.
3886 Analytical Chemistry, Vol. 82, No. 9, May 1, 2010

Figure 4. Incubation of substrate (Pyr)Box-Ala-Tyr-Tyr-Abu-ANB-NH2 (3) in human serum: (A) time 0, (B) 1 h after incubation, (C) 24 h after
incubation. MALDI-TOF analysis indicates that peak 1 corresponds to intact substrate, peak 2 to (Pyr)Box-Ala-Tyr-Tyr-Abu, and peak 3 to the
amide of ANB. The signals with retention times between 22 and 26 min originate from serum.
Mass Spectrometry Analysis. Mass spectra were recorded using
a Biflex III MALDI TOF mass spectrometer (Bruker, Germany)
and R-cyano-4-hydroxycinnamic acid as a matrix.
patients (12) with diagnosed WG were assayed using (Pyr)Box-
Ala-Tyr-Tyr-Abu-ANB-NH2 (3). A Nalgene 96-well plate was used.
Into each well, 200 µL of serum was mixed with 150 µL of buffer
(100 mM Tris-HCl, pH 7.5, supplemented with 500 mM NaCl)
followed by 3 µM (1 µL) of substrate. The fluorescence
emission at 360 nm was measured up to 180 s. Each sample
was run in triplicates.
Serum Preparation and c-ANCA Determination. The serum
samples come from 5 mL of blood drawn directly from the vein
into the sterile tube. After 20-30 min (blood clotting), each sample
was centrifuged at 4000 rpm for 5 min at RT. The supernatant
was collected and stored at 4 °C.
c-ANCA presence was determined by two methods: indirect
immunofluorescence (IFT) using ethanol-fixed neutrophils and
anti c-ANCA antibodies (EUROIMMUN AG, Germany) and an
anti-PR3 ELISA kit that comes from ORGENTEC Diagnostika
GmbH, Germany.
Serum Stability Assay. A 500 µL amount of human serum from
a Wegener-negative donor was mixed with 500 µL of 0.2 M Tris
HCl buffer pH 8.3 and placed into an Eppendorf tube, followed
by the addition of 10 µL of an appropriate substrate at a
concentration of 3 mg/mL. After 1 and 24 h, a 50 µL sample of
the mixture was analyzed by HPLC. An RP-HPLC Pro Star system
(Varian, Australia) equipped with a Kromasil 100 C8 column (8 ×
250 mm) (Knauer, Germany) and a UV-vis detector was used.
A linear gradient from 10 to 90% B during 45 min was applied
(A: 0.1% TFA; B: 80% acetonitrile in A). The analyzed peptides
were monitored at 226 nm.
Additionally, human serum was supplemented with 10 µL of
NHE or PR3 stock (0.1 mg/mL) solution. The activity of the
mixture was later evaluated using appropriate substrate(s).
Fluorescence Studies. The fluorescence assay was per-
formed using a FluoroMax 4 fluorometer (Horiba Instruments,
Inc., Ann Arbor, MI). For selected substrates, fluorescence
increase and energy transfer efficiency were determined according
to eqs 1 and 2, respectively, where F0 is the fluorescence of the
intact peptide and F1 is the fluorescence of the product after
complete hydrolysis. F0 and F1 values were calculated after the
subtraction of buffer autofluorescence (Fbuff) from both the
initial (Fini) and final fluorescence (Fmax).
RESULTS AND DISCUSSION
For all 3-(2-benzoxazol-5-yl)-L-alanine derivatives, 7-methoxycou-
marin-4-ylacetic acid (MCA) and 2-aminobenzoic acid (ABZ) (used
as a references12), the emission and absorption spectra were
collected. In each case, overlap of the emission spectrum of the
donors and the absorption spectrum of the acceptor (ANB-NH2) (see
Figure 2) was observed. For each donor-acceptor pair, the wave-
length of the excitation and emission was determined (see Table 1).
The substitution of ABZ by MCA and (X)Box-Ala at the
N-terminus of the previously designed PR3 substrate ABZ-Tyr-
Fmax - Fbuff
Finit - Fbuff
F1
F0
)
(1)
12
Tyr-Abu-ANB-NH2 results in a series of peptides displaying
F0
F1
FRET. The physicochemical properties of the obtained com-
pounds are summarized in Table 1. In all cases the energy
transfer efficiency was high, ranging from 80 (for MCA
derivate) to 93% for (2-im)Box-Ala-containing peptides. All
synthesized compounds possess outstanding spectral properties
energy transfer efficiency (%) ) 1 -
× 100% (2)
(
)
Measurement of PR3 Activity in Human Serum. The human
sera that come from both c-ANCA-negative (32) and -positive
Analytical Chemistry, Vol. 82, No. 9, May 1, 2010 3887

Figure 5. Incubation of substrate (Pyr)Box-Ala-Tyr-Tyr-Ser-ANB-NH2 in human serum: (A) time 0, (B) 1 h after incubation, (C) 24 h after
incubation. MALDI-TOF analysis indicates that peak 1 corresponds to intact substrate. The signals with retention times between 22 and 26 min
originate from serum.
and could be utilized in the enzymatic assay. The determined
values of the specificity parameter (kcat/KM) obtained for PR3
depend strongly on the introduced N-terminal benzoxazolyl
alanine moiety. For the reference substrate ABZ-Tyr-Tyr-Abu-
ANB-NH2, kcat/KM reached almost 2 × 105 M-1 s-1 12 whereas
,
the analogue with MCA exhibited a value more than 1 order
of magnitude lower. Among the four substrates containing Box-
Ala derivates, two (compounds 2 and 3) displayed a higher
specificity parameter. The most active substrate, 3, with
(Pyr)Box-Ala exhibited an almost 8-fold increase, 1.5 × 106 M-1
s
-1, due to an increase of both catalytic constant (kcat) (3-fold
increase) and affinity (KM) (4-fold increase) of the substrate
toward the enzyme. This is not the case for compounds 1 and
4 where this parameter (KM) was affected significantly (de-
crease of affinity 8 and 30 times, respectively). There is a
correlation between specificity constant and the size of the
chemical group introduced to Box-Ala (see Figure 1); small
heterocyclic moieties (pyrrole, imidazole) optimize the specificity
constant. The results presented above clearly indicate that
substrate 3 is the most efficient PR3 substrate; therefore, in
further experiments we focused our attention on this substrate.
PR3 is released from azurophile granules along with HNE and
CG. Prolonged (up to 24 h) incubation of compound 3 with these
proteases results in absence of proteolysis. These data indicate that
peptides in the presence of these three neutrophil proteases (HNE,
CG, PR3) are selectively hydrolyzed by PR3 (see Figure 3).
A sensitivity assay was performed for substrate 3. The
following equation was obtained Y ) -9.417 + 2.66e+11 × X with
R ) 0.997. The results obtained reveal that fluorescence of
substrate (Pyr)Box-Ala-Tyr-Tyr-Abu-ANB-NH2 could be easily
detected at an amount of proteinase 3 as low as 2.9 × 10-11 M,
which is 10-fold lower than that for ABZ-containing peptide 6
reported previously by our group.12
Figure 6. Analysis of results from 42 serum patients (30 negative
and 12 positive ANCA samples).
and ANB-NH2 is observed (Figure 4). Incubation of peptide
(Pyr)Box-Ala-Tyr-Tyr-Ser-ANB-NH2 (previously determined as
unhydrolyzed by PR3) under the same conditions resulted in
no observable degradation (Figure 5) or boost in fluorescence
(data not shown). This indicates that compound 3 can be utilized
as a tool to monitor the proteinase 3 activity in human serum.
Additionally, the selectivity of the substrate (3) was confirmed
by measurements of the chromophore release in serum which
was supplemented with 1 µL of 0.34 µM HNE or PR3. For each
serum sample, the activity of the appropriate proteinase was mea-
sured using an NHE-specific substrate (Suc-Ala-Ala-Pro-Val-pNA) or
PR3 (substrate 3). For the HNE- and PR3-supplemented serum
samples, there was a significant increase of the absorbance (data
not shown). Serum sample supplementation was made in two
independent experiments; one for HNE and the second for PR3.
We determined PR3 activity in 42 samples that originated from
healthy (non-Wegener) (30 patients) and Wegener’s diagnosed
patients. The results of this assay are presented in Figure 6. Using
substrate 3, we detected measurable fluorescence in all analyzed
samples. Based on the titration curve, the range of the PR3
concentration in all human serum samples of healthy patients was
Because peptide 3 undergoes selective proteolysis, it was
subjected to a serum stability assay which may contain other
enzyme(s) affecting its stability, but this would not allow accurate
determination of PR3 in body fluids. HPLC and further mass
spectrometry analysis of obtained fragments indicate that only
cleavage corresponding to proteolysis of the bond between Abu
3888 Analytical Chemistry, Vol. 82, No. 9, May 1, 2010

from 64.6 to 301.9 µM but with one sample at 651.5 µM. In the
serum of c-ANCA-positive patients, we observed a significant
increase in PR3 activity that varied from 512.5 to 1028.0 µM. The
obtained values of proteinase 3 concentration are in good agree-
ment with data provided by Schnabel et al.25 using an immu-
nochemical (ELISA) method. However, these authors did not
observe a correlation between c-ANCA and PR3 in the tested
material. The immunochemical method used only detects the free
PR3; thus, several types of antibodies are needed to detect free
PR3, PR3-inhibitor complex, and PR-c-ANCA complex. This
shortcoming of the Schnabel et al.25 method is not a factor in our
study because the low molecular weight chromogenic/fluorogenic
compounds (in excess) used will dissociate to the noncovalent
species.
fluorophore. This peptide displays dual chromogenic and fluoro-
genic properties and enabled us to detect as low as 28.7 pmol
proteinase 3. Moreover, this substrate was successfully applied
for determination of the PR3 level in human serum obtained from
WG-negative and WG-positive patients. We believe that such a
substrate could be utilized in preliminary Wegener’s diagnosis.
ACKNOWLEDGMENT
We acknowledge Monika Łe¸gowska for critical reading of this
manuscript. This work was supported by Ministry of Science and
Higher Education under grants 1600/B/H03/2009/36 (A.L.) and
0212/H03/2007/33 (K.G.).
In conclusion, a sensitive and selective substrate of proteinase
3 with new molecular probe was developed that serves as a
Received for review February 15, 2010. Accepted March
12, 2010.
(25) Schnabel, A.; Csernok, E.; Braun, J.; Gross, W. L. Am. J. Respir. Crit. Care
Med. 2000, 161, 399–405
.
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