Design, synthesis, and enzymatic evaluation of novel ZnO quantum

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Design, synthesis, and enzymatic evaluation of novel ZnO quantum dot-based assay for detection of proteinase 3 activity Jadwiga Popow-Stelmaszyk, Beata Bajorowicz, Anna Malankowska, Magdalena Wysocka, Tomasz Klimczuk, Adriana Zaleska-Medynska, and Adam Lesner Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.8b00100 • Publication Date (Web): 11 Apr 2018 Downloaded from http://pubs.acs.org on April 12, 2018

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Bioconjugate Chemistry

Spherical NH2-terminated ZnO quantum dots possessing strong emission centered at 530 nm have been conjugated with peptide probe sensitive for PR3. Conjugation of peptide probe with ZnO quantum dots allow to determine PR3 activity with detection limit equal to 1.3 pmol. 588x207mm (300 x 300 DPI)

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 † § § ‡ ∥ 16 Jadwiga Popow-Stelmaszyk , Beata Bajorowicz , Anna Malankowska , Magdalena Wysocka , Tomasz Klimczuk , 17 18 Adriana Zaleska-Medynska§, Adam Lesner† 19 20 † 21 Laboratory of Analysis and Biochemical Nanodiagnostic, Department of Environmental Technology, Faculty of 22 23 Chemistry, University of Gdansk, Gdansk 80-308, Poland 24 25 26 § Laboratory of Photocatalysis, Department of Environmental Technology, Faculty of Chemistry, University of 27 28 29 Gdansk, Gdansk 80-308, Poland 30 31 32 ‡ Laboratory of Medical Chemistry, Department of Biomedical Chemistry, Faculty of Chemistry, University of 33 34 Gdansk, Gdansk 80-308, Poland 35 36 37 38 ∥ Department of Solid State Physics, Faculty of Applied Physics and Mathematics, Gdansk University of Technology, 39 40 Gdansk 80-233, Poland 41 42 43 44 45 46 47 KEYWORDS 48 49 50 QD-based probe, PR3 detection, FRET mechanism, ZnO quantum dots, proteinase 3 51 52 53 54 55 56 57 58 59 ACS Paragon Plus Environment 60

Design, synthesis, and enzymatic evaluation of novel ZnO

quantum dot-based assay for detection of proteinase 3 activity

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ABSTRACT Herein, the synthesis and application of functionalized quantum dot-based protease probes is described. Such probes are composed of nontoxic ZnO nanocrystals decorated by amino groups followed by linker and labeled peptide attachment. Spherical NH2-terminated ZnO quantum dots (QDs) with the average size ranging from 4 to 8 nm and strong emission centered at 530 nm were prepared using the sol-gel method. The fluorescence of ZnO QDs was quenched by the BHQ1 moiety present on the N-terminal amino group of the peptide. The enzymatic cleavage of the peptide mediated by the proteinase 3 (PR3) bond resulted in an increase in the QD probe fluorescence. This observation was verified using both model and biological systems; and the picomolar detection limit was found to be more than 30 times lower than that of the previously reported internally quenched peptide (a decrease in detection limit from 43 to 1.3 pmol was observed).

INTRODUCTION Quantum dots (QDs) are highly fluorescent semiconductor nanocrystals exhibiting unique optical and electronic characteristic owing to the quantum confinement effects. The recent progress in surface functionalization with organic ligands as well as the controlled synthesis of high-quality QDs enables their use in cell targeting, bioimaging, drug delivery, and disease diagnosis.1-3 QDs possess unique properties, such as broad absorption spectra, narrow and symmetric size-dependent emission, high quantum yield, and stability against photobleaching, which make them advantageous over traditional organic dyes and fluorescent proteins for biosensing applications.1,

4, 5

Similar to

organic fluorophores, semiconductor nanocrystals can act as energy donors with organic dyes as energy acceptors in fluorescence resonance energy transfer (FRET) assays.6 Various FRET-based QD biosensors have been reported thus far, including the detection of glucose,7 melamine,8 kaempferol,9 nucleic acids,10, dopamine,15 avidin,16 salicylic acid,17 DNA,18 protease,4,

19, 20

11

aflatoxin,12 trypsin,9,

13, 14

glutathione,21 caspase 3,22 and protein kinase.23 The

most commonly used QDs for biosensors are CdTe,8, 9, 12, 17, 21 CdS,11 graphene QDs,10, 13-15, 23 ZnS-CdSe,16 CdSe/CdS core/shell,18 CdSe/ZnS core/shell,7, 19, 22 and CdSeS/ZnS core/shell.20 So far, the most of reported ZnO NPs-based biosensors are for the detection of phenol24, 25, H2O226, 27, glucose28, urea29 and cholesterol30, 31. ACS Paragon Plus Environment

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Neutrophils play an essential role against microbial infections.32 The host defense by neutrophils, however, results in tissue damage. Once recruited and activated at the site of microbial invasion, neutrophils can release significant amounts of proteases and oxidants into the extracellular space.33 Human neutrophil elastase (HNE), proteinase 3 (PR3), cathepsin G (CG),34 and the newly described neutrophil serine protease 435 represent the proteolytic repertoire of neutrophils. These four closely related proteases are called neutrophil serine proteases (NSPs). They are stored as active enzymes in azurophil granules and are released upon the activation of the neutrophil.36 The active form of PR3 is deposited not only in azurophil granules but also on the membrane of secretory vesicles.37 There are numerous extensive reviews focused on PR3 functions in normal and aberrant organisms.38 In general, PR3-elevated activity is linked to inflammation,39 cancer,40 and granulomatosis with polyangiitis (GPA), formerly called Wegener granulomatosis.41 PR3 thus seems to be a major target for neutrophil-activating ANCA in systemic vasculitis. All of the above inspired research groups including ours have attempted to build peptide probes for detecting the PR3 activity. Such probes utilize a broad panel of fluorogenic reporter groups, including 2aminobenzoic acid (ABZ), rhodamin, and coumarin derivatives.42, 43 However, thus far, no QD-based assay has been developed for the detection of the PR3 activity. The aim of this study was the design and synthesis of a ZnO QD-based internally quenched peptide substrate and its evaluation in biological system(s). ZnO QDs have been selected as a non-toxic material, as opposed to cadmium-based QDs, widely described in the literature.11, 44-48 Novel peptide–QD conjugates revealed efficient FRET between ZnO QDs and the black-hole quencher (BHQ) moiety. As a result of the cleavage of the peptide substrate caused by PR3, the energy transfer was disrupted, and the fluorescence emission intensity increased depending on the enzyme concentrations. The general formula of the design construct is shown in Figure 1.


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Figure 1. Composition and mechanism of action of QD PR3 sensor. BHQ corresponds to 4′-(2-Nitro-4-toluyldiazo)2′-methoxy-5′-methyl-azobenzene-4″-(N-ethyl)-N-ethyl-2-cyanoethyl-(N,N-diisopropyl)-phosphoramidite. QD is an amino-functionalized ZnO quantum dot. The peptide sequence Tyr-Tyr-Abu-Gln-Asp-Pro is exclusively cleaved by PR3, and it is connected to QD via functionalized linker oxyethylene linker. The arrow indicates the cleaved peptide bond.

RESULTS AND DISCUSSION Structural analysis of NH2-terminated ZnO QDs The morphology of the synthesized ZnO–NH2 QDs (ZnO QDs conjugated with aminopropyltriethoxysilane (APTES)) as well as ZnO–L–P–Q (ZnO–NH2 QDs modified with the sequence of PEG as a linker (L), peptide (P), and quencher (Q)) was studied by transmission electron microscopy (TEM) (Figure 2). ZnO QD-modified NH2 groups have a spherical shape with the average size ranging from 4 to 8 nm (Figures 2a–c). Figures 2d–f shows the TEM images of ZnO–L–P–Q; ZnO QDs are coated with a thin amorphous layer, suggesting the presence of PEG, peptide, as well as quencher on the surface ZnO QDs. The distribution of the elements in the samples (Energydispersive X-ray spectroscopy analysis, not presented here) confirmed a higher amount of carbon in the sample ZnO– L–P–Q than in ZnO–NH2. ACS Paragon Plus Environment

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Figure 2. TEM images of (a-c) ZnO QD-modified NH2 and (d–f) ZnO–L–P–Q.

The surface modification and conjugation of ZnO QDs with APTES, PEG, as well as PEG, peptide, and quencher were confirmed by Fourier transform infrared (FTIR) spectroscopy (Figure 3a). APTES is alkoxysilane with an –NH2 side group and has been widely applied to provide colloids with stability and functionalized surfaces. The capping of APTES to the surface ZnO QDs was established by the presence of the band at around 1000 cm−1, which was assigned to the Si–O groups.49 The absorption peak at 3417 cm−1 could be attributed to the stretching vibration of NH2.50 The peak at 2930 cm−1 was attributed to the symmetric methylene stretch (–CH2–) of the capping agent.51, 52 The bands at approximately 1450 and 1400 cm−1 were due to the C–H bending vibrations. A prominent absorption peak was observed at 465 cm−1 corresponding to the Zn–O stretching vibration.53 The appearance of the intense characteristic bands of PEG at 2890 and 1100 cm−1 indicates on the conjugation with PEG. The peak at 1638 cm−1 could be attributed to the amide group indicating the conjugation of PEG with peptide and peptide with quencher.54 ACS Paragon Plus Environment

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Intensity (arb. u.)

Transmittance

1 2 3 The X-ray diffraction pattern (XRD) of ZnO–NH2 QDs was consistent with the characteristic peaks of the typical 4 5 ZnO hexagonal quartzite structure (Figure 3b).55 The high purity of the ZnO sample was confirmed by the absence of 6 7 8 additional reflections in the pattern. The obtained diffraction peaks were broad, indicating the very small size of 9 10 nanocrystals and the presence of NH2 groups on the surface of ZnO. The estimated lattice parameters a = 3.253 Å and 11 12 c = 5.213 Å were very close to the reported values of a = 3.24 Å and c = 5.20 Å by Mayer et al.56 13 14 15 16 17 (a) (b) 18 ZnO 19 2930 P63mc (#186) 20 1000 a = 3.253 Å 21 3417 22 c = 5.213 Å 23 2890 14501400 1100 24 25 1400 2925 1448 26 1638 27 ZnO-NH2 28 ZnO-L 29 ZnO-L-P-Q 30 31 32 4000 3500 3000 2500 2000 1500 1000 500 20 30 40 50 60 70 80 -1 33 Wavenumber (cm ) 2Θ (deg) 34 Figure 3. (a) FTIR spectra of ZnO-NH , ZnO-L and ZnO-L-P-Q samples; (b) XRD pattern of ZnO-NH2 QDs. 2 35 36 37 38 39 40 41 Optical properties of ZnO-based system 42 43 44 The UV–VIS absorption spectra of ZnO QDs and ZnO–L–P–Q in ethanol solutions are presented in Figure 4a. For 45 51-53 46 ZnO-modified NH2, absorption below 350 nm was observed and this result was in agreement with the literature. 47 48 The edge of absorption in the sample ZnO–L–P–Q showed a slight blue shift as compared to the sample ZnO QDs. 49 50 The emission spectrum upon the UV excitation at 320 nm is shown in Figure 4b. A strong emission centered at 51 52 53 530 nm was observed for the sample ZnO QDs. The pictures in Figure 4b are the photographs showing the ZnO–NH2 54 55 QDs in aqueous ZnO–NH2 solutions under daylight (left) and UV illumination (right). Under UV excitation, the 56 57 aqueous solution emitted white light. The quantum yield of ZnO nanocrystals was measured using a solution of 58 59 reference material. The fluorescence QY of ZnO QDs was ACSthe Paragon Plus Environment 60 rhodamine 6G in ethanol (QY = 95%) as

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Absorbance (a.u.)

1 2 3 approximately 27%. As shown in Figure 4b, for ZnO–L–P–Q, a decrease in the fluorescence spectrum was observed. 4 5 The fluorescence of ZnO–NH2 QDs was ascribed to their surface defects, while the quenching of fluorescence 6 7 49 8 indicated a successful loading sequence of PEG, linker, and quencher onto the ZnO–NH2 QD surfaces. 9 10 11 12(a) (b) 13 14 15 16 17 ZnO-L-P-Q 18 ZnO-NH2 19 20 21 22 23 24 25 26 27 250 300 350 400 450 500 550 600 650 700 750 800 28 Wavelength (nm) 29 30 31 32 33 Figure 4. (a) UV-VIS spectra of ZnO–NH2 and ZnO–L–P–Q, (b)PL spectra of ZnO–NH2 and ZnO–L–P–Q with the 34 excitation at a wavelength of 320 nm and photographs of aqueous ZnO–NH2 solutions under daylight (left) and UV 35 illumination (right). 36 37 38 39 Analysis of proteinase 3 activity 40 41 To synthesize a QD-based probe that would allow us to assay the PR3 activity, we decided to utilize ZnO QDs as the 42 43 fluorescent donors. The QDs were functionalized by the addition of amino groups onto their surface. To such 44 45 modified QDs, the chemically inert linker PEG O2Oc (8-amino)-3,6-dioxaoctanoic acid)) was attached as the Fmoc 46 47 48 (9-fluorenylmethyloxycarbonyl) -derivate using simple peptide chemistry methods. After the successful removal of 49 50 the Fmoc group, QDs were coupled with the BHQ–Tyr–Tyr–Abu–Asn–Thr–Pro–OH peptide that was recently 51 52 developed by our group as one of the most selective peptide substrates of PR3. To secure efficient quenching, we 53 54 55 decided to introduce on the N-terminal group of the peptide the methylated dabcyl derivate (called black-hole 56 57 quencher one or BHQ-1). Its absorption spectra were an excellent fit to the emission spectra of the functionalized 58 59 ZnO QD. After effective coupling, the probe was ready for testing. Its physiochemical analysis (TEM, FTIR, DRSACS Paragon Plus Environment 60

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UV-VIS, and PL spectroscopy) along with photochemical studies suggests that on the surface of the functionalized QDs was a peptide layer, indicating efficient coupling. The incubation of the obtained probe (100 µg/mL) with the experimental enzyme (PR3) at the initial concentration of 10 nM resulted in an increase in the intense fluorescence due to the cleavage of the peptide bond between Abu and Asn. Further, the chromatography analysis of the cleavage products indicated the presence of a BHQ–Tyr–Tyr–Abu–OH fragment (retention time 23.12 min.), as shown in Figure 5A. Mass spectrometry analysis confirmed its molecular weight since m/z 901.1 was found that corresponds to protonated

form

of

mentioned

peptide

fragment

(Fig

5B).

Figure 5. A) RP HPLC and B) MS spectra of supernatant collected from QD-PR3 probe after 1 h incubation with PR3.


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Figure 6. Fluorescence increase observed for QD probe incubated with PR3 or neutrophil lysates in absence and presence of selective PR3 inhibitor. The significant linear increase in the fluorescence of QD PR3 probe incubated with 10 nM of proteinase 3 in appropriate buffer was observed for first 30 min of incubation (see Figure 6). No fluorescence increase was recorded for system lacking enzyme or pure quantum dots with no peptide attached. In the presence of the recently developed PR3 inhibitor (100 nM),57 we observed a significant reduction of fluorescence, reaching 97% as compare to untreated system (containing QD PR3 probe and enzyme only). This finding indicated that the fluorescence increase was proportional to the enzymatic activity of protease 3.

The significant linear increase in the fluorescence of QD PR3 probe incubated with 10 nM of proteinase 3 in appropriate buffer was observed for first 30 min of incubation (see Figure 6). No fluorescence increase was recorded ACS Paragon Plus Environment

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for system lacking enzyme or pure quantum dots with no peptide attached. In the presence of the recently developed PR3 inhibitor (100 nM),57 we observed a significant reduction of fluorescence, reaching 97% as compare to untreated system (containing only QD PR3 probe and enzyme). This finding indicated that the fluorescence increase was proportional to the enzymatic activity of protease 3. To verify whether the newly developed probes interacted with the enzyme studied in an exclusive manner, we incubated them with 10 nM and 50 nM of other enzymes of the neutrophil serine protease family: elastase and cathepsin G. An insignificant fluorescence (2%–3% above the noise level) increase was observed in all of the systems tested. Such results encouraged us to evaluate our probe in a biological system. We decide to use neutrophil lysate as a rich source of neutrophil serine proteases. The incubation of the developed probe with the buffered lysed neutrophils yielded a significant fluorescence increase. In the system with the irreversibly inhibited PR3, no significant fluorescence increase was detected. This fact again confirmed the selectivity of interaction of the ZnO QD-based developed probe with proteinase 3 itself.


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Figure 7. Titration of PR3 substrate and QD-based PR3 substrate by increasing enzyme amount.

Subsequently, the 9 systems with a constant probe amount (100 µg) were incubated with a decreasing amount of PR3 ranging from 10 nmol to 0.1 pmol (Figure 7). The detectable fluorescence increase (defined as a 3:1 signal-to-noise (S/N) ratio)) was recorded for 1.3 pmol of PR3 in the assay system). This value was at least 30 times lower than that previously reported for the PR3-detecting peptide substrates.58

CONCLUSION In summary, a new QD-based probe allowing the detection of PR3 in picomoles was developed. The novel assay was based on nontoxic ZnO QDs conjugated with a peptide probe (Tyr–Tyr–Abu–Asn–Thr–Pro–OH) and an organic quencher (BHQ). The conjugation of the peptide probe with ZnO QDs allowed us to decrease the detection limit from 43 pmol to 1.3 pmol, as compared to the same type of peptide probe using 2-aminobenzoic acid as the donor of fluorescence and 3-nitro-L-tyrosine as the acceptor.50 The new probe exhibited excellent luminescence properties and could be considered a good alternative to the detection of PR3 using the standard peptide substrate. The proposed method of PR3 activity analysis was fast, cheap, and simple, allowing the selective detection of this protease even in complex biological systems such as neutrophil lysate. Such a probe could be beneficial in the detection of diseases related to/connected with the overexpression of PR3 or neutrophil activation.

EXPERIMENTAL PROCEDURES Materials and instruments Zinc acetate dihydrate (>98% Acros) was used as the precursor for the preparation of ZnO. 3Aminopropyltriethoxysilane (APTES) (>98% Sigma Aldrich), KOH (pure p.a., POCH S.A.), and ethanol (>99.8% POCH S.A.) were used for the synthesis of NH2-modified ZnO QDs. All of the chemicals were of analytical reagent grade and were used as received without further purification. The morphology of the QDs was investigated with transmission electron microscopy (STEM-EDX, FEI Europe, model TecnaiF20 X-Twin) and selected area electron ACS Paragon Plus Environment

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diffraction (SAED). The FTIR spectra were obtained on a Bruker model IF S66 FTIR spectrometer using potassium bromide disks. Powder X-ray diffraction (PXRD, Philips/PANalytical X'Pert Pro MPD diffractometer, (Cu Kα radiation λ = 1.5418 Å) was used to determine the phase composition and calculate the lattice parameters of the QDs. The photoluminescence (PL) emission spectra were recorded using a Perkin-Elmer Luminescence Spectrometer LS 50B. The samples were excited with 340-nm-wavelength light at room temperature, and the emission was scanned between 350 nm and 800 nm. The relative quantum yield (QY) was measured using a solution of rhodamine 6G in ethanol (QY = 95%) as a reference material. The UV-vis absorption spectra were recorded on an Evolution 220, Thermo Scientific spectrometer. Preparation of ZnO–NH2 QDs ZnO QDs were prepared according to the method reported by Zhao et al. with a little modification.50 Zn (Ac)2·2H2O was dissolved in ethanol by stirring and refluxed for 90 min at 68°C; then, the solution was cooled down to room temperature (Figure 8). KOH was dissolved in 5 mL of ethanol and kept in an ultrasonic bath for 40 min. The obtained KOH solution was slowly added to the Zn(Ac)2 solution and was stirred for 1 h. Next, 0.5 mL of deionized water and 0.34 mmol of APTES (dissolved in ethanol) were added into the reaction system under constant stirring. The obtained precipitate was centrifuged; it was first washed using toluene and then washed using absolute ethanol to remove the unreacted molecules. The final powders were re-dispersed in ethanol for further characterization.


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Figure 8. Schematic representation of preparation route used to obtain ZnO QD-modified NH2.

Peptide synthesis Peptide was synthesized manually via the solid-phase method using the Fmoc/tBu approach on 2-chloro-chlorotirtyl resin (RAPP Polymere, Germany). A peptide chain was elongated in cycles of deprotection and coupling. As the αamino groups of amino acids were Fmoc protected, deprotection was performed with 20% piperidine in DMF, whereas

the

chain

elongation

was

achieved

with

standard

DIPCI/HOBt

chemistry

(diisopropylcarbodiimid/hydroxybenzotraizole). Further, we used three equiv. of the protected amino acid derivative excess for the subsequent attachment of amino acids to the solid support. At the end of the peptide synthesis, the BHQ moiety (4′-(2-Nitro-4-toluyldiazo)-2′-methoxy-5′-methyl-azobenzene-4″-(N-ethyl)-N-ethyl-2-cyanoethyl-(N,Ndiisopropyl) phosphoramidite) to act as a quencher was attached to the N-terminal amino group of Tyr. After the synthesis, the fully protected peptide was cleaved from the resin under a mild condition using an AcOH/DCM/TFE (1:8:1) mixture. The purity of the synthesized compound was verified by reverse-phase high-performance liquid chromatography (RP-HPLC). For the analysis, a Pro Star system (Varian, Australia) equipped with a Supelco– Discovery Bio Wide Pore C8 column (250 mm × 4.6 mm, 5 µm, Sigma–Aldrich) and a UV–VIS detector were used. ACS Paragon Plus Environment

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A linear gradient from 10% to 90% B (A: 0.1% trifluoroacetic acid in water; B: 80% acetonitrile in A) for 40 min and a flow rate of 1 mL/min were applied. The mass spectra of the obtained compound were recorded with a Biflex III matrix-assisted laser desorption/ionization time-of-flight (MALDI–TOF) mass spectrometer using the α-cyano-4hydroxy-cinnamic acid matrix (CCA). QD functionalization To the ZnO–NH2 QDs, the inert bifunctionalized linker Fmoc-O2Oc (8-(9-fluorenylmethyloxycarbonyl-amino)-3,6dioxaoctanoic acid) was attached by applying standard peptide chemistry methods. In brief, 3 molar excess of FmocO2Oc (further abbreviated as linker (L)) was used and coupled to the amino group of the ZnO QDs by using standard DIPCI/HOBt chemistry (diisopropylcarbodiimid/hydroxybenzotraizole) until completeness of the reaction, which was monitored by using the Kaiser test. This process was followed by the attachment of the protected BHQ– Tyr(tBu)–Tyr(tBu)–Abu–Asn(Trt)–Glu(tBu)–Pro–OH (abbreviated as quencher-peptide (Q-P)) fragment by using the method described above. After coupling, the side chain groups were removed using 30% TFA in DCM. The entire mixture was evaporated, dissolved in water, and freeze dried. The obtained construct (120 mg) was subjected to structural and enzymatic studies. Enzymatic studies PR3 incubation The human enzymes proteinase 3, cathepsin G, and neutrophil elastase were obtained from Elastin Inc. (USA). Bovine β-trypsin and turkey ovomucoid third domain (OMTKY3) were purchased from Sigma Aldrich (Germany). The concentration of the bovine β-trypsin stock solution was determined by titration with nitrophenol-p-guanidino benzoate by using burst kinetics. The concentration of each enzyme was determined using a standardized solution of the common inhibitor (OMTKY3), previously titrated by a standardized solution of bovine β-trypsin, as described previously.43 Fluorescence studies During these studies, the fluorescence spectra of all of the compounds were recorded in a Clariostar microplate reader (BMG, Germany). QDs were excited at 320 nm, and their emission was observed at 530 nm. ACS Paragon Plus Environment

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Proteolytic cleavage pattern determination Further, 1 µL of the appropriate enzyme (10−7 M) in the experimental buffer (0.1-M Tris HCl) was added to 5 µL of a solution of the QD probe substrate (100 µg/mL). The HPLC analysis of this mixture was performed after the following incubation times: 0, 15 min, 1 h, and 48 h. A linear gradient from 10%–90% B within 40 min was applied (A: 0.1% TFA; B: 80% acetonitrile in A). The analyzed peptides were monitored at 226 nm. The peaks that appeared were collected and analyzed with MALDI–TOF as described above. Sensitivity curve A constant amount of the selected peptide (100 µg/mL, 0.1-M Tris HCl buffer) was added into a solution of the specific enzyme in the appropriate buffer. The concentration of the assayed enzyme ranged from 4.31 × 10−11 M to 1.73 × 10−9 M for PR3 and from 1.33 × 10−8 M to 1.33 × 10−7 M for HNE and CG. The fluorescence increase at 520 nm versus time was measured. All of the obtained values were measured against a substrate solution with no enzyme added as the control. The threshold limit for all of the measurements was the S/N ratio of 3:1. Neutrophil lysate studies Neutrophil lysate was obtained from 1 × 106 human neutrophils purified from fresh blood by using Ficol and multiple centrifugations and was donated by Dr. Korkmaz (French Institute of Health and Medical Research, Inserm, Tours, France). For each microplate experiment, 5 µL—corresponding to 1.2 × 104 cells—was used. The entire experiment was performed at 37°C. The plate was shaken before measurement for 30 s. The increase in the fluorescence of the QD donor was measured for 30 min. Inhibitory study Furthermore, 5 µL of the PR3 selective inhibitor (Bt–PYDAP(O–C6H4–4–Cl)2) at the concentration of 2 × 10−7 M was added to each well containing 10−9 M of an enzyme or cell lysate (1 × 106 cells) dissolved in 100 µL of the same buffer.57 The plate was incubated at 37°C for 20 min. The QD probe (100 µg/mL in the buffer used for the kinetic studies) was added, the plate was mixed thoroughly, and the fluorescence increase was recorded during 30 min. The emission and excitation parameters were the same as those used for the kinetic studies.


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ACKNOWLEDGMENT The author A.M. acknowledges funding from the Foundation for Polish Science (FNP). AUTHOR INFORMATION Corresponding Author *A. Zaleska-Medynska: E-mail: [email protected]

Notes The authors declare that they have no competing financial interest with respect to this study.

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