4 aminophenylmercuric acetate

Fmoc–Leu–Ala–Gly–Gly and Fmoc–Gly–Pro–Leu–Gly–Leu–Ala–Gly–Gly peptides were purchased from Lipopharm.pl (Zblewo, Poland). Oligonucleotides 5′-CGT ACG CGT ACG CGT ACG CG-3′ and 5′-CGC GTA CGC GTA CGC GTA CG-3′ were purchased from Future Synthesis (Poznań, Poland). Collagenase IV and Hank’s Balanced Salt Solution (HBSS), with calcium and magnesium, were purchased from ThermoFisher Scientific (Walthman, MA, USA). Acetonitrile (ACN), 4-aminophenylmercuric acetate (APMA), dichloromethane (DCM), 2,5-dihydroxybenzoic acid (DHB), N,N-dimethylformamide (DMF), doxorubicin hydrochloride, formic acid, isobutyl chloroformate, methanol, sodium hydroxide, tetrahydrofuran (THF), triethylamine (TEA), and Tris-HCl were purchased from Sigma-Aldrich (St. Louis, MO, USA). Column chromatography was performed using silica gel NORMASIL 60 (40–63 mesh, VWR Chemicals, Radnor, PA, USA). Thin-layer chromatography was performed using silica plates, 60G, F₂₅₄ (Sigma-Aldrich, St. Louis, MO, USA).

MMP-2 (ab81550) and MMP-9 (ab82955) were from Abcam. Before incubation with the serum sample, pro MMP-9 was activated by incubation with 4-aminophenylmercuric acetate 1 mM (Sigma, St. Louis, MO, USA) overnight at 37 °C. The digestion of vitronectin was carried out by incubation of the substrate at 37 °C for 24 h with MMPs in an enzyme-to-substrate ratio of 1:20 in 50 mMTris-HCl, pH 7.5, containing 0.15 M NaCl, CaCl₂ 10 mM, 0.05% Brij 35 and 0.02% NaN₃.

Protein content in BAL fluid was determined with the standard Bradford assay (Bio-Rad, Glattbrugg, Switzerland) [21 (link)]. The gelatinolytic activity in BAL was determined in aliquots containing 2 μg of proteins using gelatin zymography [21 (link)]. Gelatinolytic activity was quantified using the computer-assisted image analysis program of Kodak (Eastman Kodak, Rochester, NY).
Activation of latent gelatinases was performed by pre-incubation with 1 mmol/L 4-aminophenylmercuric acetate (Sigma), dissolved in dimethyl sulfoxide, in 50 mmol/L Tris-hydrochloric acid, pH 7.3, containing 200 mmol/L NaCI and 5 mmol/L CaCl₂, for 24 h at 37 °C.
Protein levels of MMP-2, MMP-9, MMP-12, TIMP-1 and TIMP-2 in BAL were measured using ELISA systems (Boster Biological Technology, Wuhan, PRC).
Cytospins of BAL were also analyzed by immunocytochemistry for the expression of MMPs using specific antibodies for MMP-2 and MMP-9 (Spring Bioscience, Pleasanton, USA) and for MMP-12, (Bioss). The staining was performed using the Bond Polymer Refine Detection kit (Leica Biosystems, Newcastle, UK). Specificity of the staining was also tested following the same incubation protocol in the absence of the antibody.

Zymogram activities were assayed using gelatin zymography, as described previously^{9 (link)}. Cells were treated with NTS only for 10 to 30 seconds and incubated for an additional 24 hours. The supernatant (100 μl) from each sample was mixed with 1 ml of 100 mM 4-aminophenylmercuric acetate (Sigma-Aldrich), and the samples were incubated for 1 hour at 37 °C. The sample was placed in sample buffer (without Mecatoeti) for 10 minutes and electrophoresed on a polyacrylamide gel at 125 V for 120 minutes at 4 °C using a Novex Xcell II system (Life Technologies, Carlsbad, CA, USA). The gels were incubated in renaturation buffer for 60 minutes at room temperature, followed by incubation for 18 hours in 100 ml of developing buffer at 37 °C under gentle shaking. The gels were then stained for 3 hours with Coomassie Brilliant Blue. After decolourization in 400 ml of methanol, 100 ml of acetic acid, and 500 ml of distilled water, images were obtained using an image analyzer.

Recombinant mouse MMP9 protein was purchased from Abcam. To obtain maximum latent MMP9, MMP9 was treated with 2.5 mM 4-aminophenylmercuric acetate (Sigma–Aldrich) in a buffer containing 50 mM Tris–HCl, pH 7.5, 1 mM CaCl₂, and 0.05% Triton X-100 at 37 °C for 1 h prior to use. The activity of MMP9 was verified with MMP9 colorimetric drug discovery kit (Enzo) according to the manufacturer's instructions.

Analysis of substrate cleavage by isolated proteases was performed at enzyme/chemokine (E:S) ratios from 1∶10,000 up to 1∶50 (mol:mol) for 16 h at 37°C in assay buffer (50 mM Tris, 200 mM NaCl, 5 mM CaCl₂, pH 7.4). MMP8 was activated by 1 mM 4-aminophenylmercuric acetate (Sigma). Digests were spotted on MALDI target plates with sinapinic acid for MALDI-TOF analysis or terminated by adding SDS-PAGE sample buffer. Reaction products were analyzed by 15% Tris-Tricine SDS-PAGE and silver stained. Specificity constants (k_cat/K_M) of cleavage were determined by densitometry as described previously [74] (link). The mass-to-charge ratios (m/z) with +1 ionization ([M+H]⁺) were determined on a Voyager-DE STR Biospectrometry Workstation (ABI). Mass spectrometry data were deconvoluted to identify the substrate cleavage sites. Molecular weight prediction was obtained using the “Compute pI/Mw tool” [75] on expasy.org.