Enzymatic Cleavage of MMP-2/-9 Octapeptide

The MMP-2/-9-cleavable octapeptide Fmoc–Gly–Pro–Leu–Gly–Leu–Ala–Gly–Gly was evaluated by enzymatic digestion with the use of collagenase IV (containing MMP-2/-9). In the first step, 1 mL of HBSS was added to 1 g of collagenase IV (205 u/mg) and mixed to complete dissolution. Activation of collagenase IV was performed using 1 mM APMA in 0.15 M NaOH for 1.5 h at 37 °C [24 (link),43 ]. The octapeptide stock solution was added to activated 1 mM collagenase IV and incubated at 37 °C. The aliquots were removed and analyzed by HPLC, between 2 and 12 h. The HPLC Dionex Ultimate 3000 (Waltham, MA, USA) was equipped with a bioZen Intact XB-C8 column (3.6 μm, 2.1 × 150 mm; Phenomenex, Aschaffenburg, Germany). The flow rate was set at 0.5 mL/min with a mobile phase consisting of solvent A (0.1% TFA) and solvent B (100% ACN). The gradient was as follows: 0–5 min, 0% of solvent B; 5–8 min, 0–30% of solvent B. The hydrolysis product was detected by MALDI TOF/TOF 5800 mass spectrometry (AB Sciex, Darmstadt, Germany).