Cathepsin b from bovine spleen

Hemin chloride, chloroquine, quinacrine, amodiaquine, mefloquine, 8-hydroxyquinoline, quinine, quinidine and the β-carbolines norharman, tryptoline, harman, and harmine were obtained from Sigma-Aldrich. Indazole compounds 1,1´-[2,2´-biphenyldiyl)bismethylene]bis(5-nitro-1H-indazol-3-ol) (DIM-32) and 1,1´-(o-xylylene)bis(5-nitro-1H-indazol-3-ol) (DIM-5) were previously synthetized^{45 (link)}. 3,3′,5,5′-Tetramethylbenzidine (TMB), 2,2′-azinobis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS) and tween 20 were obtained from Sigma and cysteine from Merck. DMSO and hydrogen peroxide (H₂O₂) were from Scharlau and Panreac, respectively. Globin was obtained from bovine hemoglobin (Sigma) by precipitation in acetone-0.1% HCl at low temperature^{89 (link),90 (link)}, and lab-stored crystallized and lyophilized bovine serum albumin (BSA) was from Sigma. cysteine proteases: papain from Carica papaya, ficin from fig tree latex and cathepsin B from bovine spleen were obtained from Sigma. The peptide Z-Phe-Arg-AMC was purchased from Bachem, and 7-amino-4-methylcoumarin (AMC) from Sigma.

Five available proteases with therapeutic importance were used: pure elastase from human leukocytes (Sigma-Aldrich, St. Louis, MO, USA E8140), cathepsin B from bovine spleen (cysteine-peptidase) (Sigma-Aldrich, C6286), α-chymotrypsin (serine peptidase) from bovine pancreas (Sigma-Aldrich, St. Louis, MO, USA C3142), collagenase from Clostridium histolyticum (metalloproteinases) (Sigma-Aldrich, St. Louis, MO, USA C2674), and thrombin from bovine plasma (serine protease) (Sigma-Aldrich, St. Louis, MO, USA T7513). Six commercially available proteases were also used: esperase from Bacillus sp. (serine-type protease) (Novozyme, Sigma-Aldrich, St. Louis, MO, USA P5860), proteinase K from Tritirachium album (serine protease) (Sigma-Aldrich, St. Louis, MO, USA P2308), subtilisin from Bacillus licheniformis (Subtilisin A is a member of the Serine S8 endoproteinase family) (Sigma-Aldrich, St. Louis, MO, USA P5380), and Aspergillus oryzae (fungal protease/peptidase complex produced by submerged fermentation of a selected strain of Aspergillus oryzae that contains both endoprotease and exopeptidase activities) (Sigma-Aldrich, St. Louis, MO, USA P6110), Bacillus licheniformis (endoprotease of the serine type) (Sigma-Aldrich, St. Louis, MO, USA P4860), and Bacillus sp. (a serine-type protease) (Sigma-Aldrich, St. Louis, MO, USA P3111).