Isolation and activity characterization of proteases from Pangasius hypophthalmus epithelial mucus for their potential use as wound healing agents

AVILA RODRÍGUEZ, MARÍA ISABELA; MIRNA LORENA SANCHEZ; JORGE BENAVIDES

Conferencia; Biopartitioninig and Purification Conference (BPP 2019); 2019

Wound healing is a multistep process in which metalloproteases (MP) and serine proteases (SP) are strongly involved. When dysregulation of these components happens, the healing process is precluded. This type of proteases, have also been described within fish epithelial mucus, acting as innate immune response against pathogenic agents. Empirical evidence has shown that epithelial mucus of the Iridescent shark (Pangasius hypophthalmus) promotes skin regeneration. By which the isolation and characterization of these proteolytic agents from P. hyphophthalmus epithelial mucus is proposed, as to identify new MP and SP suitable for wound healing care. Epithelial mucus was extracted, pooled, clarified through centrifugation (ME) and concentrated by vacuum evaporation (EME). Protein concentration and protease activity were measured in each of the preparative steps. As well, the modulating and inhibitory effect of reported activating ions (Zn2+, Ca2+, K+ and Na+) and protease inhibitors (PMSF, benzamidine, EDTA, O-phenanthroline, and iodoacetamide) were evaluated through gelatin and casein zymography. An increase in concentration was achieved after the previously described process (ME 51.50  1.7 g/ml and EME 257.50  11.6 g/mL). Liquid protease activity was not favored through this process (504.8 U/mg for ME and 5.33 U/mg for EME), yet in-gel screening was enhanced after pretreatment. Through gelatin zymography ME and EME displayed 10 active bands (235-13 kDa), while on casein-zymography one band was observed (63 kDa), were EME presented higher resolution and definition of band. Ion and inhibition characterizations were only held for EME. K+ and Na+ enhanced gelatinolytic activity of medium weight bands (63, 58 and 48 kDa), while Ca2+ depleted most protease activity. Serine and cysteine protease inhibitors (PMSF and iodoacetamide) inhibited medium weight bands. MMP inhibitors exerted light inhibition to superior weight bands (114, 90 and 71 kDa). Benzamidine only depleted 45 kDa activity. The present analysis proves the presence of the MP and SP within P. hypopthalmus epithelial mucus. As well, it could be stated that uncharacterized sample concentration through vacuum evaporation optimizes proteases screening, making it suitable for pre-treatment when characterizing new proteolytic variants in fish epithelial mucus. This positive result opens the possibility for further protease characterization and evaluation as feasible agents for wound healing care.