PURIFICATION AND BIOCHEMICAL CHARACTERIZATION OF A PROTEASE FROM THE MUCUS OF THE SCORPION FISH Scorpaena plumieri
Name: MILENNA MACHADO PIROVANI
Publication date: 09/10/2024
Examining board:
Name |
Role |
|---|---|
| HEBERTH DE PAULA | Examinador Interno |
| JULIANA BARBOSA COITINHO GONCALVES | Coorientador |
| SUELY GOMES DE FIGUEIREDO | Presidente |
| THIAGO NUNES DE MENEZES | Examinador Externo |
Summary: Proteases are the efficient executioners of a common chemical reaction, the hydrolysis of peptide bonds. These enzymes appear in all domains of life, including plants, animals, and microorganisms. Due to their involvement in various important biological processes (e.g., blood coagulation, blood pressure regulation, protein processing, and antimicrobial activity), as well as their high specificity, these enzymes have clinical and industrial biotechnological potential. Recently, using a proteomic approach – ‘shotgun’ modality – our research group identified 228 proteins in the epidermal mucus of the Brazilian scorpionfish, Scorpaena plumieri, including various classes of proteases. In this study, a gelatinolytic protease from S. plumieri skin mucus (named Sp-MGP) was purified to homogeneity through a combination of two chromatographic steps: conventional gel filtration (Sephacryl S-200 column) and anion exchange HPLC (SynChropak AX300 column). The purification process was monitored by zymography (SDS-PAGE gelatin). Physicochemical studies indicated that the purified enzyme is an approximately 79-kDa monomeric glycoprotein, as estimated by SDS-PAGE under both reducing and non-reducing conditions. Zymogram analysis of Sp-MGP showed gelatinolytic zones of activity in the gel, with optimal activity at pH 6.0 to 9.0, which is typical of serine proteases. The enzyme stored in aqueous solutions at room temperature (25–30°C), 4°C, -25°C, and -80°C for 60 days did not lose its activity. Sp-MGP was also active on the thrombin (S-2238) and kallikrein (S-2266) synthetic substrates. The activity on S- 2238 was inhibited by aprotinin and benzamidine. These data reaffirm the suggestion that the purified enzyme is a serine protease, specifically of the thrombin-like and kallikrein-like subclasses. The information generated represents the initial step in the study of a novel protease and may contribute to exploring its biotechnological potential, in addition to providing knowledge about the diversity of molecules in fish skin mucus.
