Purification, and characterization of a new pro-coagulant protein from Iranian Echis carinatus venom

• Published in: Biochemistry and biophysics reports Vol. 38; p. 101701
• Main Authors: Khodadadi, Sayeneh, Rabiei, Hadi, Sardari, Soroush, Mahboudi, Hosein, Bayatzadeh, Mohammad Ali, Vazifeh Shiran, Nader, Sardabi, Maryam, Akbari Eidgahi, Mohammad Reza, Madanchi, Hamid, Mohammadpour, Nasser
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.07.2024; Elsevier
• Subjects: Blood coagulation; Echis carinatus, viper venom-factor II activator; Pro-coagulant; Protein purification; Venom; Blood coagulation; Pro-coagulant; Echis carinatus, viper venom-factor II activator; Protein purification; Venom
• ISSN: 2405-5808; 2405-5808
• DOI: 10.1016/j.bbrep.2024.101701

This work aimed to purify the proteins that cause blood coagulation in the venom of the Iranian Echis carinatus snake species in a comprehensive manner. Gel filtration chromatography (GFC), Ion exchange chromatography (IEC), and Size Exclusion High-Performance Liquid Chromatography (SEC-HPLC) were utilized in the purification of the coagulation factors. The prothrombin clotting time (PRCT) and SDS-PAGE electrophoresis were performed to confirm the coagulative fractions. The fraction with the shortest coagulation time was selected. The components of this designated fraction were identified through matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOF) following thorough purification. Circular dichroism (CD) was employed to determine the second structure of the coagulation factor. The crude venom (CV) was analyzed and had a total protein concentration of 97%. Furthermore, the PRCT of the crude venom solution at a concentration of 1 mg/ml was determined to be 24.19 ± 1.05 s. The dosage administered was found to be a factor in the venom's capacity to induce hemolysis. According to CD analysis, the protein under investigation had a helical structure of 16.7%, a beta structure of 41%, and a turn structure of 9.8%. CHNS proved that the purified coagulant protein had a Carbon content of 77.82%, 5.66% Hydrogen, 3.19% Nitrogen, and 0.49% Sulphur. In the present investigation, a particular type of snake venom metalloproteinase (SVMP) has undergone the process of purification and characterization and has been designated as EC-124. This purified fraction shows significant efficacy as a procoagulant. Our findings have shown that this compound has a function similar to factor X and most likely it can cause blood coagulation by activating factor II (FII). •Gel filtration and anion exchange chromatography were used to fractionate the E. carinatus venom.•Size exclusion – HPLC was used to purify a new 22 kDa pro-coagulating protein.•MALDI-TOF analysis shows Disintegrin metalloproteinase was the most similar component of the purified 22 kDa pro-coagulating protein.•Functional assay shows that purified coagulant function and probably structure are similar to coagulation factor X.