Purification and characterization of Protein C activator from Agkistrodon acutus Venom

• Published in: Preparative biochemistry & biotechnology Vol. 50; no. 9; pp. 907 - 914
• Main Authors: Sun, Yao, Bao, Peng-Ju, Zhang, Gen-Bao
• Format: Journal Article
• Language: English
• Published: England Taylor & Francis 2020; Taylor & Francis Ltd
• Subjects: Agkistrodon; Agkistrodon - metabolism; Agkistrodon acutus venom; Animals; Anticoagulants; Assaying; Cardiovascular diseases; Coagulation; Electrophoresis; Electrophoresis, Polyacrylamide Gel; Enzymatic activity; Enzyme Activation - drug effects; Enzyme Activators - chemistry; Enzyme Activators - isolation & purification; Enzyme Activators - pharmacology; Gel chromatography; Gel electrophoresis; Ion exchange; Metalloproteinase; Polyacrylamide; Protein C; Protein C - agonists; Protein C - metabolism; Protein C activator; Protein purification; Proteins; Rabbits; Snake Venoms - chemistry; Snake Venoms - metabolism; Sodium dodecyl sulfate; Sodium lauryl sulfate; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Thromboplastin; Venom; Zinc metalloproteinase; Agkistrodon acutus venom; Protein C; Protein C activator
• ISSN: 1082-6068; 1532-2297
• DOI: 10.1080/10826068.2020.1768541

Protein C (PC) plays an important role in the balance of coagulation and anticoagulation. Thus, the detection of PC activity is diagnostically significant for patients with cardiovascular diseases. Presently, the key methods to detect PC activity are the chromogenic assay and activated partial thromboplastin time (APTT) test. PROTAC used in the chromogenic assay is isolated from Agkistrodon contortrix venom as protein C activator (PCA). However, the use of the chromogenic assay is limited because of the high price of PROTAC. In this study, PCA was successfully purified from Agkistrodon acutus venom (AAV) by ion-exchange and gel chromatography. PCA from AAV has a relative molecular mass of 24 kD, calculated from the measurement of 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The components of PCA were identified by MALDI-TOF/TOF-MS and mascot searches revealed that the coverage rate between PCA and zinc metalloproteinase AaPA from AAV was 21%. The chromogenic assay and APTT test were used to measure the enzymatic activity of PCA, and the results showed that PCA from AAV could specifically activate PC. In summary, the chromogenic assay described herein is highly sensitive and easy to perform.