해양 녹조류인 구멍갈파래(Ulva pertusa)로부터 혈전분해효소의 정제 및 특성분석

Abstract

ABSTRACT

Purification and characterization of fibrinolytic enzyme from green algae Ulva pertusa

ABSTRACT

Purification and characterization of fibrinolytic enzyme from green algae Ulvapertusa

Kang, Seong-Ryeoung Advisor: Prof. Kim, Sung-Jun, Ph. D. Department of Life science Graduate School of Chosun University

Cardiovascular disease is the leading cause of death throughout the world and is viewed as a global epidemic. Thrombosis the abnormal localized blood clot inside a blood vessel is one of the main causes of cardiovascular diseases. Currently available antithrombotic drugs have undesirable side effects. In this study,fibrinolytic activity of fibrinolytic protease from green algae ulvapertusa was examined using in vitro assays and was compared with plasmin. The fibrinolytic enzyme was purified by using DEAE sepharose CL-6B fast flow chromatography followed by Sephadex G-75 gel filtration and MONO Q chromatography. The final specific activity of purified enzyme was 295.units per milligram and increased 57.65 fold comparing homogenate and final recovery yield was 2.5%. Ulvapertusa exhibited remarkably higher hydrolytic activity towards fibrin. The fibrinolytic activity of ulvapertusa was stronger than that of plasmin. Moreover, UPP had some plasminogen activator like activity. These results indicate that the enzyme purified from Ulvapertusa can be a potential candidate for the treatment of thrombosis. The apparent molecular weight of purified fibrinolytic enzyme was estimated to be about 50 kDa by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). The optimal reaction pH value and temperature for the enzyme activity were pH 6.0 and 40℃, respectively. The purified enzyme had fibrigenolytic activity showing that the protease rapidly hydrolyzed the Aα - chain followed by the Bβ - chains. The purified protease also hydrolyzed fibrin, preferentially digesting the α – chain followed by the β - chains. Interestingly the γ - chains of fibrinogen and the γ-γ chains of fibrins were not hydrolyzed. The activity of the purified enzyme was totally inhibited by Fe2+, Cu2+, and moderately Mg2+, but enhanced by the additions of Cs+, Mn2+, Ca2+, Co2+, Zn2+ metal ions. The protease activity was potently inhibited by APMSF, PMSF(serine protease inhibitor), and it was found to exhibit a specificity for the chromogenic substrate S-2288, S-2444, indicating that the purified enzyme is a factor serin like protease. The N-terminal amino acid sequence of purified enzyme was identified to be NYDAATLPEELYF. Therefore, the purified enzyme from Ulvapertusa can be an effective thrombolytic source.