할미송이버섯으로부터 혈전용해효소의 정제 및 특성 연구 (Ⅱ)

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할미송이버섯으로부터 혈전용해효소 (FE-2)를 DEAE-cellulose, Mono-S column으로 분리 정제하였다. 이 효소는 분자량이 18.2 kDa 이었으며, ICP-MS (inductively coupled plasma mass spectrometer) 분석 결과 Zn²?을 포함하고 있었다. 15번째까지의 N-terminal amino acid 서열은 A-L-Y-V-G-X-S-P-X-Q-Q-S-L-L-V이고, pH 7.5에서 활성이 가장 큰 염기성 단백질 분해효소로, EDTA와 1,10-phenanthroline에 의해 활성이 감소되는 metalloprotease였다. Mg²?, Zn²?, Fe²?, Co²?의 첨가 시 활성이 증가하였으나 Hg²?의 경우 활성이 완전히 소멸되었다. 이 효소 (FE-2)는 단백질 분해효소 저해제인 E-64 (trans-epoxysuccinyl-L-leucylamido-(4-guanidino)-butane), PMSF (phenylmethylsulfonyl fluoride), pepstatin과 2-mercaptoethanol의 영향을 받지 않으며, 섬유소원 (fibrinogen)과 반응 시 Aα chain과 Bβ chain은 분해시키지만 γ chain과는 반응하지 않았다.

Fibrinolytic enzyme (FE-2) was purified from the fruiting bodies of Tricholoma saponaceum using DEAE-Cellulose chromatography and Mono-S column chromatography. The enzyme has a molecular weight of 18.2 kDa and include Zn²? ion as found by ICP/MS. The N-terminal amino acid sequence of the enzyme was A-L-Y-V-G-X-S-P-X-Q-Q-S-L-L-V. It has a pH optimum at pH 7.5, suggested that FE-2 was a neutral protease. The activity of FE-2 was highly inhibited by EDTA and 1,10-phenanthroline, indicating that the enzyme is a metalloprotease. The activity of FE-2 was increased by Mg²?, Zn²?, Fe²?, and Co²?, but the enzyme activity was totally inhibited by Hg²?. No inhibition was found with PMSF, E-64, pepstatin and 2-mercaptoethanol. The enzyme hydrolyzed both Aα and Bβ chains of human fibrinogen. The γ chain was resistant to hydrolysis by FE-2.

#Fibrin plate assay #Fibrinolytic enzyme #Tricholoma saponaceum