• 대한약침학회지
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• 제16권2호
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• pp.46-54
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• 2013

Objective: This study was undertaken to isolate a fibrin(ogen)olytic enzyme from the snake venom of Gloydius blomhoffii siniticus and to investigate the enzymatic characteristics and hemorrhagic activity of the isolated enzyme as a potential pharmacopuncture agent. Methods: The fibrinolytic enzyme was isolated by using chromatography, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and fibrin plate assay. The characteristics of the enzyme were determined by using fibrin plate assay, protein hydrolysis analysis, and hemorrhage assay. Its amino acid composition was determined. Results: The fibrin(ogen)olytic enzyme with the molecular weight of 27 kDa (FE-27kDa) isolated from G. b. siniticus venom consisted of two heterogenous disulfide bond-linked polypeptides with the molecular weights of 15 kDa and 18 kDa. When more than $20{\mu}g$ of FE-27kDa was applied on the fibrin plate, fibrinolysis zone was formed as indicating its fibrinolytic activity. The fibrinolytic activity was inhibited completely by phenylmethanesulfonylfluoride (PMSF) and ethylenediaminetetraacetic acid (EDTA) and partially by thiothreitol and cysteine. Metal ions such as $Hg^{2+}$ and $Fe^{2+}$ inhibited the fibrinolytic activity completely, but $Mn^{2+}$ did not. FE-27kDa preferentially hydrolyzed ${\alpha}$-chain of fibrinogen and slowly hydrolyzed ${\beta}$-chain, but did not hydrolyze ${\gamma}$-chain. High-molecular-weight polypeptides of gelatin were hydrolyzed partially into polypeptides with molecular weights of more than 45 kDa. A dosage of more than $10{\mu}g$ of FE-27kDa per mouse was required to induce hemorrhage beneath the skin. Conclusion: FE-27kDa was a serine proteinase consisting of two heterogeneous polypeptides, hydrolyzed fibrin, fibrinogen, and gelatin, and caused hemorrhage beneath the skin of mouse. This study suggests that the potential of FE-27kDa as pharmacopuncture agent should be limited due to low fibrinolytic activity and a possible side effect of hemorrhage.

• 대한약침학회지
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• 제17권1호
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• pp.44-50
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• 2014

Objectives: This study was undertaken to isolate a fibrinolytic enzyme from the snake venom of Gloydius blomhoffii siniticus and to investigate its enzymatic characteristics and hemorrhagic activity as a potential pharmacopuncture agent. Methods: The fibrinolytic enzyme was isolated by using chromatography, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and fibrin plate assay. The characteristics of the enzyme were investigated using fibrin plate assay, protein hydrolysis analysis, and hemorrhage assay. Its amino acid composition was determined. Results: The fibrinolytic enzyme with the molecular weight of 32kDa (FE-32kDa) from Gloydius blomhoffii siniticus showed a fibrin hydrolysis zone at the concentration of 0.2 mg/mL in the fibrin plate assay. The fibrin hydrolysis activity of the enzyme was inhibited completely by ethylenediaminetetraacetic acid (EDTA), ethyleneglycoltetraacetic acid (EGTA), and 1, 10-phenanthroline, thiothreitol and cysteine, and partially by phenylmethanesulfonylfluoride (PMSF). Metal ions such as $Fe^{2+}$ and $Hg^{2+}$ inhibited the fibrin hydrolysis completely, but $Zn^{2+}$ enhanced it. FE-32kDa hydrolyzed ${\alpha}$-chain but did not hydrolyze ${\beta}$-chain and ${\gamma}$-chain of fibrinogen. High-molecular-weight polypeptides of gelatin were hydrolyzed partially into low-molecular-weight polypeptides, but the extent of hydrolysis was limited. FE-32kDa induced hemorrhage beneath back skin of mice at the dose of $2{\mu}g$. Conclusions: FE-32kDa is a ${\alpha}$-fibrin(ogen)olytic metalloprotease that requires $Zn^{2+}$ for fibrinolytic activity and causes hemorrhage, suggesting that the enzyme is not appropriate for use as a clinical pharmacopuncture.

• BMB Reports
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• 제34권2호
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• pp.134-138
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• 2001

By adding sodium chloride (2.5%) into a Bacillus amyloliquefaciens DJ-4 culture broth, two serine-type fibrinolytic proteases with a molecular weight of 29 (subtilisin DJ-4) and 38-kDa were stimulated on the SDS-fibrin zymogram or inhibitor gels. B. amyloliquefaciens DJ-4 showed the highest proteolytic activity (5.52 plasmin NIH unit/ml) on the fibrin plate based on the molar ratio when cells were subjected to the 2.5% NaCl. Using a fibrin plate, the secreted protease from this strain in the presence of 5% NaCl showed that about 49% of the enzyme's activity remained after incubation at $60^{\circ}C$ for 30 min, but as the salt concentration was increased (10% NaCl) the activity nearly disappeared (0.14 plasmin NIH unit/ml). However, through a fibrin zymography assay, three fibrinolytic enzymes (38, 53 and 80-kDa) from the cells in the presence of 10% NaCl were detected. Also, two salt-activated serine-type fibrinolytic professes (29 and 38kDa) showed thermostability from 65 to $70^{\circ}C$ for 30 min. Furthermore, these professes also showed stability, pH 6-11. In particular, 29-kDa (subtilisin DJ-4) was very stable in the pH range of 4-11 at $4^{\circ}C$ for 48 h.

• 한국자원식물학회:학술대회논문집
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• 한국자원식물학회 2003년도 춘계 학술발표대회
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• pp.104-104
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• 2003

In this study, seven grain seeds(sorghum maize, buckwheat, soy bean, mung bean, red bean, and barngrass) and germinated seven grain seeds were examined the fibrinolytic activity through fibrin plate assay and SDS-PAGE. The results obtained were as follows : 1. In the fibrin plate assay, the extracts of maize, barngrass, sorghum and buckwheat showed fibrinolytic activity. Especially, Maize of them showed fibrinolytic activity that was almost similar to plasmin, fibrinolytic enzyme used as a positive control. 2. In the SDS-PAGE of seven grain seeds, fibrinolytic activity was remarkably shown in mung bean and red bean. 3. In the fibrin plate assay of germinated grain seeds, buckwheat(5 mm), buckwheat(10 mm) and soy bean(10 mm) showed a level of fibrinolytic activity that was about 0.3 fold than 1.0 unit of plasmin, Also maize(10 mm) of them showed a level of fibrinolytic activity that was about 0.5 fold than 1.0 unit of plasmin. As a result, maize of grain seeds was found that it has a strong fibrinolytic activity.

• 생명과학회지
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• 제20권6호
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• pp.838-844
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• 2010

토양에서 생육하는 Streptomyces corcohrussi의 혈전용해효소가 DEAE-Sephadex A-50 그리고 Sephadex G-50 젤 여과를 이용한 크로마토그라피 방법에 의해 순수분리 되었다. SDS-PAGE 분석결과, 분리된 효소는 단일 단백질이고, 그 분자량은 약 34 kDa 이라는 것을 알 수 있었다. 순수분리된 효소의 혈전용해활성은 plasminogen-rich fibrin plate에서 0.8 U/ml 이었으나, plasminogen-free fibrin plate 에서의 그 효소활성은 0.36 U/ml 이하이었다. 이러한 결과로, 순수 분리된 효소가 plasminogen activator 로 작용한다는 것을 알 수 있었다. 단백질 저해제인 $\varepsilon$-ACA, t-AMCHA 와 mercuric chloride의 존재시에 그 혈전용해활성은 24% 이하이었는데, 이러한 결과는 이들 plasmin 저해제 그리고(혹은) fibrinogen을 fibrin으로 전환시키는 과정과 관련된 fibrinogen 저해제에 의해 이 효소가 조절될 수 있음을 나타낼 수 있다. 한편으로, 중금속 이온인 $Zn^{2+}$은 그 활성을 58% 감소시켰다. 순수 분리된 효소의 최적 온도는 약 $50^{\circ}C$ 이었고, 그 효소활성의 92% 이상은 pH 5.0과 8.0 사이에서 유지되었다. 그러므로, 이러한 결과들은 하나의 강력한 혈전용해효소를 제공해서, S. corcohrussi 유래 새로운 혈전용해제의 개발에 기여하도록 한다.

• 생명과학회지
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• 제15권6호
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• pp.987-993
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• 2005

마른 볏짚으로부터 fibrinolytic enzyme (BK)을 분비하는 균주를 분리하여 동정한 결과, Bacillus subtilis속으로 분류되었다. 분리된 균주(Bacillus subtilis BK-17를 배양하여 그 배양액으로부터 에탄올 침전, ion exchange, gel filtration 등의 과정을 거쳐 fibrinolytic enzyme (BK)를 분리 및 정제하였다. 이 정제된 효소를 SDS-PACE gel 전기영동한 결과 분자량은 약 31 kDa 이었다. BK의 활성 및 안정성에 대한 pH의 영향을 조사해본 결과, pH $6\∼8$범위에서 매우 높았고, 최적 pH는 7과 8이었다. 본 효소의 활성 및 안정성에 대한 최적 온도는 $50^{\circ}C$이었으며, $20^{\circ}C\∼50^{\circ}C$범위에서는 안정성이 그대로 유지되었지만 $60^{\circ}C\∼80^{\circ}C$범위에서는 현저히 감소하여 $50^{\circ}C$에 비해 $20\%\∼40\%$의 활성을 보였다. BK에 대한 활성 역시 안정성과 비슷한 양상을 보였고, 다만 $20^{\circ}C$에서 약 $62\%$의 활성을 보였고, 그 이후 $50^{\circ}C$까지 지속적으로 증가하여 $50^{\circ}C$에서 최대의 활성을 보였다. 실험한 금속이온들 중 1 mM의 $Zn^{2+}$와 $Ca^{2+}$에서 각각 $35\%$와 $23\%$의 저해활성을 보였지만 나머지 이온들에서는 의미 있는 영향이 없었다. 동일농도의 EDTA에 대해서는 $45\%$의 저해활성을 보였기 때문에 BK는 metallo enzyme으로 생각된다. Fibrinogen-rich fibrin plate 와 plasminogen-free fibrin plate에서의 분해활성을 검토해본 결과, 두 plate에서 비슷한 분해활성을 보였다. 따라서 본 효소는 plasminogen activator type 보다는 fibrin에 직접 활성을 가지는 것으로 생각된다.

• Restorative Dentistry and Endodontics
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• 제47권2호
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• pp.17.1-17.8
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• 2022

Endodontic microsurgery is a predictable treatment option when orthograde treatment or retreatment is unsuccessful or unfeasible. However, when there is a gross compromise of periapical bone, achievement of bone regeneration after the surgical procedure may be hampered. In such cases, the application of guided tissue regeneration principles, with adjunctive use of leukocyte platelet-rich fibrin to fill the bone defect as a bone substitute and as a membrane to cover the site, provides a cost-effective solution with the benefits of accelerated physiological healing and reduced post-surgical pain and discomfort. This case report presents 2 cases of endodontic microsurgery of the upper lateral incisors with loss of buccal cortical plate, where platelet-rich fibrin was successfully applied.

• 생명과학회지
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• 제15권3호
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• pp.434-438
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• 2005

멸치 액젖을 이용하여 ammonium sulfate침전, ion exchange, gel filtration, ethanol침전등의 과정을 거쳐 혈전분해 효소(myulchikinase, MK)를 분리 및 정제하였다. 이 정제된 효소를 SDS-PACE gel 전기영동한 결과 분자량은 약 28 kDa 이었다. MK의 활성에 대한 특성을 조사한 결과 NaCl $30\%$까지 활성이 $80\%$이상 유지되는 것으로 보아 내염성 효소로 판단된다. 온도에 대한 효소활성을 조사한 결과, MK의 온도에 대한 안정성은 $40^{\circ}C$까지는 안정하였으나 $50^{\circ}C$이상의 온도에서는 급격하게 활성 떨어졌고, 최적온도는 $40^{\circ}C$였다 MK의 활성은 $pH6\~9$범위에서 매우 안정하였고, 최적 pH는 8이였다. 또한 2가 금속 양이온에 대한 효과는 $Hg^{2+} (1mM)$에 의하여 완전히 활성저해를 보였고, $Zn^{2+}(1mM)$에 의하여 약 $50^{\circ}C$의 저해되는 것을 알았다. Fibrinogen-rich plate와 Fibrinogen-free plate에서 활성을 측정 한 결과, Fibrinogen-rich plate에서는 fibrin 분해능이 있었지만 Fiberinogen-free Plate에서는 분해활성이 없는 것으로 보아 본 효소는 plasminogen activator type의 혈전용해효소로 사료된다.

• 생명과학회지
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• 제16권2호
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• pp.357-359
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• 2006

대추 추출물에서 fibrin을 가수분해 하는 생리활성 물질이 존재함을 알고 이 추출물의 열과 산에 대한 안정성 및 그 특징을 조사 하였다. Fibrin plate method 를 사용하여 측정된 fibrinolytic activity 는 대추 추출물의 첨가량이 증가할수록 높았으며, 이 추출물은 $100^{\circ}C$ 에서 10분간 열처리시에 안정한것으로 나타났다. 또한 pH 2.0, 3.0, 4.0 에서 3시간 보관후 fibrinolytic activity 를 측정한 결과 control의 80% 이상의 활성을 나타내었다. 한편 투석후 (분자량 12,000) 에는 활성이 나타나지 않았으며, 이는 대추에서 추출한 fibrin 분해 생리활성 물질이 분자량이 낮은 저분자 물질임을 알 수 있었다.

• 한국균학회지
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• 제26권4호통권87호
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• pp.589-593
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• 1998

치악산에 자생하는 야생버섯 65종에서 fibrin 분해활성을 검색하였으며, 그 결과 9종의 버섯이 활성을 나타냈다. Agrocybe sp.와 Stropharia rugosoannulata는 작은 활성률을 보였으며, Lepiota sp.와 Coprinus comatus는 plasmin 1.5 unit의 $56%{\sim}58%$의 fibrin 분해활성을 보인 반면 Collybia maculata는 plasmin 1.5 unit와 거의 같은 활성을 보였고 Armillariella mellea와 Calocybe sp.와 Lepista nuda와 Trichaptum abietinum은 plasmin 1.5 unit의 약 2배의 fibrin 분해활성을 나타냈다.