Fibrin Zymography: A Direct Analysis of Fibrinolytic Enzymes on Gels

Kim, Seung Ho; Choi, Nack Shick; Lee, Woo Yiel

doi:10.1006/abio.1998.2816

Cited by 103 publications

(52 citation statements)

References 11 publications

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“…Fibrin zymogram gel electrophoresis was carried out as described previously [18]. The separating gel solution (12%, w/v) was prepared in the presence of Wbrinogen (0.12%, w/v) and 100 l of thrombin (10 NIH U/ml).…”

Section: Zymographymentioning

confidence: 99%

Purification and characterization of a novel thermoacid-stable fibrinolytic enzyme from Staphylococcus sp. strain AJ isolated from Korean salt-fermented Anchovy-joet

Choi

Song

Chung

et al. 2008

J Ind Microbiol Biotechnol

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A novel Wbrinolytic enzyme (AJ) was puriWed from Staphylococcus sp. strain AJ screened from Korean salt-fermented Anchovy-jeot. Relative molecular weight of AJ was determined as 26 kDa by using SDS-PAGE and Wbrin zymography. Based on a 2D gel, AJ was found to consist of three active isoforms (pI 5.5-6.0) with the same N-terminal amino acid sequence. AJ exhibited optimum pH and temperature at 2.5-3.0 and 85°C, respectively. AJ kept 85% of the initial activity after heating at 100°C for 20 min on the zymogram gel. The Michaelis constant (K m ) and K cat values of AJ towards -casein were 0.38 mM and 19.73 s ¡1 , respectively. AJ cleaved the A -chain of Wbrinogen but did not aVect the B -and -chains, indicating that it is an -Wbrinogenase. The Wbrinolytic activity was inhibited by diisopropyl Xuorophosphate, indicating AJ is a serine protease. Interestingly, AJ was very stable at acidic condition, SDS, and heat (100°C), whereas it was easily degraded at neutral and alkaline conditions. In particular, AJ formed an active homo-dimer in the pH range from 7.0 to 8.0. To our knowledge, a similar combination of acid and heat stability has not yet been reported for other Wbrinolytic enzymes.

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Section: Zymographymentioning

confidence: 99%

Purification and characterization of a novel thermoacid-stable fibrinolytic enzyme from Staphylococcus sp. strain AJ isolated from Korean salt-fermented Anchovy-joet

Choi

Song

Chung

et al. 2008

J Ind Microbiol Biotechnol

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“…The purity was authenticated at 250 nm 30 . Fibrin zymography was carried out following a modified procedure described by Kim et al 31 . A separating gel (10% w/v) supplemented with 0.12% fibrinogen (w/v) solution and 12 ml of thrombin (100 NIH U/ml) was prepared.…”

Section: Sds-page and Fibrin Zymographymentioning

confidence: 99%

Xylarinase: a novel clot busting enzyme from an endophytic fungusXylaria curta

Meshram

Saxena

Paul

2016

Journal of Enzyme Inhibition and Medicinal Chemistry

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Xylarinase is a bi-functional fibrinolytic metalloprotease isolated from the culture filtrate of endophytic fungus Xylaria curta which is monomeric with a molecular mass of 33.76 kDa. The enzyme displayed both plasmin and tissue plasminogen activator like activity under in vitro conditions. It hydrolyses Aa and Bb chains of the fibrinogen. Optimal fibrinolytic activity of xylarinase is observed at 35 C, pH 8. Ca 2+ stimulated the fibrinolytic activity of xylarinase while Fe 2+ and Zn 2+ inhibited suggesting it to be a metalloprotease. The K m and V max values of xylarinase were 240.9 mM and 1.10 U/ml for fibrinogen and 246 mM and 1.22 U/ml for fibrin, respectively. Xylarinase was found to prolong the activated partial thromboplastin time and prothrombin time. The N-terminal sequence of xylarinase (SNGPLPGGVVWAG) did not show any homology with previously known fibrinolytic enzymes. Thus xylarinase is a novel fibrinolytic metalloprotease which could be possibly used as a new clot busting enzyme.

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“…The sample to generate unfolding patterns of SB consisted of 25 mM buffer, 20% glycerol, 150 g SB and 0.002% Bromophenol Blue. Effect of substrate variation on the unfolding of SB was followed using fibrin 20 instead of BSA. Briefly, fibrinogen (0.12%) and thrombin (10 NIH units/ml) were copolymerized with acrylamide and urea with the in situ generation of fibrin.…”

Section: Transverse Urea Gradient Zymographymentioning

confidence: 99%

Application of Transverse Urea Gradient Zymography for Structural and Functional Characterization of Proteolytic Enzymes

Sharma

Bhattacharyya

2016

Current Science

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Inactivation of an enzyme as it begins to unfold, along with the conformational perturbations which follow, can provide an insight into dynamics of the unfolding pathway. Urea gradient electrophoresis combined with zymography is a sensitive technique which provides a continuous visual profile of a proteolytic enzyme undergoing denaturation and inactivation simultaneously. Trypsin has been used as a reference protease to validate and standardize the method by correlating inactivation profile generated in zymography with a solution state assay. Stem bromelain, a cysteine endopeptidase was used as a case study to evaluate this methodology. The method highlighted the effect rendered by the substrate on the stability of the proteolytic domain of the enzyme, as it undergoes urea-induced unfolding. Transverse urea gradient zymography combined with molecular modelling of stem bromelain, where the disulphide bonds have been reduced, indicated that the evolutionary retention of Cys 23 -Cys 63 could be attributed to localized stabilization imparted by this bond to the catalytic site. This method encompasses various dimensions to extend the understanding of structure-function relationship in denaturant-induced unfolding pathways of proteases.Keywords: Protein unfolding, stem bromelain, urea gradient, zymography.TRANSVERSE urea gradient zymography (TUGZ) is an extended protocol based on the incorporation of transverse urea gradient in substrate polyacrylamide gels. Urea gradient electrophoresis provides a qualitative estimate of urea-induced conformational transitions whereas zymography identifies latent proteolytic activity under denaturing conditions [1][2][3][4][5][6] . Thus, the combination of two standard techniques can provide a comparative analysis of the unfolding behaviour of proteases having complex structures under variable conditions. There are fragmented reports where TUGZ was applied to verify stability of proteases against urea denaturation or to characterize proteolytic activity in a test sample 7,8 . In order to monitor the functional changes resulting from denaturant-induced unfolding, well-studied proteases such as trypsin, chymotrypsin and collagenase were selected which could validate and establish the general applicability of this method 9,10 . Poststandardization, the method was used to evaluate the behaviour of stem bromelain in the presence of urea.Crude extracts from the stem and fruit of pineapple plant (Ananas comosus) are called 'stem bromelain' and 'fruit bromelain' respectively. These bromelains contain a number of cysteine proteases having similar physiochemical properties along with other enzymes like peroxidase and enzyme inhibitors. These cysteine proteases are also called stem bromelain (SB) and fruit bromelain respectively, according to their sources. These cysteine proteases have highest abundance in the source where one component dominates. SB, a cysteine protease of 23.8 kDa, belongs to the ( + ) protein family 11 . It is the major enzyme present in pineapple stem extract. Some of th...

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Fibrin Zymography: A Direct Analysis of Fibrinolytic Enzymes on Gels

Cited by 103 publications

References 11 publications

Purification and characterization of a novel thermoacid-stable fibrinolytic enzyme from Staphylococcus sp. strain AJ isolated from Korean salt-fermented Anchovy-joet

Purification and characterization of a novel thermoacid-stable fibrinolytic enzyme from Staphylococcus sp. strain AJ isolated from Korean salt-fermented Anchovy-joet

Xylarinase: a novel clot busting enzyme from an endophytic fungusXylaria curta

Application of Transverse Urea Gradient Zymography for Structural and Functional Characterization of Proteolytic Enzymes

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