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( Nack Shick Choi ),( Ki Hyun Yoo ),( Kab Seog Yoon ),( Pil Jae Maeng ),( Seung Ho Kim ) 생화학분자생물학회 2004 BMB Reports Vol.37 No.3
In general, a SYPRO Ruby dye is well known as a sensitive fluorescence-based method for detecting proteins by one-or two-dimensional SDS-PAGE (1-DE or 2-DE). Based on the SYPRO Ruby dye system, the combined two-dimensional fibrin zymography (2-D FZ) with SYPRO Ruby staining was newly developed to identify the Bacillus sp. proteases. Namely, complex protein mixtures from Bacillus sp. DJ-4, which were screened from Doen-Jang (Korean traditional fermented food), showed activity on the zymogram gel. The gel spots on the SYPRO Ruby gel, which corresponded to the active spots showing on the 2-D FZ gel, were analyzed by a matrix-assisted laser desorption ionization time of flight (MALDI-TOF) mass spectrometric analysis. Five intracellular fibrinolytic enzymes of Bacillus sp. DJ-4 were detected through 2-D FZ. The gel spots on the SYPRO Ruby dye stained 2-D gel corresponding to 2-D FZ were then analyzed by MALID-TOF MS. Three of the five gel spots proved to be quite similar to the ATP-dependent protease, extracellular neutral metalloprotease, and protease of Bacillus subtilis. Also, the extracellular proteases of Bacillus sp. DJ-4 employing this combined system were identified on three gels (e.g., casein, fibrin, and gelatin) and the proteolytic maps were established. This combined system of 2-D zymography and SYPRO Ruby dye should be useful for searching the specific protease from complex protein mixtures of many other sources (e.g., yeast and cancer cell lines).
( Nack Shick Choi ),( Jeung Ho Hahm ),( Pil Jae Maeng ),( Seung Ho Kim ) 생화학분자생물학회 2005 BMB Reports Vol.38 No.2
Effects of common electrophoretic reagents, reducing agents (β-mercaptoethanol [BME] and DTT), denaturants (SDS and urea), and non-ionic detergent (Triton X-100), on the activity and stability of bovine plasmin (b-pin) and human plasmin (h-pin) were compared. In the presence of 0.1% SDS (w/v), all reagents completely inhibited two pins, whereas SDS (1%) and urea (1 M) denatured pins recovered their activities after removal of SDS by treatment of 2.5% Triton X-100 (v/v). However, reducing agents (0.1 M of BME and DTT) treated pins did not restore their activities. Based on a fibrin zymogram gel, five (from b-pin) and four (from h-pin) active fragments were resolved. Two pins exhibited unusual stability in concentrated SDS and Triton X-100 (final 10%) and urea (final 6 M) solutions. Two bands, heavy chain-2 (HC-2) and cleaved heavy chain-2 (CHC-2), of b-pin were completely inhibited in 0.5% SDS or 3 M urea, whereas no significant difference was found in h-pin. Interestingly, 50 kDa (cleaved heavy chain-1, CHC-1) of b-pin and two fragments, 26 kDa (light chain, LC) and 29 kDa (microplasmin, MP), of h-pin were increased by SDS in a concentration dependent manner. We also found that the inhibition of SDS against both pins was reversible.