Choi Nack-Shick, Yoo Ki-Hyun, Yoon Kab-Seog, Maeng Pil Jae, Kim Seung-Ho
Proteome Research Laboratory, Korea Research Institute of Bioscience and Biotechnology, Daejeon 305-333, Korea.
J Biochem Mol Biol. 2004 May 31;37(3):298-303. doi: 10.5483/bmbrep.2004.37.3.298.
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).
一般来说，SYPRO Ruby染料是一种众所周知的基于荧光的灵敏方法，用于通过一维或二维SDS-PAGE（1-DE或2-DE）检测蛋白质。基于SYPRO Ruby染料系统，新开发了结合SYPRO Ruby染色的二维纤维蛋白酶谱法（2-D FZ）来鉴定芽孢杆菌属蛋白酶。也就是说，从韩国传统发酵食品豆酱中筛选出的芽孢杆菌属DJ-4的复杂蛋白质混合物在酶谱凝胶上显示出活性。通过基质辅助激光解吸电离飞行时间（MALDI-TOF）质谱分析对SYPRO Ruby凝胶上与二维纤维蛋白酶谱（2-D FZ）凝胶上显示的活性斑点相对应的凝胶斑点进行分析。通过二维纤维蛋白酶谱法检测到芽孢杆菌属DJ-4的五种细胞内纤溶酶。然后通过MALID-TOF MS分析SYPRO Ruby染料染色的二维凝胶上与二维纤维蛋白酶谱相对应的凝胶斑点。五个凝胶斑点中的三个被证明与枯草芽孢杆菌的ATP依赖性蛋白酶、细胞外中性金属蛋白酶和蛋白酶非常相似。此外，利用该组合系统鉴定了芽孢杆菌属DJ-4在三种凝胶（如酪蛋白、纤维蛋白和明胶）上的细胞外蛋白酶，并建立了蛋白水解图谱。这种二维酶谱法和SYPRO Ruby染料的组合系统应该有助于从许多其他来源（如酵母和癌细胞系）的复杂蛋白质混合物中寻找特定的蛋白酶。
