BMB Reports

Korean Society for Biochemistry and Molecular Biology (생화학분자생물학회)
권호 표지
DOI QR코드

DOI QR Code

A Method for Direct Application of Human Plasmin on a Dithiothreitol-containing Agarose Stacking Gel System

  • Choi, Nack-Shick (Systemic Proteomics Research Center, Korea Research Institute of Bioscience and Biotechnology) ;
  • Chung, Dong-Min (Systemic Proteomics Research Center, Korea Research Institute of Bioscience and Biotechnology) ;
  • Yoon, Kab-Seog (Systemic Proteomics Research Center, Korea Research Institute of Bioscience and Biotechnology) ;
  • Maeng, Pil-Jae (Department of Microbiology, Chungnam National University) ;
  • Kim, Seung-Ho (Systemic Proteomics Research Center, Korea Research Institute of Bioscience and Biotechnology)
  • Published : 2005.11.30
Download PDF
⟨ Previous Next ⟩

Abstract

A new simplified procedure for identifying human plasmin was developed using a DTT copolymerized agarose stacking gel (ASG) system. Agarose (1%) was used for the stacking gel because DTT inhibits the polymerization of acrylamide. Human plasmin showed the lowest activity at pH 9.0. There was a similar catalytically active pattern observed under acidic conditions (pH 3.0) to that observed under alkaline conditions (pH 10.0 or 11.0). Using the ASG system, the primary structure of the heavy chain could be established at pH 3.0. This protein was found to consist of three fragments, 45 kDa, 23 kDa, and 13 kDa. These results showed that the heavy chain has a similar structure to the autolysed plasmin (Wu et al., 1987b) but there is a different start amino acid sequence of the N-termini.

Keywords

References

  1. Brennan, J. P., Wait, R., Begum, S., Bell, J. R., Dunn, M. J. and Eaton, P. (2004) Detection and mapping of widespread intermolecular protein disulfide formation during cardiac oxidative stress using proteomics with diagonal electrophoresis. J. Biol. Chem. 279, 41352-41360 https://doi.org/10.1074/jbc.M403827200
  2. Choi, N. S., Hahm, J. H., Maeng, P. J. and Kim, S. H. (2005) Comparative study of enzyme activity and stability of bovine and human plasmins in electrophoretic reagents, ${\beta}$- mercaptoethanol, DTT, SDS, Triton X-100, and urea. J. Biochem. Mol. Biol. 38, 177-181 https://doi.org/10.5483/BMBRep.2005.38.2.177
  3. Choi, N. S. and Kim, S. H. (2000) Two fibrin zymography methods for analysis of plasminogen activators on gels. Anal. Biochem. 281, 236-238 https://doi.org/10.1006/abio.2000.4572
  4. Kim, S. H. and Choi, N. S. (1999) Electrophoretic analysis of protease inhibitors in fibrin zymography. Anal. Biochem. 270, 179-181 https://doi.org/10.1006/abio.1999.4080
  5. Kim, S. H., Choi, N. S. and Lee, W. Y. (1998) Fibrin zymography: a direct analysis of fibrinolytic enzymes on gels. Anal. Biochem. 263, 115-116 https://doi.org/10.1006/abio.1998.2816
  6. Schaller, J., Moser, P. W., Dannegger-Muller, G. A. K., Rosselet, S. J., Kampfer, U. and Rickli, E. E. (1985) Complete amino acid sequence of bovine plasminogen: comparison with human plasminogen. Eur. J. Biochem. 149, 267-278 https://doi.org/10.1111/j.1432-1033.1985.tb08921.x
  7. Matsudaira, P. (1987) Sequence from picomole quantities of proteins electroblotted onto polyvinylidene difluoridemembranes. J. Biol. Chem. 262, 10035-10038
  8. Wu, H. L., Shi, G. Y. and Bender, M. L. (1987a) Preparation and purification of microplasmin. Proc. Natl. Acad. Sci. USA 84, 8292-8295
  9. Wu, H. L., Shi, G. Y., Wohl, R. C. and Bender, M. L. (1987b) Structure and formation of microplasmin. Proc. Natl. Acad. Sci. USA 84, 8793-8795

Cited by

  1. Protein reducing agents dithiothreitol and tris(2-carboxyethyl)phosphine anodic oxidation vol.23, 2012, https://doi.org/10.1016/j.elecom.2012.06.027