In this section, proteases are categorized into three modes of actions: (1) serine protease; (2) cysteine protease; (3) metalloprotease.1)
Serine proteases are inhibited by AEBSF, benzamidine, etc.2) Cysteine proteases are inhibited by compounds which react with SH groups such as 2-iodoacetamide or 2-iodoacetic acid.3) Metalloproteases are inhibited by chelating agents, EDTA or 1,10-phenanthroline.4)
In the course of protein extraction, proteolysis is considered to be a major problem because it leads to decreasing yields. Addition of protease inhibitors helps to avoid the proteolysis and improves recovery of the desired protein.5) In the experiment of immunoprecipitation, protease inhibitors are also used to avoid decomposition of antigens or antibodies by proteolytic impurities.6) Protease inhibitors which are frequently used in biochemical research are illustrated in this section.
Thiol protease inhibitors, 2-iodoacetamide and 2-iodoacetic acid, cannot be used in protein extraction, because they form covalent bonds to the SH group of the proteins. In protein research, they are used as an alkylation reagent to derivatize the proteins which are extracted from an electrophoresis gel for mass spectrometric analysis.7)
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Enzyme Inhibitors [Biochemicals and Reagents]
References
- 1) G. G. Guilbault, in Handbook of Enzymatic Methods of Analysis, Clinical and Biochemical Analysis, A series of Monographs and Textbook, ed. by M. K. Schwartz, Marcel Dekker, Inc., New York, 1976.
- 2) (a) P. Walsmann, M. Richter, F. Markwardt, Acta Biol. Med. Germ. 1972, 28, 577.
- (b) M. Mares-Guia, E. Shaw, W. Cohen, J. Biol. Chem. 1967, 242, 5777.
- 3) R. Arnon, Methods Enzymol. 1970, 19, 226.
- 4) H. Matsubara, Methods Enzymol. 1970, 19, 642.
- 5) (a) K. Weber, J. R. Pringle, M. Osborn, Methods Enzymol. 1972, 26, 3.
- (b) J. Sambrook, D. W. Russell, in Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Labolatory Press, New York, 2001.
- 6) P. L. R. Bonner, in Protein Purification, Taylor & Francis, New York, 2007.
- 7) W. R. Gray, in Protein Function: A Practical Approach, 2nd ed., ed. by T. E. Creighton, Oxford University Press, New York, 1997, Chap. 7, p. 165.
- 8) Handbook of Inhibitors, ed. by T. Akiyama, K. Kohu, Yodosha, 2006.