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When should protease inhibitors be added? Which protease inhibitors should be used? Will EDTA interfere with Complex IV activity?
Asked on Sep 21 2011
Thank you for contacting us. Please find further information below about the use of protease inhibitors: When should protease inhibitors be added? Protease inhibitors should be added during the extraction procedure. Which protease inhibitors should be used? We recommend any off the shelf cocktail of protease inhibitor Will EDTA interfere with Complex IV activity? The role of Magnesium, Zinc and Sodium ions in the activity of cytochrome c oxidase (to my knowledge) is not yet fully understood and therefore the outcome of adding a chelator of this ions is all but a guess. Cytochrome c oxidase is composed of 13 subunits, 3 of which (I, II and III) form the catalytic core of the enzyme. The other subunits are involved in assembly, stability and regulation of the complex. There is Mg bound at the interface of subunits I and II of complex IV. Zn is bound to subunit Vb. There is Sodium bound to subunit I at a site which also accepts calcium and this has been shown to affect the spectral properties of the haeme a3 (which is critical for activity). Typically the activity buffer to carry out a CIV activity assay, contains a very small amount of EDTA. My suggestion is for the customer not to add more than 5 or 10uM EDTA final concentration to avoid any potential artifacts. We will add this to the protocol for other customers, thanks for bringing this to our attention. Please let me know if I can be of further help.
Answered on Sep 21 2011