Dear colleagues, I have been trying to get crude extracts for the determination of the activity of antioxidant enzymes in seeds of a native tree species (Bignoniaceae). Embryos were separated and ground with liquid N to a fine powder. When adding typical buffers found in research publications for this purpose, either in the presence of DTT, EDTA, ascorbic acid, PVPP, triton 0.5% and protease inhibitors (PMSF), the extract (initially colorless) becomes gradually greenish (it is not due to the presence of pigments in the embryo!). The same occurs with the tissue powder (which is initially “white”) when kept in ice or at -18 ºC instead of liquid N, it turns greenish and then progressively darker. As far as I can understand, this seems to be a phenomenon similar to the tissue “browning” caused by the oxidation of polyphenolics (or other compounds?), even though, I insist, the first products are greenish (copper ions involved??). I have been trying different extraction procedures, including steps with some organic solvents such as chloroform, ethanol or methanol (the embryo tissue is also plenty of lipids), but despite in some occasions the aqueous phase was almost colorless, I could not detect any enzyme activity (also in cases where proteins should be present according to the Bradford test). If germinating embryos are used, the greening/browning process after pulverization with liquid N is much faster if the tissue powder is not kept in liquid N or after adding the extraction buffer. Everything points to some kind of inactivation of the enzymes due to this oxidation process (or whatever it is!). Any idea of what may be happening and how might I get extracts with active antioxidant enzymes? Sorry for the long explanation!
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