Hello everybody,
I have a problem assaying PAL activity in Euphorbia plant, when I do the assay I got the abosrbance at 290 nm increased for the first 10 min and then decreased progressively with time up to 1 h. I happened for two different volumes of the extract added. As a control reaction, I used extracts from maize leaves and Ficus leaves, but they behaved similarly. The reaction was made in microplate with total volume 220 ul containing 100 mM Tris-HCl buffer pH 8.5, 50 mM L-Phenylalanine and 50 or 100 ul extract included. incubation was at 37 degrees. Control was same assay mix but without any extract.
Thanks
Thank you ... the procedure was in a reaction volume of 200 ul in a microplate I mixed 50 or 100 ul of extract with 50 mM final conc. L-Phenyl alanine in 100 mM Tris-HCl buffer buffer pH 8.5, mixed well and incubated at 37 degrees in a microplate reader with every 2 min raeding interval at 290 nm. As I mentioned the reading increased for about 10 min and then dropped progressively being at the end less than original reading at 0 time. control was reaction mix without extract and its absorbance remained constant
Hello Abu, perhaps your (crude) extract contains other enzymes, using the arising cinnamic acid as their substrate... So, the product is progressively metabolized to other intermediates of the cinnamate pathway, no longer absorbing at 290 nm...
Best regards
Enrico
Thanks Enrico ... yes this could happen but it does not explain why the reading go down even below the original value at zero time
Sorry, I had not taken into the due account that (essential) detail. Maybe other enzymes could act directly on phenylalanine (e.g. unspecific transaminases...?) therefore complicating your system and anyway causing an unexpected absorbance decrease...
Your question reminded me of this paper. Zhang, X., Gou, M., & Liu, C. J. (2013). Arabidopsis Kelch repeat F-box proteins regulate phenylpropanoid biosynthesis via controlling the turnover of phenylalanine ammonia-lyase. The Plant Cell Online, 25(12), 4994-5010.
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