Enzyme source was prepared with phosphate buffer, triton x and pvp. Now with dilution buffer as phosphate buffer for peroxidase and tris buffer for Pal. Can we calculate the enzyme activity? Will there be any difference? And why?
From your question i could infer that the enzyme source for PAL and peroxidase is common. Since PAL works better at alkaline pH generally between 7.8-8.5, you need to use Tris buffer to find out the activity of PAL. The substrate for PAL would be phenylalanine which would be broken to Ammonia and Trans Cinnamate in the presence of this enzyme and you would moniter the formation of Cinnamate by taking absorbance at 270 nm.
However the peroxidase works better in neutral range you need to use Phosphate buffer for your activity. You can use a number of substrates as donars for peroxidase activity which form chromophoric products.
Hi
You can find a paper attached here in which they use 0.1% sodium phosphate buffer for the extraction of enzyems and from this single extract they analyse three enzymes (Phenylalanine ammonia lyase, Peroxidase and Polyphenol oxidase) activity. I think this may help you to save time and extra effort. See the attachment or you go for the paper "Rhizobacterial bioformulation for the effective management of Macrophomina root rot in mungbean" from the google.
Yes, you can determine activities of PAL as well as peroxidase with the same crude enzyme extract in phosphate buffer. However, make sure that the molarity of reaction buffer must be more (at least two to four times) than extraction buffer (i.e. phosphate buffer in which you extracted the protein). Unfortunately in the paper referred by Sudheer Yadav, authors stated that they used 0.1 M extraction buffer (phosphate buffer), and 0.01 M reaction buffer (phosphate buffer) for determining peroxidase activity & 0.05 M (50 mM) buffer (Tris-HCl buffer) for PAL activity. Authors were fortunate that both editor as well as reviewers overlooked this blunder.
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