enzyme activity - (Jun/11/2002 )
I whish to test enzyme activity. But before I don't know how to extract the enzyme from a vegetable. Do I need to have enzyme purification or is it enough to precipitate all the proteins and to use them for measuring enzyme activity?
thank you for help
To extract enzymes from vegetable, it is helpful for you to know how your target enzymes are categorized. If you want to know Ascorbate peroxidase kinetics, you can take it into account to extrat total protein without centrifugation because it is bound in membrane. the Composition of buffer used for extraction may vary accoring to the target enzyme property, worker's taste...
It is very convenent for you to extract total protein with assay buffer which is used for target enzyme reaction. Because enzymes are labile to heat, protease and proteinase, oxidizing reagent,... people often add DTT and/or EGTA(EDTA) and/or PMSF in extraction buffer. In some case, DTT in crude extract affects enzyme reaction, as it is a strong reducing agent. If you are interested in a enzyme, firstly looking up Enzymolygy journal for the assay method. And then you can search jeounals for specific protocol in pubmed.
(Edited by Jinhan Hong at 8:42 pm on June 12, 2001)
In addition to previous thread, I must say introduction for plant enzyme assay.
Plant tissue is so rigid that you can use a homogenizer or pestle and mortal to grind plant material in liquid nitrogen. After homogenization and elimination of debris by centrifugation at 15000g for 15min, the supernatant can be subjected to common protocol.