[kemi1]

Hi everybody
I have two questions about enzyme activity. 1. If I want to purify an enzyme though some steps which aresolubilization, DE-52 column, hydroxyapaite column, Sephacryl S-200 column and I want to measure the enzyme activity, After an DE-52 column I have a fraction. How shall I measure the activite? Shall I put an substrate to the enzyme and measure the Absorbance?
I know the definition of the enzyme activity but how can I measure the enzyme acitivity both practically and theoretically ?
Thanks !   

[Yggdrasil]

In general, the activity assay depends on the protein, but it will generally involve incubating the enzyme with it substrate and measuring the rate of disapearance of substrate/rate of appearance of product using a spectrophotometer.  The activity assay you use will be the same at all points during the experiment, so you would use the same activity assay to measure the activity of your crude lysate and your column fractions.  As your preparation becomes more pure, the specific activity of your sample (units of activty/mg of protein) should increase, reflecting the removal of contaminating proteins from your sample.