protein concentration factor - (Jan/15/2010 )

Hi all,

what is the effect of putting too much protein in enzyme assay? it is good to put as much protein as possible to make sure the reaction is complete. why we need to optimize and choose from the optimization.

You have to tell us more about your assay to get a usefull aswer. Most of the time is not necessary to maximize the concentrations of the reagens but in your assay who knows?

I'm conducting UDP-glucuronyl transferase (UGT) enzyme assay. The enzyme source is from microsomes I prepared my self from male SD rats with standard procedure (spun twice, the second time at 100,000g for 60 minute, pellet resuspended in phosphate buffer with 20% glycerol, kept at -80 degree). I'm refering to bock method, which only used 0.2 mg/ml protein, incubation time =15 minute. As for me, the reaction seem not change when I'm doing optimization by increasing the protein concentration. the product fromation is very less, from 0.5 mM substrate, only 0.02 mM product form. and when calculating enzyme activity, the variation between replicate was very high(eg: 0.02mM product will give activity = 6.67 nmol product/min/mg protein and 0.05mM product will give activity = 16.67 nmol/min/mg protein. one day I tried to add undiluted microsomes (which is very thick) and much product are formed with less variation between replicates. I'm not satisfied since I saw many articles published this works with small amount of protein conc (lesh than 2 mg/ml) and they got good result. What is wrong with my assay??

In theory, doubling the enzyme should double the reaction rate. Practically, its easier to see this with a purified enzyme but not often seen with an enzyme extract which is far more complicated. Its possible that when you add more extract other things in it are inhibiting your enzyme, and that is why you do not see an increaes in activity.

I would check the specific activity of your enzyme under the conditions that other labs measure it. If its extremely low, say 10-fold less or more than others, than your preparation of enzyme extract maybe bad, and you should make another preparation.

Another possibility is that your substrate is of poor quality. Maybe try to get another sample from a different company.