Hi there,

1. The X-axis scale of your graphics is not linear (ie. the distance between X value is always the same whereas the actual variation between protein concentration is not). Format the graphics as a cloud and you will see that the curve is linear and saturates for high concentrations.

2. It looks like you are measuring enzyme intrinsic fluorescence and not any activity (fluorescence is constant from time zero and proportional to enzyme concentration). Saturation seems to come from your detection system (as soon as signal is above 50000 units.

Lag phase should be related to the time the equipment (which one) starts to measure. The enzyme is always active and no lag phase should be "real". But see 2 below.

1. For the second issue, looking a the graph in the left what I see is that no enzyme activity occurs. One posible option is that the enzyme is very active (and the subtrate exhausted an the lowest concentration of enzyme ad very quickly) but a second possibility is that the method is not working.

2. "Going" to the graph in the right, your enzyme or the medium/solvent is fluorescnet. The higher the concentration the higher the fluorescence.

Hello,

@Dominique, @Rafael: Thank you for your comments. Attached is the check for intrinsic fluorescence/fluorescence of the medium for my enzyme at time 0. I used increasing conc. of enzyme at time 0 for samples containing protein+substrate and for samples containing only protein. The substrate conc. is same for all the samples.

Only protein samples do not show any fluorescence, thus, the protein/medium should not be a contributing factor.

If the enzyme was very active, then for the previous expt (previous attached data sheet), with increasing time, the lower enzyme conc. should show increase in activity (reaching upto atleast 60,000units as is the case for 1-2uM enzyme). What do you think?

In general, the assay is fluorescence based. I use recombinant purified protein. The substrate is attached to a fluorophore (fluorescence is quenched here). After the enzyme has acted upon the substrate, the fluorophore is released from the substrate (from another enzymatic reaction), which results in increase in fluorescence units.

What else can I do to check the activity of my enzyme?

Thank you.

Dear Megha

I try to understand everything to help. In your recent message I do not understand the last part:

"After the enzyme has acted upon the substrate (OK until here), the fluorophore is released from the substrate (from another enzymatic reaction ???), which results in increase in fluorescence units (???)

My substrate is a peptide bound to a fluorophore. It's intensity is quenched when conjugated to the peptide substrate. But when the enzyme catalyzes the substrate, the modified substrate is now susceptible to protease cleavage. As a result, the fluorophore becomes free and no more quenched. Thus, with increase in product formation, the fluorescence should increase.

Hi again,

What I still don't understand is that your fluorescence at t=0 is not zero (it should actually be normalized to zero...) and that it also does vary with enzyme concentration though you said enzyme and buffer exhibit no fluorescence... So the question is how do you proceed for t=0 (do you add inactivated enzyme to the sample?)? Sample without enzyme should give the same result as the sample with enzyme if there is no reaction...

Is it possible that the enzyme concentration-dependent fluorescence at time zero is due to a change in fluorescence of the substrate when the enzyme just binds to it, rather than when the substrate is cleaved by the enzyme? This might be possible to observe as an increase in fluorescence polarization. Also, you could try following the reaction by another means, such as HPLC or UPLC, to see if the peptide is actually degraded.

Is the first time point, which you are calling time zero, really at time zero, or is there a certain amount of time elapsed between mixing the substrate with the enzyme and making the first measurement? If the latter is the case, then the amount of hydrolysis that occurs during this time interval is responsible for the enzyme concentration-dependent fluorescence increase, and it should be directly proportional to the enzyme concentration, which is what you see.

Hi Adam,

The first time point is actual 0min. I add the substrate simultaneously to all the enzyme conc, and I quench the reaction to take the reading simultaneously (a lag of max 30-40sec in case it is, between diff samples).

If the enzyme is so active at time 0, then why is it loosing activity/showing very less activity in the time course of 90min?

I think I understand 90% of everything now. If so, what I see is that enzyme plus substrate but without any enzymatic reaction is giving you fluorescence. The higher the enzyme the higher the fluorescence but without any enzymatic reaction as you see already at zero time an increase from 0 to 60,000 AU when you increase the ezyme concentration from 0 to 2. As you Megha say, at time zero there is not enzyme activity. In summary discarding that the enzyme is very active even at low concentrations, it seems that you re not measuring enzyme activity but something else that I cannot figure out.

You have not yet shown any time course in which the fluorescence signal changes with time.

Dear Megha,

have you considered your enzyme could be a (or near) single turnover enzyme? That is with a fast first step of the reaction (30-40 sec would be enough), leading to the release of your fluorophore but a second step of the reaction (regenerating the enzyme for a second turnover) so slow that you don't see it during your 90 minute-time course. This hypothesis may only be valid depending on the mechanism of your enzyme.

Hi Adam,

Attached is a time-course experiment with diff conc. of enzyme.

Hi Pierre-Yves Colin,

I do feel that my my enzyme might have a pre-steady state kinetics, but I do not have stop-flow to do it. Also, in my time-course experiments, increasing conc. of enzyme increases the reading at time=0min. But, in general if you increase enzyme conc., product formed is more. So, how can I say that my enzyme is having burst kinetics?

Increasing the enzyme concentration increases the reading at time zero because the reaction is going so fast at the high enzyme concentrations that it has already proceeded a bit before you can quench it and take the first measurement.

I don't see any evidence in your graph of a lag phase, but the data are pretty noisy, so it is hard to be sure.

Hi Adam,

So in such a case there is no evidence that it could be burst phase? In general, with an enzyme reaction, how can you get an indication that it could be burst phase?

A burst phase would probably not be observed unless the enzyme concentration were very high, stoichiometric with the concentration of product detected during the burst phase due to a single turnover of the enzyme in pre-steady-state kinetics. Your highest enzyme concentration was 1 µM. What was the concentration of product observed during the suspected burst phase?