Hi,
I am determining pronase activity using a continuous fluorescence-based micro assay with FITC-casein as substrate (25 μg/ml final concentration in the assay). Excitation and emission wavelengths were suggested by the manufacturer (and several papers). Optics from the top. I used black microplates (black bottom). Constant volume of sample, substrate, and final volume. The data was blank subtracted.
Serial dilutions of pronase were prepared, and the initial reaction velocities (RFU/min) were measured for each dilution (slope in the linear region of the progress curve). The method seems to work OK. The activity decreased linearly with enzyme dilution, indicating a well-behaved and proportional enzymatic response across the concentration range tested.
However, when specific activity (EA/μg) was calculated by dividing the activity by the corresponding protein concentration (μg/mL), an unexpected trend was observed: specific activity increased as the enzyme was diluted, particularly at lower concentrations. It should be the same since the relationship between the enzyme activity and the enzyme dilution was linear.
I know that pronase is a mixture of different enzymes, which can be the source of the issue I found. I want to establish the activity in terms of a specific activity as a reference for future experiments. Have you seen this issue? How to deal with it?
Thanks!!!!
Specific activity can be thought of as the slope of the graph of activity versus enzyme mass. You wrote that "the relationship between the enzyme activity and the enzyme dilution was linear." The slope of that line is the specific activity, after dividing by the volume of the reaction.
Hi Adam B Shapiro , thanks for your concern!
I determined the reaction velocity (arbitrary units: RFU/min) for each dilution (two-fold dilution series) as the slope of the linear part (coef correl > 0,99) of each progress curve (blanked RFU vs time). The relationship between RFU/min and the dilution factor was linear. It looks great. So far, so good. Since all the assay mixtures have the same enzyme and final volumes, the activity was expressed as per ml. Then, I divided the activity/ml by the enzyme concentration at each dilution to obtain the specific activity at each dilution, which I expected to be the same. And it wasn't!! Since I noticed an unexpected trend, it occurred to me to graph RFU/min vs specific activity, and it was like an exponential decay! I have never seen that!! I have repeated the experiment twice with similar results.
Looking forward to your feedback. Thanks!
The problem may be with using FITC-casein. It isn't a well-defined, homogeneous substrate for kinetic studies. It consists of casein that has been saturation-labeled with FITC, causing self-quenching of fluorescein. As proteolysis occurs, FITC-peptides are released, partially relieving the self-quenching. As the reaction progresses, the substrate continuously changes.
The problem may be exacerbated by pronase being a mixture of enzymes, which may differ in their substrate specificities.
My suggestion is to try the experiment with a well-defined substrate, such as a FRET-labeled synthetic peptide or a C-terminal AMC-labeled synthetic peptide.
Hi Adam B Shapiro
Thanks for the hints! I will consider the substrates you mention. I suppose they would cover at least a relevant part of the several enzymes present in pronase, and hence can help to establish a reference specific activity. Before going any further, do you think that switching to a discontinuous FITC-Casein method, such as the one provided by Sigma (PF0100), could help to avoid the unwanted issue for determining the specific activity of pronase found using the continuous method I used? Thanks!
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