Hi everyone!
My name is Nacho.
I am trying to assay lipase activity from insect midgut supernatant, and I have some problems and some doubts about it.
I do not have the pH-stat. Thus, the tritiation method was disregarded. Therefore, only colorimetric and fluorescent methods were available to me.
I understand that the colorimetric method is composed of a buffer in very low concentration, a pH indicator, and an emulsified substrate. The emulsion can be performed by sonication (with Arabic gum) or by adding a surfactant.
I assay phospholipase A according to Price et al. 2007 (in attachment) in midgut supernatant and purified insect phospholipase A1. In brief, it is a colorimetric microplate method using 2 mM HEPES, bromothymol blue, and phosphatidylcholine emulsified with 5 mM TritonX-100. I read the color change of the pH indicator at 620 nm at each min for 15 min. I had phospholipase activity considering blanks (sample without substrate) in both, supernatant and purified enzyme. However, there is a little or no difference in phospholipase activity between the different enzyme dilutions (10x, 20x). I also read the color variation at each hour for 5 hours. In that case, the activity falls along with time, even with the substrate in excess. I do not understand why the activity falls. Is there some evidence of lipases losing their activity?
I also performed a fluorescence assay method for TAG lipase in midgut supernatant using an olive oil emulsion in Arabic gum, and rhodamine B. Results were similar to the previous ones.
Summarizing, I do not know if I really have lipase activity, or if I am performing the method well.
Is there someone with lipase activity assay experience?
Thank you in advance
Reduction of enzyme activity over time is not unusual for two reasons:
Did you calibrate your method by adding small amounts of acid, so you know which proton concentration causes which colour change?
Have you verified that your enzyme is stable in the presence of the detergents? Which detergent do insects use in their gut: cholate like mammals? In that case, I'd switch and make the concentration about the same as present in the gut.
Is the pH of the buffer high enough that all fatty acids produced dissociate (pKa ~ 4.5)?
Did you use some lipase with known activity to check your assay?
Thank you very much for the tips.
Yes, we calibrated the method with HCl, and we know the proton concentration that causes the color change of the pH indicator. However, we don't know how to use this information to assay the lipase (and phospholipase).
We assay the enzyme with synthetic NP-ester, and we know that our enzyme is active. Perhaps, we can assay with NP-ester with different detergents to verified if our enzyme is stable in their presence. I know that it isn't cholate, but I still don't know what detergent do insects use in their guts.
Yes, the pH buffer is 7.5.
No, we don't have a lipase with known activity. Now, we have our purified phospholipase, which is active with NP-esters.
However, we don't see any activity assaying with a natural substrate (phosphatidylcholine) and colorimetric method.
when you hydrolyse lipids, you get alkanoate and protons, each proton is equivalent to an ester bond cleaved. The velocity is mol product formed per second, i.e., katal (or a small fraction thereof, nkat or fkat). The specific activity (kat/mg of enzyme) is a measure of purity. The turnover number, molecules of product formed by a molecule of enzyme in one second, allowes the comparison of, say, isoenzymes.
- Badges
- Science topic
- Similar topics
- Microbiology
- Assays