I am not an expert on this.

Maybe phospholipids are not accessible in the micelles or lipase got deactivated?

Hi Murrugapan, try this buffer: 250 mM Tris-HCl, 500 mM NaCl, 5mM CaCl2, pH 8.9. pH may be most important

A few thoughts on this issue: 1. PLA2 generally works better in slightly alkalotic environment (@ pH=~8 or so); 2. in most cases, calcium is necessary to make the enzyme work (agree with Mykhaylo here!); 3. Beware of the TLC plate material and the solvent you use to separate phospholipids from lysophospholipids (I assume the lyso-form is what you want to see; otherwise you may need to find a way of eluting fatty acid moieties with TLC).

Just my $ 0.02.

I agree with above. Bee venom is similar to snake venom in Ca requirement and higher pH (8-9). According to my experience this type of phospholipases, they need to be intercalated into bilayer or micelles just to reach a proper ester bond of PL. They are fragile to alteration of the physical state of the suprastructure to be digested. I mean, that the "fluidity" of PL significantly alter the activity of PLA2s.

Concluding, maybe your detergent molecules affect the activity of your PLA2 by themself. Did you check the activity on detergent-free system (eg. liposomes)? Snake venom PLA2 can be "inhibited" by addition of various amphiphiles to the liposomal system. But this "inhibition" is only due to the increase of the lag time. I have observed in a continous titration system (pHstat) almost 100 times increase of lag time, but aferwards it started correctly working. If your enzyme will work in the control system in will be a proof that your detergent is a kind of inhibitor. On the other hand, try longer times of incubation.

The file attache is just an example.

I agree with the above. You should consult the provider of the bee venom for the optimal pH and salt concentrations.. With regards to separating the lyso lipids from the intact phospholipids, a solvent system of chloroform/methanol/acetic acid/water ; 80:18:12:5 (by vol.) on silica gel 60 separates most lyso-PLs from the intact lipids. You may also see the released fatty acids accumulating near solvent front when you compare the digest to the undigested phospholipids. Check the position of the fatty acid products by running a standard of oleic acid.

Thank you Dr.Howard Goldfine . Your solvent system worked very well to detect lyso phospholipds from the intact lipds. It looks like the enzyme is active in cleaving only phosphatidyl choline and phosphatidyl ethanolamine at pHs 7.4 to 9.0 after 2hrs incubation.