lipase activity assay - p-nitrophenyl myristate - Turbidity of my substrate solution (May/08/2008 )
hi to everybody,
I want to assay lipase potential of a purified enzyme. I use para-nitrophenyl myristate as substrate, dissolved in 2-propanol to a concentration of 16.5 mM (solution A) -I sonicate for 5 min at 30°C
I also prepare a solution of 50 mM Na phosphate buffer ph 7.2, or ph 8.0 containing 0.4% Triton X-100 (Solution .
in order to have my substrate solution, I combine 1 mL solution A + 9 mL of solution B, but at this step it forms aggregates, making impossible to read at spectrophotometer
Does somebody know how to prepare a clear solution with this substate?
Do I have to use a different solvent instead of 2-propanol? any idea is welcome.
hope to hear you soon
Do the aggregates clear after a couple of hours? This may be just the emulsion formed when PNP-myristate contacts water. The lipase then hydrolyzes the PNP-myristate. You might need to add some deoxycholate (bile acid).
What happens if you leave out the PNP-myristate and just add the propanol to the Kphos? Try just the propanol with the triton-x, that might be the problem. (you want to find out which ar ethe two immiscible species in the mix).
Other than that, check "Data for Biochemical Research," (Dawson, Elliot, Elliot & Jones. Oxford press) for solubility details. At 20deg C, looks like you will get up to 32.5g myristate in 100ml of CHCl3. Or get 23.5g in 100ml EtoH. or 15.3g in 100ml EtAcetate. As with the propanol-KPhos, assess the miscibility without myristate first (saves wasting precious sample).
A wild alternative is to use the old spectroscopic method: lipase plus olive oil (or linoleic if you are unsure about the consistency of the olive oil from bottle to bottle). Use an emulsion of the olive oil (i.e. pop it in water) and incubate at pH7.8 for 3hrs at 37deg C. This liberates fatty acids which you titrate to pH10.5 with NaOH (50mmol/l) or to a light blue shade with thymolphthalein indicator.