Simplified methods for the synthesis of 2-hexadecanoylthio-1-ethylphosphorylcholine and for the determination of phospholipase A2 activity.
A simple and straight forward method was developed for the synthesis of 2-hexadecanoylthio-1-ethyl phosphorylcholine (HEPC). The new procedure, which used p-toulenesulfonate instead of 2-bromoethyl phosphorylcholine, not only reduced the reaction time but also allowed the reaction to proceed under mild conditions. Using HEPC as a substrate, we have also developed a microplate assay for measuring phospholipase A2 activity which is rapid and will be useful for analyzing a large number of samples in a very short time. The applicability of this assay method for assessing phospholipases A2 from two different sources and determining their kinetic constants is also demonstrated. This method can also be extended for measuring lipases and lysophospholipases using a suitable thioester. Thus, both synthesis and assay methods will be useful in basic and applied research on phospholipases and related enzymes.
Bhat
M K
MK
AFRC Institute of Food Research, Department of Protein Engineering, Reading Laboratory, UK.
Mueller-Harvey
I
I
Sumner
I G
IG
Goodenough
P W
PW
D000818:Animals; D002122:Calcium Chloride; D003840:Deoxycholic Acid; D004492:Edetic Acid; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008244:Lysophosphatidylcholines / Q000138:chemical synthesis*; D008832:Microchemistry / Q000379:methods; D017830:Octoxynol; D010179:Pancreas / Q000201:enzymology; D010741:Phospholipases A / Q000032:analysis*; D054467:Phospholipases A2; D010767:Phosphorylcholine / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000737:chemistry; D011092:Polyethylene Glycols; D012910:Snake Venoms / Q000201:enzymology; D013053:Spectrophotometry / Q000379:methods; D013552:Swine; D013696:Temperature
D008244:Lysophosphatidylcholines; D012910:Snake Venoms; D003840:Deoxycholic Acid; D010767:Phosphorylcholine; D011092:Polyethylene Glycols; C019574:thioglycollecithin; D017830:Octoxynol; D004492:Edetic Acid; D010741:Phospholipases A; D054467:Phospholipases A2; D002122:Calcium Chloride