Determination of enzymatic activity and properties of secretory phospholipase A2 by capillary electrophoresis.
A capillary electrophoretic (CE) system coupled with a diode array UV detector was used for the assay of secretory phospholipase A2 (sPLA2) activity. This method is based on monitoring both the breakdown of substrates and the formation of products simultaneously using micellar electrokinetic chromatographic techniques. Under our developed separation conditions, we analyzed the substrates and products quantitatively, and investigated enzyme activity as a function of reaction time and presence of enzyme activator or inhibitor. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry was also utilized to confirm the phosphatidylcholine, a substrate of sPLA2. In order to test the feasibility of the developed method for measurement of enzymatic activity, we compared it to the conventional radioactive assay method for sPLA2. On the basis of our results, the conventional method can be complemented, or even replaced, by this new CE method which possesses the advantages of short analysis time, use of non-radiolabeled and inexpensive substrates, simple measurement of enzymatic activity, and exact quantitation of substrate and product.
Choi
S
S
Bioanalysis and Biotransformation Research Center, Korea Institute of Science and Technology, Cheongryang, Seoul, South Korea.
Lee
Y S
YS
Na
D S
DS
Yoo
Y S
YS
D016718:Arachidonic Acid / Q000032:analysis; D001881:Borates / Q000737:chemistry; D002021:Buffers; D003840:Deoxycholic Acid / Q000032:analysis; D019075:Electrophoresis, Capillary / Q000379:methods*; D006863:Hydrogen-Ion Concentration; D010713:Phosphatidylcholines / Q000032:analysis; D010741:Phospholipases A / Q000032:analysis* / Q000378:metabolism; D054467:Phospholipases A2; D011849:Radioactive Tracers
D001881:Borates; D002021:Buffers; D010713:Phosphatidylcholines; D011849:Radioactive Tracers; D003840:Deoxycholic Acid; D016718:Arachidonic Acid; D010741:Phospholipases A; D054467:Phospholipases A2