Phospholipase A2 assay using an intramolecularly quenched pyrene-labeled phospholipid analog as a substrate.
A phospholipid analog 1-palmitoyl-2-6(pyren-1-yl)hexanoyl-sn-glycero-3-phospho-N- (trinitrophenyl)aminoethanol (PPHTE) in which pyrene fluorescence is intramolecularly quenched by the trinitrophenyl group was used as a substrate for pancreatic phospholipase A2. Upon phospholipase A2 catalyzed hydrolysis of this molecule pyrene monomer fluorescence emission intensity increased as a result of the transfer of the pyrene fatty acid to the aqueous phase. Optimal conditions for phospholipase A2 hydrolysis of PPHTE were similar to those observed earlier for other pyrenephospholipids (T. Thuren, J. A. Virtanen, R. Verger, and P. K. J. Kinnunen (1987) Biochim. Biophys. Acta 917, 411-417). Although differential scanning calorimetry revealed no thermal phase transitions for PPHTE between +5 and +60 degrees C the Arrhenius plot of the enzymatic hydrolysis of the lipid showed a discontinuity at 30 degrees C. The molecular origin of this discontinuity remains at present unknown. To study the effects of dimyristoylphosphatidylcholine (DMPC) phase transition at 23.9 degrees C on phospholipase A2 reaction PPHTE was mixed with DMPC in a molar ratio of 1:200 in small unilamellar vesicles. The hydrolysis of DMPC-PPHTE vesicles was measured by following the increase in pyrene monomer fluorescence emission due to phospholipase A2 action on PPHTE. Below the phase transition of DMPC the enzymatic reaction exhibited a hyperbolic behavior. At the transition as well as at slightly higher temperatures a lag period was observed. The longest lag period was approximately 20 min. Above 26 degrees C no lag time could be observed. However, the reaction rates were slower than below the phase transition temperature.(ABSTRACT TRUNCATED AT 250 WORDS)
Thuren
T
T
Department of Medical Chemistry, University of Helsinki, Finland.
Virtanen
J A
JA
Somerharju
P J
PJ
Kinnunen
P K
PK
D002118:Calcium; D004983:Ethanolamines; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D010179:Pancreas / Q000201:enzymology; D010712:Phosphatidic Acids; D010714:Phosphatidylethanolamines; D010740:Phospholipases / Q000032:analysis*; D010741:Phospholipases A / Q000032:analysis*; D054467:Phospholipases A2; D010743:Phospholipids; D011721:Pyrenes; D013050:Spectrometry, Fluorescence
D004983:Ethanolamines; D010712:Phosphatidic Acids; D010714:Phosphatidylethanolamines; D010743:Phospholipids; D011721:Pyrenes; C056297:1-palmitoyl-2,6-(pyren-1-yl)hexanoyl-sn-glycero-3-phospho-N-(trinitrophenyl)aminoethanol; D010740:Phospholipases; D010741:Phospholipases A; D054467:Phospholipases A2; D002118:Calcium