Conjugated polyelectrolyte based real-time fluorescence assay for phospholipase C.
A fluorescence turnoff assay for phospholipase C (PLC) from Clostridium perfringens is developed based on the reversible interaction between the natural substrate, phosphatidylcholine, and a fluorescent, water-soluble conjugated polyelectrolyte (CPE). The fluorescence intensity of the CPE in water is increased substantially by the addition of the phospholipid due to the formation of a CPE-lipid complex. Incubation of the CPE-lipid complex with the enzyme PLC causes the fluorescence intensity to decrease (turnoff sensor); the response arises due to PLC-catalyzed hydrolysis of the phosphatidylcholine, which effectively disrupts the CPE-lipid complex. The PLC assay operates with phospholipid substrate concentrations in the micromolar range, and the analytical detection limit for PLC is <1 nM. The optimized assay provides a convenient, rapid, and real-time sensor for PLC activity. The real-time fluorescence intensity from the CPE can be converted to substrate concentration by using an ex situ calibration curve, allowing PLC-catalyzed reaction rates and kinetic parameters to be determined. PLC activation by Ca2+ and inhibition by EDTA and fluoride ion are demonstrated using the optimized sensor.
Liu
Yan
Y
Department of Chemistry, University of Florida, P.O. Box 117200, Gainesville, Florida 32611-7200, USA.
Ogawa
Katsu
K
Schanze
Kirk S
KS
D000818:Animals; D000838:Anions / Q000737:chemistry; D002384:Catalysis; D002417:Cattle; D003016:Clostridium perfringens / Q000201:enzymology; D004573:Electrolytes / Q000138:chemical synthesis / Q000737:chemistry*; D005453:Fluorescence; D006868:Hydrolysis; D007700:Kinetics; D010742:Phospholipid Ethers / Q000737:chemistry / Q000378:metabolism; D011108:Polymers / Q000138:chemical synthesis / Q000737:chemistry*; D012995:Solubility; D012996:Solutions; D010738:Type C Phospholipases / Q000032:analysis / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism*; D014867:Water / Q000737:chemistry
C052298:1,2-dihexadecyl-sn-glycero-3-phosphocholine; D000838:Anions; D004573:Electrolytes; D010742:Phospholipid Ethers; D011108:Polymers; D012996:Solutions; D014867:Water; D010738:Type C Phospholipases