Determination of the Kinetic Parameters for Phospholipase C (Bacillus cereus) on Different Phospholipid Substrates Using a Chromogenic Assay Based on the Quantitation of Inorganic Phosphate

Abstract The kinetic parameters of the phosphatidylcholine-preferring phospholipase C from Bacillus cereus (PLC Bc ) have been evaluated for phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine substrates with a new assay based on the quantitation of inorganic phosphate (Pi). Treatment of the phosphomonoester product of the PLC Bc -catalyzed hydrolysis of these phospholipids with alkaline phosphatase releases Pi. This Pi forms a complex with ammonium molybdate that is then reduced by ascorbic acid to provide a blue molybdenum chromogen with an absorbance maximum at 700 nm. This highly sensitive assay may be used to determine accurately less than 5 nmol of Pi in solution. Performing the assay in 96-well plates provides a rapid and convenient method to evaluate a variety of phospholipids as substrates for PLC Bc. The assay has been utilized to ascertain the kinetic constants for the PLC Bc -catalyzed hydrolysis of 1,2-dihexanoyl- sn -glycero-3-phosphocholine, 1,2-di-hexanoyl- sn -glycero-3-phosphoethanolamine, and 1,2-dihexanoyl- sn -glycero-3-phospho- l -serine. It is found that these compounds are substrates for the enzyme with their V max s being in the order of phosphatidylcholine > phosphatidylethanolamine > phosphatidylserine.