An improved assay for the determination of phospholipase C activity

• Published in: European journal of lipid science and technology Vol. 109; no. 5; pp. 469 - 473
• Main Authors: Durban, Markus A., Bornscheuer, Uwe T.
• Format: Journal Article
• Language: English
• Published: Weinheim WILEY-VCH Verlag 01.05.2007; WILEY‐VCH Verlag; Wiley-VCH
• Subjects: alkaline phosphatase; assay; Bacillus cereus; Biological and medical sciences; Fat industries; Food industries; Fundamental and applied biological sciences. Psychology; molybdate; Phospholipase C; Oils and fats industry; Bacillus cereus; Bacillales; molybdate; assay; Bacillus; Bacteria; alkaline phosphatase; Bacillaceae; Phospholipase C
• ISSN: 1438-7697; 1438-9312
• DOI: 10.1002/ejlt.200700027

Phospholipases C (PLC, EC 3.1.4.3) are enzymes that specifically hydrolyze the C‐O‐P bond in phospholipids, yielding sn‐1,2(2,3)‐diacylglycerides and the phosphate residue bearing the corresponding headgroup. The biochemical characterization of PLC requires methods for the reliable determination of their activity. Here, an assay is described in which the phosphate residue released by the PLC is cleaved with an alkaline phosphatase. The phosphate formed is then extracted with n‐butanol and quantified as phosphomolybate complex. The applicability of this method is demonstrated for a concentration range from 10 nM to 10 mM for a range of phospholipids bearing different headgroups in an aqueous and a two‐phase system. The method has the additional advantage that the crude enzyme can be used without the need for purification.