title: Activity-based targeting of secretory phospholipase A2enzymes: A fatty-acid-binding-protein assisted approach.
abstract: Syntheses and enzymological characterization of fluorogenic substrate probes targeting secretory phospholipase A2(sPLA2) for detection and quantitative assays are presented. Three fluorogenic phosphatidylcholine analogs PC-1, PC-2, and PC-3 each containing the duo of 7-mercapto-4-methyl-coumarin fluorophore and 2,4-dinitroanaline quencher on either tail were synthesized from (R)-3-amino-1,2-propanediol and R-(-)-2,2-dimethyl-1,3-dioxolane-4-methanol. These small reporter groups are advantageous in preserving natural membrane integrity. Phosphocholine was incorporated into the sn-3 position of the glycerol backbone. Acyl amino group at the sn-1 position in PC-1 and PC-2 is meant to block sPLA1. The sn-1 and sn-2 positions of the glycerol backbone in PC-1 have a quencher terminated 12-carbon chain and fluorophore terminated 11-carbon chain respectively. PC-2 has a quencher terminated 3-carbon chain at the sn-2 and chain terminating fluorescent reporter at the sn-1 positions. PC-3 resembles PC-1 except for an ester instead of amide at the sn-1 position, because of which it is more similar to natural phospholipids than PC-1. It was designed to elucidate the effect of replacing the ester group with amide by comparing its hydrolysis rate with that of PC-1. Design principles apply to synthesis of other labeled phospholipids. Enzymological characterization using bee-venom sPLA2was performed by a fatty-acid-binding-protein fluorescence assay and by pH-Stat method in which the amount of fatty acid released by hydrolysis is given by the amount of base required to maintain a constant pH of 8.0. Hydrolytic activity toward PC-1 and PC-3 were each about 238±25μmol/mg/min and 537μmol/mg/min on unmodified phospholipid. Ester to amide change did not affect hydrolysis rates. Activity toward PC-2 was about 45-μmol/mg/min. PC-1 and PC-3 show potential for targeted real-time spectrophotometric assay of sPLA2.
authors.affiliation: Department of Chemistry and Biochemistry, California State University, Northridge, CA 91330-8268, USA. Electronic address: akeshavarz@ucmerced.edu.
authors.forename: Amir
authors.initials: A
authors.lastname: Keshavarz
authors.affiliation: Department of Chemistry and Biochemistry, California State University, Northridge, CA 91330-8268, USA.
authors.forename: Ligia
authors.initials: L
authors.lastname: Zelaya
authors.affiliation: Department of Physics and Center for Biological Physics, California State University, Northridge, CA 91330-8268, USA.
authors.forename: Jasmeet
authors.initials: J
authors.lastname: Singh
authors.affiliation: Department of Physics and Center for Biological Physics, California State University, Northridge, CA 91330-8268, USA. Electronic address: radha.ranganathan@csun.edu.
authors.forename: Radha
authors.initials: R
authors.lastname: Ranganathan
authors.affiliation: Department of Chemistry and Biochemistry, California State University, Northridge, CA 91330-8268, USA.
authors.forename: Joseph
authors.initials: J
authors.lastname: Hajdu
mesh_terms: D004789:Enzyme Activation; D005227:Fatty Acids / Q000737:chemistry / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000032:analysis* / Q000737:chemistry*; D015394:Molecular Structure; D010713:Phosphatidylcholines / Q000032:analysis / Q000737:chemistry*; D054497:Phospholipases A2, Secretory / Q000032:analysis* / Q000378:metabolism*
chemical_list: D005227:Fatty Acids; D005456:Fluorescent Dyes; D010713:Phosphatidylcholines; D054497:Phospholipases A2, Secretory
keywords: Fluorescent phospholipid substrates; Phospholipase A(2) assay; Phospholipid syntheses