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Activity-based targeting of secretory phospholipase A2 enzymes: 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 sPLA2 was 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.

journal_name

Chem Phys Lipids

journal_title

Chemistry and physics of lipids

authors

Keshavarz A,Zelaya L,Singh J,Ranganathan R,Hajdu J

doi

10.1016/j.chemphyslip.2016.11.006

subject

Has Abstract

pub_date

2017-01-01 00:00:00

pages

38-48

eissn

0009-3084

issn

1873-2941

pii

S0009-3084(16)30183-9

journal_volume

202

pub_type

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Activity-based targeting of secretory phospholipase A2 enzymes: A fatty-acid-binding-protein assisted approach.