A Comparison Between ADIFAB and Reporter Vesicles in the Measurement of PLA2 Activity

Abstract

ADIFAB is a fluorescent probe used to quantify the production of free fatty acids (FFA) in aqueous dispersion. ADIFAB is frequently used to measure the hydrolysis activity of phospholipase A2 (PLA2) in a variety of systems that range from synthetic phospholipid to apoptotic cells. PLA2 activity catalyzes the sn-2-ester bond of phospholipids, releasing a FFA and lysophospholipid. Changes in the emission wavelength of ADIFAB from 430nm to 505 nm produced by the interaction with FFA, can be measured by calculating the normalized difference between the two intensities (Generalized Polarization), serving as a quantifying method for PLA2 activity. In biological applications, it has been shown that the emission spectrum of ADIFAB is sensitive to a variety of ions such as calcium and carboxylates. This sensitivity can produce strong pitfall in measurement of FFA production. In this work we compare ADIFAB with a method presented by Davidsen et al. (BBA.1609 (2003) 95-101) to quantify the FFA production by PLA2 activity. This method use large unilamellar vesicles composed of 1,2-di-O-octadecyl-sn-glycero-3-phosphocholine as PLA2 activity reporter vesicles. Vesicles contain self-quenching concentrations of calcein, which is liberated by the exposure to PLA2 hydrolysis products. The kinetics of calcein liberation is closely correlated with PLA2 activity. The correlation curves shows that the FFA production reported by the two methods is comparable, reproducible, and quantitative. Also, the reporter vesicle method is less sensitive to different buffer conditions of the sample, showing great osmotic stability and high sensitivity to FFA production. In particular, we performed measurements of PLA2 activity in Staphylococcus aureus and Escherichia coli only the reported vesicle is able to detect the hydrolysis process. In presence of bacteria, and absence of PLA2,the ADIFAB spectra is red-shifted, making it insensitive to additional FFA production.