Single lipid vesicle assay for characterizing single-enzyme kinetics of phospholipid hydrolysis in a complex biological fluid.
Artikel i vetenskaplig tidskrift, 2013

Imaging of individual lipid vesicles is used to track single-enzyme kinetics of phospholipid hydrolysis. The method is employed to quantify the catalytic activity of phospholipase A2 (PLA2) in both pure and complex biological fluids. The measurements are demonstrated to offer a subpicomolar limit of detection (LOD) of human secretory PLA2 (sPLA2) in up to 1000-fold-diluted cerebrospinal fluid (CSF). An additional new feature provided by the single-enzyme sensitivity is that information about both relative concentration variations of active sPLA2 in CSF and the specific enzymatic activity can be simultaneously obtained. When CSF samples from healthy controls and individuals diagnosed with Alzheimer's disease (AD) are analyzed, the specific enzymatic activity is found to be preserved within 7% in the different CSF samples whereas the enzyme concentration differs by up to 56%. This suggests that the previously reported difference in PLA2 activity in CSF samples from healthy and AD individuals originates from differences in the PLA2 expression level rather than from the enzyme activity. Conventional ensemble averaging methods used to probe sPLA2 activity do not allow one to obtain such information. Together with an improvement in the LOD of at least 1 order of magnitude compared to that of conventional assays, this suggests that the method will become useful in furthering our understanding of the role of PLA2 in health and disease and in detecting the pharmacodynamic effects of PLA2-targeting drug candidates.

Författare

Seyed Tabaei

Chalmers, Teknisk fysik, Biologisk fysik

Michael Rabe

Chalmers, Teknisk fysik, Biologisk fysik

Henrik Zetterberg

Göteborgs universitet

Vladimir Zhdanov

Chalmers, Teknisk fysik, Kemisk fysik

Fredrik Höök

Chalmers, Teknisk fysik, Biologisk fysik

Journal of the American Chemical Society

0002-7863 (ISSN) 1520-5126 (eISSN)

Vol. 135 38 14151-8

Ämneskategorier

Medicinska grundvetenskaper

Neurovetenskaper

DOI

10.1021/ja4046313

PubMed

23957250