Single Lipid Vesicle Assay for Characterizing Single-Enzyme Kinetics of Phospholipid Hydrolysis in a Complex Biological Fluid Full article
Journal |
Journal of the American Chemical Society
ISSN: 0002-7863 , E-ISSN: 1520-5126 |
||||||||
---|---|---|---|---|---|---|---|---|---|
Output data | Year: 2013, Volume: 135, Number: 38, Pages: 14151-14158 Pages count : 8 DOI: 10.1021/ja4046313 | ||||||||
Tags | Assays; Cerebrospinal fluid; Enzyme activity; Hydrolysis; Phospholipids; Sensitivity analysis | ||||||||
Authors |
|
||||||||
Affiliations |
|
Funding (3)
1 | Swedish Research Council | |
2 | Stiftelsen för strategisk forskning | |
3 | VINNOVA |
Abstract:
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.
Cite:
Tabaei S.R.
, Rabe M.
, Zetterberg H.
, Zhdanov V.P.
, Höök F.
Single Lipid Vesicle Assay for Characterizing Single-Enzyme Kinetics of Phospholipid Hydrolysis in a Complex Biological Fluid
Journal of the American Chemical Society. 2013. V.135. N38. P.14151-14158. DOI: 10.1021/ja4046313 WOS Scopus РИНЦ
Single Lipid Vesicle Assay for Characterizing Single-Enzyme Kinetics of Phospholipid Hydrolysis in a Complex Biological Fluid
Journal of the American Chemical Society. 2013. V.135. N38. P.14151-14158. DOI: 10.1021/ja4046313 WOS Scopus РИНЦ
Dates:
Submitted: | May 8, 2013 |
Published online: | Sep 11, 2013 |
Published print: | Sep 25, 2013 |
Identifiers:
Web of science | WOS:000330162900034 |
Scopus | 2-s2.0-84884833348 |
Elibrary | 20453340 |
Chemical Abstracts | 2013:1300874 |
Chemical Abstracts (print) | 159:480297 |
OpenAlex | W2093813389 |