Capillary electrophoresis-laser-induced fluorescence (CE-LIF) assay for measurement of intracellular D-serine and serine racemase activity.
An enantioselective capillary electrophoresis-laser-induced fluorescence (CE-LIF) method for the analysis of D-serine (D-Ser) in cellular matrices has been developed. The assay involves derivatization with FITC followed by CE-LIF using 0.5 mM hydroxyl propyl-β-cyclodextrin in borate buffer [80 mM, pH 9.3]. The method was able to resolve D-Ser and L-Ser with an enantioselectivity (α) of 1.03 and a resolution (R(s)) of 1.37. Linearity was established from 0.25 to 100.00 μM. The assay was also able to enantioselectively resolve 6 additional amino acid racemates. The method was applied to the determination of intracellular D-Ser concentrations in PC-12, C6, 1312N1, and HepG2 cell lines. This method was used to determine the concentration-dependent increases in D-Ser and associated EC₅₀ values produced by L-Ser and the concentration-dependent decreases in d-Ser and associated IC₅₀ values produced by glycine, a competitive inhibitor of serine racemase (SR). Western blot analysis determined that the PC-12 and C6 cell lines contained monomeric and dimeric forms of SR while the 1321N1 and HepG2 cells contained only the monomeric form. Although the SR dimer has been identified as the active form of the enzyme, all four of the tested cell lines expressed enzymatically active SR.
Laboratory of Clinical Investigation, National Institute on Aging, National Institutes of Health, Baltimore, MD 21224, USA.
Nagendra S
NS
Singh
Rajib K
RK
Paul
Megan
M
Sichler
Ruin
R
Moaddel
Michel
M
Bernier
Irving W
IW
Wainer
D000818:Animals; D015153:Blotting, Western; D019075:Electrophoresis, Capillary / Q000379:methods*; D056945:Hep G2 Cells; D006801:Humans; D007834:Lasers; D019998:Racemases and Epimerases / Q000032:analysis*; D012015:Reference Standards; D012694:Serine / Q000032:analysis*; D013050:Spectrometry, Fluorescence / Q000379:methods*; D000099300:Serine Racemase
D019998:Racemases and Epimerases; D012694:Serine; D000099300:Serine Racemase