Capillary electrophoresis method for determination of D-serine and its application for monitoring of serine racemase activity.
Serine racemase (SR) is an enzyme responsible for the biosynthesis of D-serine, the coagonist of the N-methyl-D-aspartate receptor, in the brain. Therefore, it has been suggested as a possible therapeutic target for the treatment of various neurodegenerative diseases. To develop a potent inhibitor of SR, a simple, sensitive, fast, and robust assay is needed. In this paper, a new CE method for the determination of D-serine is described. Serine enantiomers are resolved in the form of o-phthaldialdehyde (OPA)/2-mercaptoethanol (2-ME) derivatives in an alkaline BGE composed of 50 mM sodium tetraborate, pH 9.7, and containing 40 mM 2-hydroxypropyl-gamma-CD as a chiral selector. The problem of time-limited stability of OPA/2-ME derivatives has been overcome by employing in-capillary derivatization of the sample, i.e., the derivatization reaction was carried out directly in the separation capillary in the first phase of the CE run. UV-absorption detection at 230 nm allowed concentration detection limit of 3 microM. Baseline resolution of D- and L-serine derivatives was achieved in less than 10 min. This fact, together with the simple sample pretreatment, allowed application of the method to medium-throughput monitoring of SR activity, such as the screening of potential SR inhibitors. A good agreement was achieved between the developed CE method and the previously established HPLC method for determination of the inhibition constant, K(i), of a new SR inhibitor, L-erythro-3-hydroxyaspartate.
Koval
Dusan
D
Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Prague.
Jirásková
Jana
J
Strísovský
Kvido
K
Konvalinka
Jan
J
Kasicka
Václav
V
D000818:Animals; D002851:Chromatography, High Pressure Liquid; D019075:Electrophoresis, Capillary / Q000379:methods*; D051379:Mice; D019998:Racemases and Epimerases / Q000378:metabolism*; D012694:Serine / Q000031:analogs & derivatives* / Q000032:analysis*; D013237:Stereoisomerism; D000099300:Serine Racemase
D019998:Racemases and Epimerases; D012694:Serine; D000099300:Serine Racemase