Analysis of gamma-carboxyglutamic acid content of protein, urine, and plasma by capillary electrophoresis and laser-induced fluorescence

When the properties of an analyte are known, the separation system can be d esigned to make the analyte of interest migrate at either a much faster ora much slower velocity compared to other molecules in the sample matrix. A s imple and sensitive method to analyze the gamma-carboxyglutamic acid (Gla) content of protein, urine, and plasma was developed using capillary electro phoresis with laser-induced fluorescence detection (CE-IIF), The separation method is designed according to the specific properties of three amino aci ds of interest. The number of Gla residues from three vitamin K-dependent p roteins were estimated by quantifying the amount of fluorescein thiocarbamy l derivative of Gla after alkaline hydrolysis and fluorescein isothiocyanat e labeling. Human prothrombin, blood coagulation factor X, and bovine osteo calcin were calculated to have 10.0 +/- 0.7, 11.0 +/- 0.6, and 2.1 +/- 0.1 Gla residues. per mole of protein, respectively, which agreed well with ami no acid sequencing data. The analysis of free Gla content in urine and plas ma was also demonstrated by this method. It was demonstrated that submicrog rams of protein can be characterized by CE-LIF.