On-line monitoring of enzymatic conversion of adenosine triphosphate to adenosine diphosphate by micellar electrokinetic chromatography.
Capillary electrophoresis can be a valuable tool for the on-line monitoring of bioprocesses. The enzymatic conversion of nucleotide adenosine triphosphate (ATP) to adenosine diphosphate (ADP) by hexokinase (HK) was monitored in the bioreactor interfaced by a laboratory-built microsampler to a capillary electrophoresis unit. The use of this specially designed sampling device enabled rapid consecutive injections to be performed without high-voltage (HV) interruptions. No additional sample preparation was required. The method of micellar electrokinetic chromatography, employing reversed electroosmotic flow (EOF) by cationic surfactant and reversed polarity mode provided a good resolution and short analysis time of less than 5 min. The samples were injected electrokinetically, using -25 kV voltage for 3 s and detected by their UV absorbance at 254 nm. The analytes were detected at a microg/ml level with a reproducibility of about 7%. To demonstrate the potential of CE in understanding the processes of biological interest, such as nucleotide degradation and metabolism, the investigation of the efficiency and the time course of the enzymatic transformation was carried out.
Kulp
Maria
M
Faculty of Science, Tallinn Technical University, Ehitajate Tee 5, 19086 Tallinn, Estonia. maria@argus.chemnet.ee
Kaljurand
Mihkel
M
D000244:Adenosine Diphosphate / Q000096:biosynthesis*; D000255:Adenosine Triphosphate / Q000378:metabolism*; D020374:Chromatography, Micellar Electrokinetic Capillary / Q000379:methods*; D006593:Hexokinase / Q000378:metabolism*; D007700:Kinetics; D015203:Reproducibility of Results
D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D006593:Hexokinase