DETERMINATION OF CREATINE-KINASE ACTIVITY AND PHOSPHOCREATINE IN OFF-LINE AND ONLINE MODES WITH CAPILLARY-ELECTROPHORESIS

Assays for phosphocreatine (PC) and creatine kinase (CK) were performe d using capillary electrophoresis (CE) both off-line and on-line in ap proach. Both substrate and enzyme assays were based solely on the phos phorylation reaction involving the conversion of adenosine diphosphate (ADP) to adenosine triphosphate (ATP) which was separated and monitor ed at 256 nm. Polymer mediated stabilization with PEG-8000 can extend the lifetime of the enzyme activity to at least 73 h. Although the off -line procedure required a 10 min batch incubation time of the reactio n mixture, low detection limits at the 10 mu M PC and 1 U/l CK levels are achievable. For the single injection on-line method, sample throug hput for one assay is 7 min excluding wash and equilibration steps and detection limits of 25 mu M PC and 8 U/l CK are possible. Because of the mu M detection limits, only simple dilution of spiked PC and CK se rum samples before analysis is needed. A new technique of on-line mult iple injections of both substrate and enzyme into the capillary filled with co-factor and buffer was also demonstrated. Sample throughput fo r the multiple injection method is 9 min for five samples, excluding w ash and equilibration steps. Because linearity and quantitation were o nly fair, the multiple injection method would be more useful for the r apid profiling of samples. (C) 1998 Elsevier Science B.V. All rights r eserved.