Adaptation of an HPLC system for transient-state enzyme kinetic experiments: pulse-flow method

• Published in: Journal of biochemical and biophysical methods Vol. 21; no. 2; pp. 145 - 153
• Main Authors: Tsygankov, A.Yu, Motorin, Yu.A., Wolfson, A.D., Kirpotin, D.B.
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.07.1990
• Subjects: Automated assays; beta-Galactosidase - metabolism; Chromatography, High Pressure Liquid - instrumentation; Enzyme Activation; Enzyme kinetics; Enzymes - metabolism; Equipment Design; high-performance liquid chromatography; Kinetics; Magnesium - physiology; Mg 2+-activation; Pulsatile Flow; Pulse-flow method; β-Galactosidase; Mg 2+-activation; Pulse-flow method; Enzyme kinetics; Automated assays; β-Galactosidase
• ISSN: 0165-022X; 1872-857X
• DOI: 10.1016/0165-022X(90)90061-G

The components of an HPLC system can be easily reasembled into an instrument for transient-state enzyme kinetic experiments based on the continuous-flow technique. The scheme for reassembly, operation and data evaluation is described in details for the Pharmacia FPLC system. The working quality of the suggested scheme was tested using a well-studied process of bacterial β-galactosidase activation by Mg 2+ ions. The found parameters were in a good agreement with the literature data. The pulse-flow mode was developed for the delivery of working solutions to reduce the required quantities of enzyme and/or other reaction components. The work presents new approach to HPLC modules as building blocks for sophisticated liquid flow reactors, such as automatic solid phase synthesizers, sequencers and so on.