Separation of glycolytic metabolites by column chromatography

✍ Scribed by Hans Werner Hofer

• Publisher: Elsevier Science
• Year: 1974
• Tongue: English
• Weight: 498 KB
• Volume: 61
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1016/0003-2697(74)90332-7

✦ Synopsis

Glycolytic and gluconeogenic metabolites can be separated by a simple column chromatographic procedure using a sigmoid salt gradient in the presence of borate. Highly accurate values of the radioactivity in the metabolites were obtained by the combination of the chromatographic separation with a radioactive dilution technique.

Investigations of intermediary metabolism frequently involve the use of radiolabeled compounds. For example, comparison of the specific radioactivities of glycolytic and gluconeogenic intermediates provided evidence for compartmentation of these pathways (1). Determination of the amount of radioactive label incorporated into a given metabolite requires the separation of ten or more compounds present initially as mixtures in tissue extracts and differing in their concentrations by more than two orders of magnitude.

Most of the studies published in this field (2-4) use thin-layer or paper chromatography to separate the metabolites, despite the disadvantages associated with these methods in the separation of sugar phosphates and other phosphate esters.

These difficulties can be avoided by the use of column chromatography. Elution with borate-containing buffers, as suggested by Khym and Cohn (5) opens the possibility that isomeric hexose phosphates can be separated by this technique. The application of the radioactive dilution method and of enzymatic analysis allows the precise determination of specific radioactivity in samples of convenient size.

A simple method for the separation of most glycolytic metabolites using inexpensive instrumentation is presented in this report.

Methods

Ion exchange resin. Analytical grade Dowex 1 X 8 anion exchange resin was supplied by Bio-Cal (AG 1 X 8, minus 400 mesh).

Chemicds. Buffer substances were products of Merck, Darmstadt, 54