Biospecific Affinity Chromatography in Aqueous‐Organic Cosolvent Mixtures
- 3 March 1975
- journal article
- Published by Wiley in European Journal of Biochemistry
- Vol. 52 (1) , 99-105
- https://doi.org/10.1111/j.1432-1033.1975.tb03977.x
Abstract
The effect of the weak polarity-reducing agent, ethylene glycol, on the binding of pig heart lactate dehydrogenase to N6-(6-aminohexyl)-AMP-Sepharose has been investigated. In the absence of the reagent and under the conditions used, a non-specific interaction of the enzyme with the adsorbent led to recoveries of enzyme activity as low as 60% when the enzyme was eluted from the columns with a linear NADH gradient. The inclusion of low concentrations of ethylene glycol in column irrigants considerably improved the recovery of enzyme activity with quantitative recoveries being obtained in the presence of 20-30%. Concentrations of ethylene glycol about 35% altered the native conformation of lactate dehydrogenase and led to a decreased affinity for the immobilised ligand. Under these conditions, the altered protein fluorescence and decreased ability to bind NADH could be correlated with the chromatographic behaviour of the enzyme on columns of N6-(6-aminohexyl)-AMP-Sepharose. These effects were exploited to elute the enzyme from a column of immobilised-AMP with a linear gradient of ethylene glycol.Keywords
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