Affinity Chromatography on Immobilised Nucleotides

Abstract

The effect of pH and temperature on the capacity and binding of Bacillus stearothermophilus, alcohol dehydrogenase and phosphofructokinase to N⁶‐(6‐aminohexyl)‐5′‐AMP‐Sepharose has been examined. Specific elution from the substituted AMP‐Sepharose was examined using a variety of cofactors, fragments of cofactors and substrates. A purification scheme for each enzyme on the substituted AMP‐Sepharose using nucleotides and gradients of pH and salt is presented. Interestingly, elevated temperature increased the affinity of both enzymes for N⁶‐(6‐aminohexyl)‐5′‐AMP‐Sepharose, however, the Michaelis constant for nucleotide determined at various temperatures remained constant. The effect of pH and salt concentration on the binding of B. stearothermophilus glyceraldehyde‐3‐phosphate dehydrogenase to 6‐aminohexanoyl‐NAD⁺‐Sepharose was also examined; raising the pH above 7.5 lowers the capacity of the matrix and the effect of a range of ammonium sulphate concentrations on the adsorption of the enzyme was examined. A specific purification of glyceraldehyde‐3‐phosphate dehydrogenase from partially purified extracts of this organism was achieved.