Affinity chromatography on immobilised nucleotides. Some applications to the purification of thermophilic dehydrogenases and kinases.
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.
D000249:Adenosine Monophosphate; D000429:Alcohol Oxidoreductases / Q000302:isolation & purification*; D002846:Chromatography, Affinity / Q000379:methods; D001411:Geobacillus stearothermophilus / Q000201:enzymology*; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000302:isolation & purification*; D006863:Hydrogen-Ion Concentration; D009243:NAD; D009249:NADP; D009994:Osmolar Concentration; D010732:Phosphofructokinase-1 / Q000302:isolation & purification*; D011485:Protein Binding; D012685:Sepharose; D013696:Temperature
D009243:NAD; D000249:Adenosine Monophosphate; D009249:NADP; D012685:Sepharose; D000429:Alcohol Oxidoreductases; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D010732:Phosphofructokinase-1