Separation of Pinusbanksiana Lamb. glyceraldehyde 3-phosphate (NAD) dehydrogenase from phenolic-based pigments with affinity chromatography

Abstract

Glyceraldehyde 3-phosphate (NAD) dehydrogenase was extracted from Pinusbanksiana Lamb, seed and from seedlings up to 12 days old. Enzyme activity in crude extracts of seedlings increased markedly from days 2–8 and then decreased slightly, as indicated by spectrophotometric assay. In contrast, staining of acrylamide gels after disc electrophoresis indicated that catalytically active isozymes were not present in crude extracts at and after day 6. Phenolics greatly increased in extracts from days 0–10 and polymerization of these phenolics apparently lead to binding of the resulting phenolic-based pigment to enzyme. The pigment–enzyme complex demonstrated greatly enhanced electrophoretic movement, in comparison with the individual isozymes, which caused frontal migration of the collective isozymes. Affinity chromatography restored electrophoretic mobility of isozymes equal to that obtained with enzyme from ungerminated seed. Enzyme was inseparable from pigment by several other methods.