Affinity purification and determination of enzymatic activity of recombinantly expressed aldehyde dehydrogenases.
Aldehydes are highly reactive and ubiquitous molecules involved in numerous biochemical processes and physiological responses. Many biologically important aldehydes are metabolized by the superfamily of NAD(P)(+)-dependent aldehyde dehydrogenases [aldehyde:NAD(P)(+) oxidoreductases, EC 1.2.1, ALDH]. Here we describe a straightforward protocol for purification of soluble recombinantly expressed ALDH enzyme based on metal affinity chromatography and the subsequent determination of enzymatic activity using aldehydic substrates, which is assayed spectrophotometrically at 340 nm by conversion of NAD(P)+ to NAD(P)H.
Kirch
Hans-Hubert
HH
Institute of Molecular Physiology and Biotechnology of Plants (IMBIO), University of Bonn, Kirschallee 1, Bonn, Germany. hhkirch@uni-bonn.de
Röhrig
Horst
H
D000444:Aldehyde Dehydrogenase / Q000302:isolation & purification* / Q000378:metabolism*; D002846:Chromatography, Affinity / Q000379:methods*; D032181:Craterostigma / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D057075:Enzyme Assays; D004800:Enzymes, Immobilized / Q000302:isolation & purification; D004926:Escherichia coli; D008670:Metals; D011994:Recombinant Proteins / Q000302:isolation & purification* / Q000378:metabolism*
D004800:Enzymes, Immobilized; D008670:Metals; D011994:Recombinant Proteins; D000444:Aldehyde Dehydrogenase