Affinity chromatography of putrescine oxidase from Micrococcus rubens and spermidine dehydrogenase from Serratia marcescens.
Putrescine oxidase [EC 1.4.3.4], putrescine : oxygen oxidoreductase (deaminating) (flavin-containing), from Micrococcus rubens and spermidine dehydrogenase from Serratia marcescens were adsorbed on amine-Sepharose 4B in which one of the terminal amino groups of diamine or triamine was covalently bound to Sepharose 4B leaving the other terminal amino group(s) free. The affinities of these enzymes for the amine-Sepharose 4B increased on increasing the chain length of the methylene groups in the immobilized amines and fell upon addition of the substrate. The affinity of putrescine oxidase modified with 1-ethyl-3-(3-dimethylamino-propyl)-carbodiimide (EDC) was reduced in comparison with that of the native enzyme so far as 1,12-diaminododecane-Sepharose 4B was concerned. From these results, it can be concluded that the interactions between the enzyme and the amine-Sepharose result from specific affinities mediated through the active sites of the enzymes. It is suggested that spermidine dehydrogenase as well as putrescine oxidase has as anionic point and a hydrophobic region in the active site. On the basis of these results, the applicability of the enzyme affinities to purification procedures was examined. When partially purified enzymes were subjected to affinity chromatography, the following results were obtained. Putrescine oxidase gave a purification factor of 40-fold with about 100% recovery on a 1,12-diaminododecane-Sepharose column. In the case of spermidine dehydrogenase, the purification factor and recovery on a 1,8-diaminooctane-Sepharose column were about 1,200-fold and 86%, respectively. By introducing affinity chromatography as a purification step, each enzyme could be purified more simply and with higher recovery.
D002846:Chromatography, Affinity; D046911:Macromolecular Substances; D008837:Micrococcus / Q000201:enzymology*; D008970:Molecular Weight; D000587:Oxidoreductases Acting on CH-NH Group Donors / Q000302:isolation & purification*; D011700:Putrescine; D012706:Serratia marcescens / Q000201:enzymology*; D013095:Spermidine
D046911:Macromolecular Substances; D000587:Oxidoreductases Acting on CH-NH Group Donors; D013095:Spermidine; D011700:Putrescine