title: Large-scale preparation and reconstitution of apo-flavoproteins with special reference to butyryl-CoA dehydrogenase from Megasphaera elsdenii. Hydrophobic-interaction chromatography.
abstract: A new method is described for the large-scale reversible dissociation of flavoproteins into apoprotein and prosthetic group using hydrophobic-interaction chromatography. Lipoamide dehydrogenase from Azotobacter vinelandii and butyryl-CoA dehydrogenase from Megasphaera elsdenii are selected to demonstrate the usefulness of the method. In contrast to conventional methods, homogeneous preparations of apoproteins in high yields are obtained. The apoproteins show high reconstitutability. The holoenzymes are bound to phenyl-Sepharose CL-4B at neutral pH in the presence of ammonium sulfate. FAD is subsequently removed at pH 3.5-4.0 by addition of high concentrations of KBr. Large amounts of apoenzymes (200-500 mg), showing negligible residual activity, are eluted at neutral pH in the presence of 50% ethylene glycol. The holoenzyme of lipoamide dehydrogenase can be reconstituted while the apoprotein is still bound to the column or the apoenzyme can be isolated in the free state. In both cases the yield and degree of reconstitution of holoenzyme is more than 90% of starting material. Apo-lipoamide-dehydrogenase exists mainly as a monomer in solution and reassociates to the native dimeric structure in the presence of FAD. The apoenzyme is stable for a long period of time when kept in 50% ethylene glycol at -18 degrees C. Steady-state fluorescence-polarization measurements of protein-bound FAD indicate that reconstituted lipoamide dehydrogenase possesses a high stability which is governed by the low dissociation rate constant of the apoenzyme-FAD complex. The holoenzyme of butyryl-CoA dehydrogenase cannot be reconstituted when the apoenzyme is bound to the column. However, stable apoprotein can be isolated in the free state yielding 50-80% of starting material, depending on the immobilization conditions. The coenzyme A ligand present in native holoenzyme is removed during apoprotein preparation. The apoenzyme is relatively stable when kept in 50% ethylene glycol at -18 degrees C. From kinetic and gel filtration experiments it is concluded that the reconstitution reaction of butyryl-CoA dehydrogenase is governed by both the pH-dependent hydrodynamic properties of apoenzyme and the pH-dependent stability of reconstituted enzyme. At pH 7, the apoenzyme is in equilibrium between dimeric and tetrameric forms and reassociates to a native-like tetrameric structure in the presence of FAD. The stability of reconstituted enzyme is strongly influenced by the presence of CoA ligands as shown by fluorescence-polarization measurements. The degree of reconstitution of butyryl-CoA dehydrogenase is more than 80% of the original specific activity under certain conditions.(ABSTRACT TRUNCATED AT 400 WORDS)
authors.affiliation: Department of Biochemistry, Agricultural University, Wageningen, The Netherlands.
authors.forename: W J
authors.initials: WJ
authors.lastname: Van Berkel
authors.forename: W A
authors.initials: WA
authors.lastname: Van den Berg
authors.forename: F
authors.initials: F
authors.lastname: Müller
mesh_terms: D001059:Apoproteins / Q000302:isolation & purification*; D001419:Bacteria / Q000201:enzymology; D001665:Binding Sites; D044926:Butyryl-CoA Dehydrogenase; D002850:Chromatography, Gel; D008058:Dihydrolipoamide Dehydrogenase / Q000378:metabolism*; D004800:Enzymes, Immobilized; D044943:Fatty Acid Desaturases / Q000378:metabolism*; D005182:Flavin-Adenine Dinucleotide; D005415:Flavins / Q000302:isolation & purification; D005420:Flavoproteins / Q000378:metabolism*; D005454:Fluorescence Polarization; D012685:Sepharose / Q000031:analogs & derivatives; D013056:Spectrophotometry, Ultraviolet; D014461:Ultracentrifugation
chemical_list: D001059:Apoproteins; D004800:Enzymes, Immobilized; D005415:Flavins; D005420:Flavoproteins; D005182:Flavin-Adenine Dinucleotide; C033452:Phenyl-Sepharose CL-4B; D012685:Sepharose; D044943:Fatty Acid Desaturases; D044926:Butyryl-CoA Dehydrogenase; D008058:Dihydrolipoamide Dehydrogenase