Premium
Rapid purification of rabbit reticulocyte lipoxygenase for electron paramagnetic spectroscopy characterization of the non‐heme iron
Author(s) -
Carroll Richard T.,
Muller Jessica,
Grimm Jennifer,
Dunham William R.,
Sands Richard H.,
Funk Max O.
Publication year - 1993
Publication title -
lipids
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.601
H-Index - 120
eISSN - 1558-9307
pISSN - 0024-4201
DOI - 10.1007/bf02536646
Subject(s) - chemistry , reticulocyte , chromatography , electron paramagnetic resonance , isoelectric focusing , gel electrophoresis , polyacrylamide gel electrophoresis , lysis , enzyme , biochemistry , nuclear magnetic resonance , physics , messenger rna , gene
An efficient three‐step purification technique has been developed for the reticulocyte 15‐lipoxygenase from rabbit. Ammonium sulfate fractionated reticulocyte lysate was purified by size exclusion chromatography and preparative scale isoelectric focusing. The entire procedure was complete in less than eight hours and was carried out in batches which typically yielded 10 mg of purified enzyme. The identity and purity of the enzyme were evaluated by N ‐terminal sequencing, sodium dodecylsulfate polyacrylamide gel electrophoresis and specific activity determinations. The enzyme contained approximately one g‐atom iron per mole of protein. The iron was present in an electron paramagnetic spectroscopy (EPR) silent, presumably high spin iron(II), form in the isolated enzyme. Treatment with one equivalent of 13‐hydroperoxy‐9( Z ),11( E )‐octadecadienoic acid resulted in the appearance of an EPR signal around g6.