EPR spectroscopy and electrospray ionization mass spectrometry reveal distinctive features of the iron site in leukocyte 12-lipoxygenase

The procedure for the expression and purification of recombinant porcine leukocyte 12-lipoxygenase using Escherichia coli [K.M. Richards, L.J. Marnett, Biochemistry 36 (1997) 6692–6699] was updated to make it possible to produce enough protein for physical measurements. Electrospray ionization tande...

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Bibliographic Details
Published in:Archives of biochemistry and biophysics 2009-10, Vol.490 (1), p.50-56
Main Authors: Rapp, Johanna, Xu, Shu, Sharp, Allan M., Griffith, Wendell P., Kim, Yong-Wah, Funk, Max O.
Format: Article
Language:English
Subjects:
Amino Acid Sequence
Animals
Arachidonate 12-Lipoxygenase - chemistry
Arachidonate 12-Lipoxygenase - genetics
Arachidonate 12-Lipoxygenase - metabolism
Arachidonic acid
Electron Spin Resonance Spectroscopy - methods
Escherichia coli - genetics
Hydrogen-Ion Concentration
Iron
Iron - metabolism
Leukocytes - metabolism
Lipid Peroxides - pharmacology
Lipoxygenase
Molecular Weight
Oxidation-Reduction
Polyunsaturated fatty acid metabolism
Recombinant Proteins - chemistry
Recombinant Proteins - isolation & purification
Recombinant Proteins - metabolism
Spectrometry, Mass, Electrospray Ionization - methods
Sus scrofa
Temperature
Transformation, Genetic
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Summary:The procedure for the expression and purification of recombinant porcine leukocyte 12-lipoxygenase using Escherichia coli [K.M. Richards, L.J. Marnett, Biochemistry 36 (1997) 6692–6699] was updated to make it possible to produce enough protein for physical measurements. Electrospray ionization tandem mass spectrometry confirmed the amino acid sequence. The redox properties of the cofactor iron site were examined by EPR spectroscopy at 25 K following treatment with a variety of fatty acid hydroperoxides. Combination of the enzyme in a stoichiometric ratio with the hydroperoxides led to a g4.3 signal in EPR spectra instead of the g6 signal characteristic of similarly treated soybean lipoxygenase-1. Native 12-lipoxygenase was also subjected to electrospray ionization mass spectrometry. There was evidence for loss of the mass of an iron atom from the protein as the pH was lowered from 5 to 4. Native ions in these samples indicated that iron was lost without the protein completely unfolding.
ISSN:0003-9861
1096-0384
DOI:10.1016/j.abb.2009.08.004