title: Chemical preparation of an isotopically enriched superoxide dismutase and its characterization as a standard for species-specific isotope dilution analysis.
abstract: The development of methods to analyze accurately and precisely individual metalloproteins is of increasing importance. Here we describe for the first time the chemical preparation and characterization of an isotopically enriched metalloenzyme containing two different metal isotopes. Its evaluation as a standard in species-specific isotope dilution analysis by HPLC coupled to inductively coupled plasma mass spectrometry is carefully evaluated. Our model enzyme bovine superoxide dismutase (SOD) contains both Cu and Zn and is remarkably stable at high temperatures and even under denaturing conditions. The enzyme's metal cofactors were removed under a range of different conditions and replaced with isotopically enriched 65Cu and 68Zn. Depending on the conditions, various isotopic ratios differing from the natural Cu and Zn abundances were obtained for the reconstituted enzyme. Both the wild type and isotopically enriched enzyme had the same migration pattern on native 1D-PAGE. Using an enzyme activity test, we showed that the incorporated 65Cu was bound to the right SOD-binding site, since the measured activity correlated directly with the amount of Cu incorporated. Mixing the native and the isotopically enriched enzyme standard with free enriched 65Cu and 68Zn or a metal chelator did not result in any exchange or loss of the metals from the enzyme at neutral pH. This verifies the stability of the enzyme metal center under the chosen conditions. The isotopically enriched enzyme standard was spiked into a wild type SOD solution to evaluate its use for species-specific isotope dilution experiments. To our knowledge, this is the first report of the chemical preparation of a metalloenzyme containing two different isotopically enriched metals. We provide evidence that the incorporated isotopically enriched metals are bound to the right binding site of SOD using an specific enzymatic activity assay.
authors: College of Physical Science, Department of Chemistry, University of Aberdeen, Meston Walk, Aberdeen, UK AB24 3UE.
authors: Christian L
authors: CL
authors: Deitrich
authors: Andrea
authors: A
authors: Raab
authors: Barbara
authors: B
authors: Pioselli
authors: Jane E
authors: JE
authors: Thomas-Oates
authors: Jörg
authors: J
authors: Feldmann
mesh_terms: D000818:Animals; D002417:Cattle; D002851:Chromatography, High Pressure Liquid / Q000379:methods / Q000592:standards; D003300:Copper / Q000737:chemistry; D007553:Isotope Labeling; D007554:Isotopes; D013058:Mass Spectrometry / Q000379:methods / Q000592:standards*; D009942:Organometallic Compounds / Q000032:analysis*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013045:Species Specificity; D013482:Superoxide Dismutase / Q000032:analysis*; D013997:Time Factors; D015032:Zinc / Q000737:chemistry; D015033:Zinc Isotopes / Q000737:chemistry
chemical_list: D007554:Isotopes; D009942:Organometallic Compounds; D015033:Zinc Isotopes; D003300:Copper; D013482:Superoxide Dismutase; D015032:Zinc