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.

Evaluation of gel electrophoresis conditions for the separation of metal‐tagged proteins with subsequent laser ablation ICP‐MS detection

“…Generally it can be said that all results achieved need to be considered very carefully in order to judge whether they are reliable or whether the signals derived from GE-LA-ICP-MS analysis are from contaminants or artefacts. A fully quantitative approach would benefit from the use of isotopically labelled metalloproteins [40,41]. When spiked and equilibrated with the sample, the isotopically labelled species allows metal loss during sample preparation and separation to be compensated for.…”

Inductively Coupled Plasma–Mass Spectrometry (ICP-MS) for Quantitative Analysis in Environmental and Life Sciences: A Review of Challenges, Solutions, and Trends

“…The production of the isotopically enriched metalloproteins is usually carried out under in vitro conditions, via incubation of the apo protein (metal-free) with a spike solution, which contains the isotopically enriched metal. [123][124][125] Recently the in vivo synthesis of isotopically enriched metal proteins such as 57 Feferritin 126,127 or 65 Cu-plastocyanin 128 has also been successfully demonstrated, just to give a few examples. Despite the inherent advantage of such speciesspecific standards it is important to ensure that during the demetallation process, which is necessary for the in vitro generation of the apo protein, no degradation of the protein occurs.…”

Species specific isotope dilution versus internal standardization strategies for the determination of Cu, Zn-superoxide dismutase in red blood cells

“…The isotopically enriched SOD was prepared using a combination of previously reported methods by Deitrich et al 17 and Yamazaki et al 28 In brief, the commercial protein standard (either human or bovine) was dissolved in 10 mM ammonium acetate pH 6.3. These stock solutions were then transferred into 3-mL dialysis membranes with a 5000 Da molecular weight cutoff (MWCO) (Spectra/Pro CE Flot-A-Lyzer, Spectrum Europe B.V.).…”

“…17,28 Fig . 3 shows the chromatographic profiles of the isotopes 63 Cu and 65 Cu of isotopically enriched SOD from bovine erythrocytes.…”