Pulsed Electron Paramagnetic Resonance Spectroscopy of ³³S‑Labeled Molybdenum Cofactor in Catalytically Active Bioengineered Sulfite Oxidase

Molybdenum enzymes contain at least one pyranopterin dithiolate (molybdopterin, MPT) moiety that coordinates Mo through two dithiolate (dithiolene) sulfur atoms. For sulfite oxidase (SO), hyperfine interactions (<i>hfi</i>) and nuclear quadrupole interactions (<i>nqi</i>) of magnetic nuclei (<i>I</i> ≠ 0) near the Mo­(V) (d¹) center have been measured using high-resolution pulsed electron paramagnetic resonance (EPR) methods and interpreted with the help of density functional theory (DFT) calculations. These have provided important insights about the active site structure and the reaction mechanism of the enzyme. However, it has not been possible to use EPR to probe the dithiolene sulfurs directly since naturally abundant ³²S has no nuclear spin (<i>I</i> = 0). Here we describe direct incorporation of ³³S (<i>I</i> = 3/2), the only stable magnetic sulfur isotope, into MPT using controlled <i>in vitro</i> synthesis with purified proteins. The electron spin echo envelope modulation (ESEEM) spectra from ³³S-labeled MPT in this catalytically active SO variant are dominated by the “interdoublet” transition arising from the strong nuclear quadrupole interaction, as also occurs for the ³³S-labeled exchangeable equatorial sulfite ligand [Klein, E. L., et al. Inorg. Chem. 2012, 51, 1408−1418]. The estimated experimental <i>hfi</i> and <i>nqi</i> parameters for ³³S (<i>a</i>_iso = 3 MHz and <i>e</i>²<i>Qq</i>/<i>h</i> = 25 MHz) are in good agreement with those predicted by DFT. In addition, the DFT calculations show that the two ³³S atoms are indistinguishable by EPR and reveal a strong intermixing between their out-of-plane p_<i>z</i> orbitals and the d_<i>xy</i> orbital of Mo­(V)

Heparin does not affect sFlt-1 protein complex size but interferes with sFlt-1 binding to negatively charged surfaces.

<p>Serum samples before and after low molecular weight heparin treatment were subjected to velocity gradient centrifugation. Western blotting of fractions 5–18 was performed using Flt-1 specific antibody (<b>A</b>). Serum samples before and after addition of 10 mg/l enoxaparin were subjected to cation exchange chromatography. In non-treated serum samples 70.14% of sFlt-1 binds to the column, whereas the remaining 29. 86% are found in the flow through. After enoxaparin treatment only 56.88% of sFlt-1 is bound and 43.11% appear in the flow through (p = 0.0386) (<b>B</b>).</p

sFlt-1 and PlGF serum levels increase after the administration of low molecular weight heparin.

<p>2-1 levels increase to 1.26 fold on average (95% CI (interval before LMWH) 0,9627–1,040; 95% CI (interval after LMWH) 1.103–1.418; p = 0.0045) (<b>A</b>), PlGF levels increase to 1.15 fold on average (p = 0.0126) (<b>B</b>), the resulting increase of sFlt-1/PlGF ratio does not reach statistical significance (<b>C</b>).</p

Administration of low molecular weight heparin results in elevated urinary sFlt-1 levels.

<p>Urinary sFlt-1 levels (pg/mg Crea) are increased 2 hours after administration of enoxaparin in 7 out of 10 patients (<b>A</b>). Subgroup analysis according to proteinuria at presentation (cut-off of 300 mg protein/g creatinine) reveals that patients with a proteinuria <300 mg/g respond with a significantly higher increase of urinary sFlt-1 after heparin treatment (p = 0.0365) (<b>B</b>).</p

Patient characteristics on admission.

<p>Physical parameters and laboratory values were assessed upon admission in all patients. DM1 = diabetes mellitus type 1; IUGR = intra uterine growth restriction; PE = preeclampsia; PIH = pregnancy induced hypertension.</p