FIGURE 3.

DSF screening reveals multiple iron-chelating SCN ligands in restrictive human urine. a, urine fractionation scheme. Four urine specimens supporting high SCN activity (restrictive; red squares) and four supporting low activity (permissive; blue circles) were pooled and extracted by Chrom P SPE prior to HPLC fractionation. An example chromatogram is shown for the 50% methanol elution where blue, red, green, and orange traces show absorbance at 240, 280, 350, and 435 nm, respectively. DSF analysis of HPLC fractions (reconstituted to ∼80-fold concentration over starting urine) was conducted with 3.2 μg/ml additional FeCl₃ to identify ferric siderocalin complexes in the pooled restrictive (b) or permissive (c) urine fractions. Columns show the change in T_m (ΔT_m) for each fraction relative to solvent control. HPLC fractions are presented consecutively as bars along the x axis; error bars represent S.E.M. Fractions of interest from each SPE elution are highlighted, and a representative DSF plot is shown below (fraction in dotted line; SCN control in solid line).