## 3 Results and Discussion
### Serum--Heme Binding Study  
After identification of serum components, the following concern was the specificity of detection: the selectivity towards bound heme was already confirmed with a 0% recovery of free heme (Supplementary Data, Figure S2), but univocal detection also needed confirmation. In Figure 3 the differential absorption of HSA, serum, and IgG at 280 and 405 nm (heme absorption maximum) is shown. All three samples absorb strongly at 280 nm but show no absorption at 405 nm. The lack of signal at 405 nm found for serum confirms that this setup can allow to work in a'single experiment' setting, where individual proteins can be used for further detailed studies after a general behaviour is observed. In fact, serum itself, sorted in its component, is a clear negative control and any difference in heme binding can be promptly and selectively detected. Figure 2: Separation profile of whole serum (black), HSA (blue) and IgG under the same analysis conditions. The same amount of serum was treated with increasing amounts of heme to obtain heme final concentrations of 5, 10, 20, 40 \(\upmu\)M and analyzed with HF5-mutidetection (Figure 4). As the concentration of heme increased, it was possible to observe the formation of an intense peak at 405 nm reflecting the retention time of band 1. The interaction between heme and band 1 was predominant, while a very low signal could be found in band 1\({}^{\prime}\)s tail, and at around 13 min tailing into band 3 (Figure 4, inset). These signals can be attributed to SA aggregates, hemopexin, and HDL [56; 57; 58] and other type of immunoglobulins such as IgA and IgM [59; 60]. Interestingly, no interaction was observed in correspondence of band 2, attributed to IgG. Though a few works reporting interactions between heme and immunoglobulins are found in literature, it is important to notice that they do not regard interaction studies in physiological conditions and concern specific classes of antibodies, often specifically made [61]. On the contrary, the formation of stable complexes between blood proteins and heme is not fully understood and this setup can contribute to increase the knowledge on preferred heme-clearing pathways. As confirmation and to correctly interpret the interactions between SA and heme, mixtures of SA or IgG and the same amount of heme previously employed were also analyzed. In the case of SA, interaction was not only confirmed, but also yielded peak integrals identical to what was found in correspondence of band 1 (Figure 5a). In this case, as SA dimer and oligomers were also present, it was possible to notice how all aggregated states of SA interacted with heme at the same time and at all concentrations. Figure 4: Overlay of absorption at 405 nm of serum treated with increasing heme concentrations (5–10–20–40 uM). Black trace: serum profile at 280 nm. Inset: zoom of the fractograms overlay to observe low signals at the tail of band 1 and at 13–17 min. Figure 3: Differential absorption at 280 nm and 405 nm for (**a**) HSA (**b**) IgG and (**c**) serum. Instead, for what concerns IgG, no signal at 405 nm was detected at the tested concentrations and thus we increased the amount of heme until a visible signal was detectable; this only happened when final heme concentration reached 100 \(\upmu\)M (Figure 5b), which is beyond clinical interest [62]. Lastly, and as an additional investigation on the possible competition and binding equilibria in serum, we also searched for interactions between IgG-enriched serum and heme; in fact, some literature reports IgG-heme binding in case of the opposite imbalance to that shown above [32]. Serum was enriched to reach a final IgG concentration of 20, 25 and 30 \(\upmu\)M, in the range of moderate-to-severe clinical conditions, to also simulate co-morbidity to hemolytic diseases. Then, heme was added to the sample which was submitted to HF5-multidetection analysis.