_applied sciences_  
_Article_  
**Tracking Heme-Protein Interactions in Healthy and Pathological Human Serum in Native Conditions by Miniaturized FFF-Multidetection**  
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Footnote 1: The  
An excess of free heme is toxic for the organism. High levels of redox-active iron (contained in heme) can catalyze the formation of Reactive Oxygen Species (ROS). ROS damage lipid membranes, proteins, nucleic acids, activate cell signaling pathways and oxidant-sensitive, proinflammatory transcription factors, alter protein expression, and perturb membrane channels [6, 7]. Due to its lipophilic nature, heme is able to intercalate in the lipidic membrane causing cellular lysis [8]. Finally, heme is strongly pro-inflammatory since it induces the recruitment of leukocytes and consumes nitric oxide, thus impairing vascular function. The production of ROS and the membrane damage caused directly and indirectly by heme make it a strong hemolytic agent. Several pathological conditions are associated with hemolysis, including sickle cell anemia, thalassemia, malaria, paroxysmal nocturnal hemoglobinuria, etc. [9]. Other vascular diseases caused by a detrimental effect of heme on the vessel wall are atherosclerosis and vascular calcifications [10]. Due to free heme toxicity, blood serum is equipped with mechanisms to prevent extracellular heme toxicity. Among them, a key function is covered by the soluble scavengers of free hemoglobin and heme, Haptoglobin (Hp) [11] and Hemopexin (Hx) [8], respectively. Hp acts on the dangerous free hemoglobin released by hemolysis processes by capturing it and generating a complex which is sent to the reticulo-endothelial system for its elimination [12]. When the buffering capacity of plasma Hp is exceeded, hemoglobin is quickly oxidized to methemoglobin, which releases free heme [13]. Heme is then bound by Hx. Hx is a 57-kDa acute phase plasma glycoprotein, able to bind an equimolar amount of heme and to deliver it to hepatocytes where the heme-Hx complex is internalized by receptors LRP 1 (LDL receptor-related protein 1) mediated endocytosis [5]. However, when hemolysis levels are too high or comorbidities reduce the bioavailability of hemopexin, other proteins act as scavenging heme agents, namely serum albumin (SA), LDL, HDL, and Immunoglobulins [14]. The interest upon heme-protein affinity is not only correlated to restoring homeostasis properties. It has been observed that heme-exposed IgGs, following an appropriate incubation procedure, can exhibit new and promising properties (such as polineactivity), and with this regard some of the serum immunoglobulins interact with heme [15]. Literature describing what happens during these incubation procedures is very diverse. Some studies suggest that heme, due to its pro-ox nature, induces chemical modification of IgGs without the formation of a stable complex [16, 17]. Others instead showed the existence of binding, and formation of stable complexes, between heme and antibodies [14, 18, 19]. However in each of these studies, the drawback of having simultaneous bound and unbound heme led to difficult quantification and lab-heavy functionalization steps (immobilization, derivatization). Some approaches to determine biomolecule-biomolecule interactions exploit coinimmunoprecipitation [18, 19]. Others use imaging such as atomic force microscopy, based on force spectroscopy (AFM-FS) [20, 21], magnetic tweezers and dynamic light scattering [22, 23]. Energy transfer such as Forster resonance energy transfer (FRET) [24, 25], Bioluminescence resonance energy transfer (BRET) [26] and fluorescence cross-correlation spectroscopy (FCCS) [27, 28] are also employed. In the case of serum-heme interactions, it is to be noted that the experimental difficulties are numerous, and the matrix is very complex.