Interactions of Haptoglobin with Monomeric Globin Species: Insights from Molecular Modeling and Native Electrospray Ionization Mass Spectrometry

Abstract

Haptoglobin (Hp) binds free hemoglobin (Hb) dimers to prevent negative consequences of Hb circulation in the extracellular environment. Although both monomeric Hb and myoglobin (Mb) species also present potential risks, their interactions with Hp have not been extensively studied. Mb is homologous to both the α- and β-chains of Hb and shares many conserved Hb/Hp interface residues, yet whether Hp binds Mb remains unclear. To address this, computational biology tools were used to predict the interactions required for Hp to bind monomeric globins, and the predicted association was tested using native electrospray ionization mass spectrometry (ESI-MS). The Hb/Hp crystal structure was used as the template to create molecular models of two Mb molecules bound to an Hp heterodimer (Mb2/Hp). Molecular modeling suggests that Mb can bind at the Hp α-chain binding site, where 73% of the globin/Hp interactions are conserved. By contrast, several ionic β-chain residues involved in complementary electrostatic interactions with Hp correspond to residues with the opposite charge in Mb, suggesting unfavorable electrostatic Hp/Mb interactions at the β-chain binding site. As shown by native ESI-MS, isolated monomeric Hbα subunits can form 2:1 complexes with Hp heterotetramers in the absence of Hb β-chains. Native ESI-MS also confirmed that Mb can bind to Hp heterotetramers in solution with stoichiometries of 1:1 and 2:1 at physiological pH and ionic strength. The affinity of Hp for Mb appears to be diminished relative to that of Hb α-chains. Our in silico experiments rationalize this change and demonstrate that molecular modeling of protein/protein interactions is a valuable aid for MS experiments.

Changes in the Acute-Phase Protein Concentrations and Activities of Some Enzymes in Pigs Following the Repair of Experimentally Induced Articular Cartilage Defects Using Two Types of Biocement Powder

“…While SAA was characterized by marked and rapid increase upon stimulation, Hp was defined as moderate acute-phase protein that usually has a slower and mild increase compared to SAA. Hp has an important function to bind myoglobin derived from traumatized muscle cells and hemoglobin from damaged erythrocytes to facilitate degradation and avoid oxidative stress caused by hemoglobin [37,38]. A trend of decreasing values was observed from the 14th post-operative day, reaching the preoperative concentrations 1 month after surgery, which suggests normal post-operative healing, the absence of inflammatory processes and restoration of homeostasis.…”

The α-globin chain of hemoglobin potentiates tissue plasminogen activator induced hyperfibrinolysis in vitro

“…One would expect the presence of α-globin chains and β-globin chains in very similar concentrations if hemolysis alone was the impetus for their release into the circulation as seen in autoimmune hemolytic anemia and hemolytic crises in patients with sickle cell anemia, thalassemia, glucose-6-phosphate dehydrogenase deficiency, or hereditary spherocytosis, and the monomeric α-globin chain is cleared directly by haptoglobin, whereas the β-globin chain is not. 48,49 The relative increase in the α-globin chain as denoted by the 1 to 2 ratio of the α-globin chain to the β-globin chain in the HF group is curious and may be due to its release from the vascular wall in which it is present in the vascular endothelium at the myoendothelial junctions in which it functions to control vascular tone. 50,51 Severe injury induces the release and accumulation of intracellular proteins from damaged tissue or hemolysis into the circulation, which may alter hemostasis and may be grouped by the state of fibrinolysis: SD, HF, and physiologic.…”

A subset of calcium‐binding S100 proteins show preferential heterodimerization

“…Native nano‐ESI MS experiments were carried out on a Synapt G1 HDMS time‐of‐flight instrument (Waters, Milford, MA, USA). NanoESI was conducted using gold‐coated emitters made from borosilicate glass capillaries .…”