Abstract
Hemopexin is a serum glycoprotein that binds heme with high affinity and delivers heme to the liver cells via receptor‐mediated endocytosis. A hinge region connects the two non‐disulfide‐linked domains of hemopexin, a 35‐kDa N‐terminal domain (domain I) that binds heme, and a 25‐kDa C‐terminal domain (domain II). Although domain II does not bind heme, it assumes one structural state in apo‐hemopexin and another in heme‐hemopexin, and this change is important in facilitating the association of heme‐hemopexin with its receptor. In order to elucidate the structure and function of hemopexin, it is important to understand how structural information is transmitted to domain II when domain I binds heme. Here we report a study of the protein‐protein interactions between domain I and domain II using analytical ultracentrifugation and isothermal titration calorimetry. Sedimentation equilibrium analysis showed that domain I associates with domain II both in the presence and absence of heme with K~d~ values of 0.8 μM and 55 μM, respectively. The interaction between heme‐domain I and domain II has a calorimetric enthalpy of +11 kcal/mol, a heat capacity (Δ__C~P~) of ‐720 cal/mol‐K, and a calculated entropy of +65 cal/mol‐K. By varying the temperature of the centrifugation equilibrium runs, a van't Hoff plot with an apparent change in enthalpy (Δ__H) of ‐3.6 kcal/mol and change in entropy (AS) of +8.1 cal/mol‐K for the association of apo‐domain I with domain II was obtained. The association is, however, not strongly dependent on ionic strength. The values of the thermodynamic parameters obtained suggest that different types of interactions predominate in the presence and absence of heme: hydrophobic interactions between heme‐domain I and domain II and likely a balanced mix of different forces (e.g., van der Waals and hydrophobic interactions) between apodomain I and domain II.