In vitro binding of leukotriene B 4 (LTB 4) to human serum albumin: Evidence from spectroscopic, molecular modeling, and competitive displacement studies

Abstract

Circular dichroism (CD) and UV absorption spectroscopy were utilized for the first time to investigate the interaction between leukotriene B 4 (LTB 4) and human serum albumin (HSA) in vitro. The weak intrinsic CD signal of LTB 4 was enhanced fivefold in the presence of HSA. The red-shifted, hypochromic, and reduced vibrational fine structure of the ligand/protein UV absorption spectrum indicated complexation of the two molecules in solution. Results obtained from CD titration experiments were subjected to non-linear regression analysis to estimate the binding parameters ( K a = 6.7 × 10 4 M −1, n = 1). Palmitic acid strongly decreased the induced CD signal of the LTB 4/HSA complex, suggesting the role of a high-affinity fatty acid HSA binding site in the leukotriene complexation. Molecular modeling calculations based on the crystal structure of HSA predicted that the long-chain fatty acid site that overlaps with drug binding site II in subdomain IIIA was the most likely binding location for LTB 4. Using the drug site II-specific marker ligand rac-ibuprofen, this prediction was confirmed with induced-CD displacement measurements. To the authors’ knowledge, the current study represents the first demonstration of binding of LTB 4 to HSA in vitro and has implications for the biological transport of this important pro-inflammatory mediator in vivo.