In vitro binding of leukotriene B4 (LTB4) to human serum albumin: evidence from spectroscopic, molecular modeling, and competitive displacement studies.
Circular dichroism (CD) and UV absorption spectroscopy were utilized for the first time to investigate the interaction between leukotriene B4 (LTB4) and human serum albumin (HSA) in vitro. The weak intrinsic CD signal of LTB4 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 (Ka = 6.7 x 10(4) M(-1), n = 1). Palmitic acid strongly decreased the induced CD signal of the LTB4/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 LTB4. 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 LTB4 to HSA in vitro and has implications for the biological transport of this important pro-inflammatory mediator in vivo.
Zsila
Ferenc
F
Department of Bioorganic Chemistry, Institute of Biomolecular Chemistry, Chemical Research Center, P.O. Box 17, Budapest H-1525, Hungary. zsferi@chemres.hu
Bikádi
Zsolt
Z
Lockwood
Samuel F
SF
D001665:Binding Sites; D001667:Binding, Competitive / Q000187:drug effects / Q000502:physiology; D002942:Circular Dichroism; D006801:Humans; D007052:Ibuprofen / Q000737:chemistry / Q000494:pharmacology; D007975:Leukotriene B4 / Q000737:chemistry*; D008024:Ligands; D008958:Models, Molecular; D015394:Molecular Structure; D011485:Protein Binding / Q000187:drug effects; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D012709:Serum Albumin / Q000737:chemistry* / Q000187:drug effects; D013056:Spectrophotometry, Ultraviolet; D013329:Structure-Activity Relationship
D008024:Ligands; D012709:Serum Albumin; D007975:Leukotriene B4; D007052:Ibuprofen