Effect of ibuprofen and warfarin on the allosteric properties of haem-human serum albumin - A spectroscopic study

Haem binding to human serum albumin (HSA) endows the protein with peculiar spectroscopic properties. Here, the effect of ibuprofen and warfarin on the spectroscopic properties of ferric haem-human serum albumin (ferric HSA-ha em) and of ferrous nitrosylated haem-human serum albumin (ferrous HSA-haem- NO) is reported. Ferric HSA-haem is hexa-coordinated, the haem-iron atom be ing bonded to His105 and Tyr148. Upon drug binding to the warfarin primary site, the displacement of water molecules - buried in close proximity to th e haem binding pocket - induces perturbation of the electronic absorbance p roperties of the chromophore without affecting the coordination number or t he spin state of the haem-iron, and the quenching of the H-1-NMR relaxivity . Values of K-d for ibuprofen and warfarin binding to the warfarin primary site of ferric HSA-haem, corresponding to the ibuprofen secondary cleft, ar e 5.4 +/- 1.1 x 10(-4) rm and 2.1 +/- 0.4 x 10(-5) M, respectively. The aff inity of ibuprofen and warfarin for the warfarin primary cleft of ferric HS A-haem is lower than that reported for drug binding to haem-free HSA. Accor dingly, the K-d value for haem binding to HSA increases from 1.3 +/- 0.2 x 10(-8) m in the absence of drugs to 1.5 +/- 0.2 x 10(-7) M in the presence of ibuprofen and warfarin. Ferrous HSA-haem-NO is a five-coordinated haem-i ron system. Drug binding to the warfarin primary site of ferrous HSA-haem-N O induces the transition towards the six-coordinated haem-iron species, the haem-iron atom being bonded to His105. Remarkably, the ibuprofen primary c left appears to be functionally and spectroscopically uncoupled from the ha em site of HSA. Present results represent a clear-cut evidence for the drug -induced shift of allosteric equilibrium(a) of HSA.