Effect of ibuprofen and warfarin on the allosteric properties of haem–human serum albumin

Abstract

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–haem) and of ferrous nitrosylated haem–human serum albumin (ferrous HSA–haem‐NO) is reported. Ferric HSA–haem is hexa‐coordinated, the haem‐iron atom being bonded to His105 and Tyr148. Upon drug binding to the warfarin primary site, the displacement of water molecules − buried in close proximity to the haem binding pocket − induces perturbation of the electronic absorbance properties of the chromophore without affecting the coordination number or the spin state of the haem‐iron, and the quenching of the 1H‐NMR relaxivity. Values of Kd for ibuprofen and warfarin binding to the warfarin primary site of ferric HSA–haem, corresponding to the ibuprofen secondary cleft, are 5.4 ± 1.1 × 10−4 m and 2.1 ± 0.4 × 10−5 m, respectively. The affinity of ibuprofen and warfarin for the warfarin primary cleft of ferric HSA–haem is lower than that reported for drug binding to haem‐free HSA. Accordingly, the Kd value for haem binding to HSA increases from 1.3 ± 0.2 × 10−8 m in the absence of drugs to 1.5 ± 0.2 × 10−7 m in the presence of ibuprofen and warfarin. Ferrous HSA–haem‐NO is a five‐coordinated haem‐iron system. Drug binding to the warfarin primary site of ferrous HSA–haem‐NO induces the transition towards the six‐coordinated haem‐iron species, the haem‐iron atom being bonded to His105. Remarkably, the ibuprofen primary cleft appears to be functionally and spectroscopically uncoupled from the haem site of HSA. Present results represent a clear‐cut evidence for the drug‐induced shift of allosteric equilibrium(a) of HSA.

Evidence for pH-dependent multiple conformers in iron(II) heme–human serum albumin: spectroscopic and kinetic investigation of carbon monoxide binding

“…Furthermore, this behavior, which underlies the occurrence of multiple conformations of iron(II) heme-HSA, may have great impact on the function of heme-HSA as a metabolite and drug transporter, since this proton-linked modulation is reflected in its capability of interacting with molecules circulating in the bloodstream, as previously demonstrated [3,8,20,22,[29][30][31][32]. Therefore, HSA, not only acting as a heme carrier but also displaying transient heme-based properties, represents a case for ''chronosteric effects'' [62], which opens the scenario toward the possibility of a time-and metabolite-dependent multiplicity of roles for HSA.…”

“…According to Sudlow's nomenclature, bulky heterocyclic anions bind preferentially to Sudlow's site I (located in subdomain IIA), whereas Sudlow's site II (located in subdomain IIIA) is preferred by aromatic carboxylates with an extended conformation ( Fig. 1) [3,11,[18][19][20][21][22][23][24]. Among others, HSA is able to bind 7 equiv of long-chain fatty acids (FAs) at multiple binding sites (labeled FA1 to FA7) ( Fig.…”

“…At pH [ 8.0 and in the absence of ligands, HSA exhibits the basic form, which is characterized by a high affinity for some ligands (e.g., heme). Ligands that bind with the highest affinity to one of the conformational states may allosterically modulate the HSA transition [3,8,20,22,[29][30][31][32].…”

Reversible two-step unfolding of heme–human serum albumin: a 1H-NMR relaxometric and circular dichroism study

“…Heme-HSA shows a considerably high r 1 value (approximately 25 s -1 mM -1 ), compared with other heme-proteins such as hemoglobin and myoglobin [11,19,32,[48][49][50][51]. Nuclear magnetic relaxation dispersion studies of heme-HSA revealed a strong paramagnetic contribution due to a cluster of water molecules buried near the heme that may be employed to follow a number of events including conformational transitions [16]. Addition of GnCl up to 5.0 M brings about a progressive decrease of r 1 (Fig.…”

“…One of the FA binding sites (FA1) has evolved to selectively bind heme with high affinity (K d = 1.0 9 10 -8 M [11]) with the tetrapyrrole ring arranged in a D-shaped cavity limited by Tyr138 and Tyr161 residues that provide p-p stacking interaction with the porphyrin and supply a donor oxygen (from Tyr161) for the Fe(III) heme iron [10][11][12][13][14]. In turn, heme binding to HSA endows the protein with heme-based reactivity [4,15] and spectroscopic properties [16][17][18][19][20][21].…”