Relaxometric characterization of human hemalbumin

Abstract

Hemalbumin [i.e., Fe(III)-protoporphyrin IX-human serum albumin; Fe(III)heme-HSA] is an important intermediate in the recovery of heme iron following hemolysis. Relaxometric data are consistent with the occurrence of a hexacoordinated high-spin Fe(III) center with no water in the inner coordination sphere. The relatively high relaxation enhancement observed for an aqueous solution of Fe(III)heme-HSA (r1p=4.8 mM(-1)s(-1) at 20 MHz, pH 7, and 25 C) is ascribed to the occurrence of a strong contribution from water molecules in the second coordination sphere. Structural analysis of the putative binding region has been performed by a Monte Carlo simulated annealing procedure, which allowed us to identify His105 and Tyr148 as axial ligands. The role of a tyrosinate as the sixth Fe(III)heme ligand is supported by the pH-dependent analysis. Interestingly, when Fe(III) is replaced by Mn(III), the occurrence of a fast exchanging water molecule at pH values close to neutrality is detected. As the pH is increased, the Mn(III) containing system behaves analogously to Fe(III)heme-HSA. At higher pH, the phenolate ligand is eventually displaced by OH- from both Fe(III) and Mn(III) centers. Support for the proposed bonding scheme has been gained also from competitive binding assays for the sixth coordination site by fluoride, azide, and imidazole ligands.

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.…”

“…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 was prepared by adding the appropriate volume of the stock heme solution (i.e., 1.2 9 10 -2 M heme in 1.0 9 10 -1 M NaOH) to the 1.0 9 10 -3 M HSA solution (1.0 9 10 -1 M phosphate buffer, pH 7.0) to obtain a final heme-HSA concentration ranging between 5.0 9 10 -6 and 1.0 9 10 -3 M. The heme to HSA ratio ranged between 1:10 and 9:10. The excess of HSA ensured that the heme was bound to the HSA primary binding site only [11]. The 1.0 9 10 -1 M myristate solution was prepared by adding 1.0 9 10 -4 mol sodium myristate to 1.0 ml of 1.0 9 10 -1 M NaOH.…”

“…The intensity of the signal is proportional to the magnetization at the end of the evolution interval [35]. The reproducibility in T 1 measurements was ±0.5% [11]. The temperature was controlled by a Stelar VTC-91 airflow heater and checked in the sample cavity with a mercury thermometer.…”

“…In sites FA1-FA5 the carboxylate moiety of FAs is anchored by electrostatic/polar interactions; in contrast, sites FA6 and FA7 do not display clear evidence of polar interactions that keep in place the carboxylate head of the FA, thus suggesting that sites FA6 and FA7 are low-affinity FA binding sites [3,4,[6][7][8][9][10]. 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].…”