Binding and relaxometric properties of heme complexes with cyanogen bromide fragments of human serum albumin

The spectroscopic and reactivity properties of hemin complexes formed with cyanogen bromide fragments B
(residues 1–123), C (124–298), A (299–585), and D (1–298) of human serum albumin (HSA) have been investigated. The
complex hemin-D exhibits binding, spectral, circular dichroism, and reactivity characteristics very similar to those of
hemin-HSA, indicating that fragment D contains the entire HSA domain involved in heme binding. The characteristics of the
other hemin complexes are different, and a detailed investigation of the properties of hemin-C has been carried out because
this fragment contains the HSA binding region of several important drugs. Hemin-C contains a low-spin Fe(III) center, with two
imidazole ligands, but the complex undergoes a reversible structural transition at basic pH leading to a high-spin, fivecoordinated
Fe(III) species. This change determines a marked increase in the relaxation rate of water protons. Limited
proteolysis experiments and mass spectral analysis carried out on fragment C and hemin-C show that the region encompassing
residues Glu-208 to Trp-214 is protected from activity of proteases in the complex and, therefore, is involved in the
interaction with hemin. A structural model of fragment C enables us to propose that His-242 and His-288 are the axial ligands
for the Fe(III) center.