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, five-coordinated 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.
D001665:Binding Sites; D055592:Biophysical Phenomena; D001703:Biophysics; D002942:Circular Dichroism; D003488:Cyanogen Bromide / Q000494:pharmacology*; D006418:Heme / Q000737:chemistry*; D006427:Hemin / Q000737:chemistry*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D008956:Models, Chemical; D008958:Models, Molecular; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011522:Protons; D012709:Serum Albumin / Q000737:chemistry*; D021241:Spectrometry, Mass, Electrospray Ionization; D013053:Spectrophotometry
D011522:Protons; D012709:Serum Albumin; D006418:Heme; D006427:Hemin; D003488:Cyanogen Bromide