Use of amphotericin B as optical probe to study conformational changes and thermodynamic stability in human serum albumin.
The binding of polyene antibiotic amphotericin B to serum albumin was studied using absorption, fluorescence, and circular dichroism techniques. A hypochromic effect was observed in the absorption spectrum of amphotericin B in the presence of albumin with maxima at 366 nm, 385 nm, and 408 nm, which correspond to the absorption of the monomeric form of amphotericin B. A modification on the circular dichroism spectrum of amphotericin B in the presence of albumin was observed at bands 329 nm and 351 nm (excitronic interaction), which suggests that only amphotericin B monomer is bound to the protein. Amphotericin B perturbs the specific markers for sites I, II, and fatty acid binding site bound to these sites, suggesting that amphotericin B interacts with a great binding area in albumin. Lysines 199 and 525 in albumin participate in the molecular interaction between amphotericin B and the protein. The absorption spectrum of amphotericin B bound to albumin was sensitive to the chemical and thermal treatment of the protein, to neutral-basic transition of albumin and to conformational changes induced by the binding of other ligands to this protein.
Romanini
Diana
D
Departamento de Química-Física and CONICET, Faculty of Biochemical and Pharmaceutical Sciences, National University of Rosario, Suipacha 570 (2000) Rosario, Argentina.
Müller
Gabriela
G
Picó
Guillermo
G
D000596:Amino Acids / Q000737:chemistry; D000666:Amphotericin B / Q000494:pharmacology*; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001665:Binding Sites; D002942:Circular Dichroism; D004305:Dose-Response Relationship, Drug; D005456:Fluorescent Dyes / Q000494:pharmacology; D006639:Histidine / Q000737:chemistry; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008024:Ligands; D008239:Lysine / Q000737:chemistry; D011487:Protein Conformation; D012709:Serum Albumin / Q000737:chemistry*; D013050:Spectrometry, Fluorescence; D013696:Temperature; D013816:Thermodynamics; D014443:Tyrosine / Q000737:chemistry; D014508:Urea / Q000494:pharmacology
D000596:Amino Acids; D000900:Anti-Bacterial Agents; D005456:Fluorescent Dyes; D008024:Ligands; D012709:Serum Albumin; D014443:Tyrosine; D006639:Histidine; D000666:Amphotericin B; D014508:Urea; D008239:Lysine