A fluorescent reporter detects details of aromatic ligand interference in drug-binding sites of human serum albumin.
Human serum albumin (HSA) transports many ligands including small aromatic molecules: metabolites, drugs etc. Phenylbutazone is an anti-inflammatory drug, which binds to the drug-binding site I of HSA. Its interaction with this site has been studied using a fluorescent dye, CAPIDAN, whose fluorescence in serum originates from HSA and is sensitive to the changes in HSA site I in some diseases. Its fluorescence in HSA solutions is strongly suppressed by phenylbutazone. This phenomenon seems to be a basic sign of a simple drug-dye competition. However, a more detailed study of the time-resolved fluorescence decay of CAPIDAN has shown that phenylbutazone lowers fluorescence without changing the total amount of bound dye. In brief, the HSA-bound dye forms three populations due to three types of environment at the binding sites. The first two populations probably have a rather strong Coulomb interaction with the positive charge of residues Arginine 218 or Arginine 222 in site I and are responsible for approximately 90% of the total fluorescence. Phenylbutazone blocks this interaction and therefore lowers this fluorescence. At the same time the binding of the third population increases considerably in the presence of phenylbutazone, and, as a result, the actual number of bound dye molecules remains almost unchanged despite the ligand competition. So, time resolved fluorescence of the reporter allows to observe details of interactions and interference of aromatic ligands in drug binding site I of HSA both in isolated HSA and in serum.
Federal Research and Clinical Center of Physical Chemical Medicine, 1-a Malaya Pirogovskaya, Moscow, 119435, Russia.
Gennady
G
Dobretsov
Federal Research and Clinical Center of Physical Chemical Medicine, 1-a Malaya Pirogovskaya, Moscow, 119435, Russia. Electronic address: esmoline@mail.ru.
Natalia
N
Smolina
Lebedev Physical Institute, 53 Leninskij Prospekt, Moscow, 119991, Russia.
Tatiana
T
Syrejshchikova
Federal Research and Clinical Center of Physical Chemical Medicine, 1-a Malaya Pirogovskaya, Moscow, 119435, Russia.
Varvara
V
Brilliantova
Moscow Research Institute of Psychiatry, 3 Poteshnaya, Moscow, 107076, Russia.
Marat
M
Uzbekov
D001665:Binding Sites; D004353:Drug Evaluation, Preclinical / Q000379:methods; D005456:Fluorescent Dyes / Q000737:chemistry*; D006801:Humans; D006841:Hydrocarbons, Aromatic / Q000737:chemistry; D007094:Imides / Q000737:chemistry*; D008024:Ligands; D009281:Naphthalenes / Q000737:chemistry*; D011485:Protein Binding; D025941:Protein Interaction Mapping / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D012709:Serum Albumin / Q000737:chemistry* / Q000378:metabolism; D013050:Spectrometry, Fluorescence / Q000379:methods*
D005456:Fluorescent Dyes; D006841:Hydrocarbons, Aromatic; D007094:Imides; D008024:Ligands; D009281:Naphthalenes; D012709:Serum Albumin; C511435:dimethylaminonaphthalic acid N-carboxyphenylimide
Fluorescent reporter CAPIDAN; Human serum albumin; Interference of aromatic ligands; Phenylbutazone