On-line version ISSN 1414-431X
Braz J Med Biol Res vol.37 no.7 Ribeirão Preto July 2004
D. Silva1, C.M. Cortez1 and S.R.W. Louro2
1Departamento de Ciências Fisiológicas, Universidade do Estado do Rio Janeiro, Rio de Janeiro, RJ, Brasil
2Departamento de Física, Pontifícia Universidade Católica do Rio de Janeiro, Rio de Janeiro, RJ, Brasil
The binding of chlorpromazine (CPZ) and hemin to bovine serum albumin was studied by the fluorescence quenching technique. CPZ is a widely used anti-psychotic drug that interacts with blood components, influences bioavailability, and affects function of several biomolecules. Hemin is an important ferric residue of hemoglobin that binds within the hydrophobic region of albumin with high specificity. Quenching of the intrinsic fluorescence of bovine serum albumin (BSA) was observed by selectively exciting tryptophan residues at 290 nm. Emission spectra were recorded in the range from 300 to 450 nm for each quencher addition. Stern-Volmer graphs were plotted, and the quenching constant estimated for BSA solution titrated with hemin at 25ºC was 1.44 (± 0.05) x 105 M-1. Results showed that bovine albumin tryptophans are not equally accessible to CPZ, in agreement with the idea that polar or charged quenchers have more affinity for amino acid residues on the outer wall of the protein. Hemin added to albumin solution at a molar ratio of 1:1 quenched about 25% of their fluorescence. The quenching effect of CPZ on albumin-hemin solution was stronger than on pure BSA. This increase can be the result of combined conformational changes in the structure of albumin caused firstly by hemin and then by CPZ. Our results suggest that the primary binding site for hemin on bovine albumin may be located asymmetrically between the two tryptophans along the sequence formed by subdomains IB and IIA, closer to tryptophan residue 212.
Key words: Chlorpromazine, Hemin, Albumin, Fluorescence quenching
Chlorpromazine (CPZ) is a phenothiazine drug widely used in psychiatric treatment, and possesses a tricyclic ring bend at the N-S axis. Similar to other anti-psychotic drugs, its chronic use may cause severe side effects (1-3), such as parkinsonism, cornea opacity and respiratory troubles, and is suspected to be related to iron sediments in blood vessels and nervous cells (4). It is also known that high acute doses of CPZ induce hyperglycemia in people with latent diabetes mellitus. The cause of many of these side effects is still unclear.
CPZ binds to three important blood components: red blood cell membranes, albumin and lipoproteins. CPZ also interacts with lipid, the bilayer and proteins of biomembranes, influencing their permeability (5,6), although the
mechanisms responsible are still in discussion. Recently, Rukhadze et al. (7) investigated the association of CPZ with bovine serum albumin (BSA), concluding that concentration of both the drug and protein influenced the absolute and relative values of the free fraction of CPZ.
Binding to plasma proteins is an important pharmacological parameter, since it frequently affects the distribution and elimination of a drug, as well as the duration and intensity of its physiological effects (8). Difficulties in predicting the clinical effects of the plasma level of a drug may reflect the biological variability in its absorption and elimination. Indeed, it is well known that the interaction of CPZ with blood components influences bioavailability and affects the function of several biomolecules (9).
On the basis of these considerations, we studied the interaction between CPZ and albumin, and determined if CPZ could influence the hemin-albumin interaction. Hemin is an important porphyrin residue of hemoglobin that binds to the hydrophobic region of albumin with high specificity (10). It is known that highly hydrophobic porphyrins can penetrate lipidic regions of membranes, while moderately hydrophobic ones preferentially distribute into polar regions of the cell (11). Porphyrins and related compounds have been used widely as therapeutic drugs. Most clinical applications have focused on fluorescence detection and photodynamic therapy of cancer (12).
Albumin represents 52-60% of the total plasma protein and plays an important role in the transport and storage of hormones, ions, fatty acids and drugs, acting by regulating their plasma concentrations (9,13). It is the major binding protein for most anionic drugs, and binds cationic drugs with moderate affinity, but more avidly than other plasma proteins such as alpha-acid glycoproteins and lipoproteins (14). Phenothiazines are cationic (basic) compounds (8).
Most drugs bind reversibly to a number of binding sites on albumin, and there is evidence of conformational changes in protein induced by its interaction with low molecular weight drugs. These changes appear to affect the secondary and tertiary structure of albumin. Hushcha et al. (15) reported that albumin globules have the most compact configuration at physiological pH, and either increasing the pH of the medium to 8.0 or decreasing it to 5.4 resulted in the increase of globule size. The interaction with CPZ causes conformational changes of albumin similar to basic transitions. Verbeeck et al. (8), using the equilibrium dialysis technique, reported that the binding of CPZ by albumin is a high capacity and low affinity interaction.
In the present study, quenching of the intrinsic fluorescence of BSA was observed by selectively exciting tryptophan residues. BSA consists of a chain of 580 amino acid residues forming a single polypeptide of known sequence, which contains three homologous a-helix domains (I-III) (13,16) and two tryptophan residues. These are located at positions 134 and 212 of the chain. Stern-Volmer graphs were plotted, and the primary binding sites of CPZ and hemin to BSA were analyzed.
We have performed in vitro studies on the interaction of CPZ and hemin with BSA. CPZ and hemin were purchased from Sigma. To prevent photodegradation, CPZ stock solutions were kept in the dark at -10ºC. Hemin stock solutions (5 mM in 20 mM NaOH) were also stored at -10ºC. Fluorescence measurements were performed with a PTI-QM1 Fluorescence System (Pontifícia Universidade Cató