The Influence of Oxidative Stress on Serum Albumin Structure as a Carrier of Selected Diazaphenothiazine with Potential Anticancer Activity

Abstract

Albumin is one of the most important proteins in human blood. Among its multiple functions, drug binding is crucial in terms of drug distribution in human body. This protein undergoes many modifications that are certain to influence protein activity and affect its structure. One such reaction is albumin oxidation. Chloramine T is a strong oxidant. Solutions of human serum albumin, both non-modified and modified by chloramine T, were examined with the use of fluorescence, absorption and circular dichroism (CD) spectroscopy. 10H-3,6-diazaphenothiazine (DAPT) has anticancer activity and it has been studied for the first time in terms of binding with human serum albumin-its potential as a transporting protein. Using fluorescence spectroscopy, in the presence of dansylated amino acids, dansyl-l-glutamine (dGlu), dansyl-l-proline (dPro), DAPT binding with two main albumin sites-in subdomain IIA and IIIA-has been evaluated. Based on the conducted data, in order to measure the stability of DAPT complexes with human (HSA) and oxidized (oHSA) serum albumin, association constant (K_a) for ligand-HSA and ligand-oHSA complexes were calculated. It has been presumed that oxidation is not an important issue in terms of 10H-3,6-diazaphenothiazine binding to albumin. It means that the distribution of this substance is similar regardless of changes in albumin structure caused by oxidation, natural occurring in the organism.

Keywords: 10H-3,6-diazaphenothiazine; human serum albumin; oxidation; spectroscopic methods.

Figure 1

Structural formula of 10H-3,6-diazaphenothiazine (DAPT).

Figure 2

Absorption spectra of unmodified (human serum albumin (HSA)) and oxidized (oxidized human serum albumin (oHSA)) human serum albumin at 5 × 10⁻⁶ mol·L⁻¹ concentration. In the insert second derivative of 5 × 10⁻⁶ mol·L⁻¹ HSA and oHSA absorption spectrum.

Figure 3

Emission fluorescence spectra of unmodified (HSA) and oxidized (oHSA) human serum albumin at 5 × 10⁻⁶ mol·L⁻¹ concentration excited at (a) λ_ex 275 nm and (b) λ_ex 295 nm.

Figure 4

Excitation fluorescence spectra of unmodified (HSA) and oxidized human serum albumin (oHSA) at 5 × 10⁻⁶ mol·L^–1 concentration. In the insert normalized excitation fluorescence spectra of oHSA and unmodified HSA.

Figure 5

Circular dichroism (CD) spectra of HSA and oHSA at 1 × 10⁻⁶ mol·L^–1 concentration.

Figure 6

Emission fluorescence spectra of (a) dGlu and (b) dPro in the presence of HSA and oHSA. [dGlu] = [dPro] 5 × 10⁻⁵ mol·L⁻¹, [HSA] = [oHSA] 1 × 10⁻⁵ mol·L⁻¹; λ_ex = 350 nm.

Figure 7

Scatchard curve for (a) dGlu-HSA, dGlu-oHSA and (b) dPro-HSA, dPro-oHSA systems; λ_ex 350 nm. [HSA] = [oHSA] 1 × 10⁻⁵ mol·L⁻¹, [dGlu] 0.5 × 10⁻⁵ mol·L⁻¹–6 × 10⁻⁵ mol·L⁻¹. In the insert the binding isotherms for dPro-HSA and dPro-oHSA systems.

Figure 8

Comparison of quenching curves of (a) dGlu and (b) dPro at 5 × 10⁻⁶ mol·L⁻¹ concentration in the presence of HSA, oHSA at 5 × 10⁻⁶ mol·L⁻¹ concentration and DAPT (2.5 × 10⁻⁶ mol·L⁻¹–8.5 × 10⁻⁵ mol·L⁻¹); the error calculated as maximum deviation does not exceed the symbols.

Figure 9

The Stern-Volmer curves of F₀/F vs. 10H-3,6-diazophenothiazine (DAPT) concentration at (a) λ_ex 275 nm and (b) λ_ex 295 nm; the error calculated as maximum deviation does not exceed the symbols.

Figure 10

The binding isotherms for DAPT-HSA and DAPT-oHSA systems at (a) λ_ex 275 nm and (b) λ_ex 295 nm.

Figure 11

The Klotz plots for DAPT-HSA and DAPT-oHSA systems at (a) λ_ex 275 nm and (b) λ_ex 295 nm.