Human Serum Albumin Aggregation/Fibrillation and its Abilities to Drugs Binding

Abstract

Human serum albumin (HSA) is a protein that transports neutral and acid ligands in the organism. Depending on the environment's pH conditions, HSA can take one of the five isomeric forms that change its conformation. HSA can form aggregates resembling those in vitro formed from amyloid at physiological pH (neutral and acidic). Not surprisingly, the main goal of the research was aggregation/fibrillation of HSA, the study of the physicochemical properties of formed amyloid fibrils using thioflavin T (ThT) and the analysis of ligand binding to aggregated/fibrillated albumin in the presence of dansyl-l-glutamine (dGlu), dansyl-l-proline (dPro), phenylbutazone (Phb) and ketoprofen (Ket). Solutions of human serum albumin, both non-modified and modified, were examined with the use of fluorescence, absorption and circular dichroism (CD) spectroscopy. The experiments conducted allowed observation of changes in the structure of incubated HSA (HSA_INC) in relation to nonmodified HSA (HSA_FR). The formed aggregates/fibrillation differed in structure from HSA monomers and dimers. Based on CD spectroscopy, previously absent βstructural constructs have been registered. Whereas, using fluorescence spectroscopy, the association constants differing for fresh and incubated HSA solutions in the presence of dansyl-amino acids and markers for binding sites were calculated and allowed observation of the conformational changes in HSA molecule.

Keywords: aggregation/fibrillation; human serum albumin; spectroscopic methods.

Figure 1

Emission fluorescence spectra of thioflavin T (ThT) (1 × 10⁻⁵ mol·L⁻¹) in the presence of unmodified human serum albumin (HSA_FR) and incubated human serum albumin (HSA_INC) at (a) 37 °C for 9 days and (b) 65 °C for 7 days; HSA concentration 1 × 10⁻⁶ mol·L⁻¹, λ_ex 440 nm.

Figure 2

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

Figure 3

Emission fluorescence spectra of unmodified human serum albumin (HSA_FR) and incubated human serum albumin (HSA_INC) at 5 × 10⁻⁶ mol·L⁻¹ concentration; (a) λ_ex 275 nm, (b) λ_ex 295 nm.

Figure 3

Emission fluorescence spectra of unmodified human serum albumin (HSA_FR) and incubated human serum albumin (HSA_INC) at 5 × 10⁻⁶ mol·L⁻¹ concentration; (a) λ_ex 275 nm, (b) λ_ex 295 nm.

Figure 4

Synchronous fluorescence spectra of HSA_FR and HSA_INC at 5 × 10⁻⁶ mol·L⁻¹ concentration; (a) ∆λ 15 nm, (b) ∆λ 60 nm.

Figure 5

CD spectra of 3 × 10⁻⁶ mol·L⁻¹ HSA_FR and HSA_INC.

Figure 6

Scatchard curve for dGlu–HSA_FR and dGlu–HSA_INC systems; λ_ex 350 nm.

Figure 7

Scatchard curve for (a) dPro–HSA_FR and (b) dPro–HSA_INC systems. The inserts show the binding isotherms; λ_ex 350 nm.

Figure 8

Scatchard curve for Phb–HSA_FR and Phb–HSA_INC systems; λ_ex 275 nm.

Figure 9

Scatchard curve for (a) Phb–HSA_FR and (b) Phb–HSA_INC systems. Inserts show the binding isotherms; λ_ex 295 nm.

Figure 9

Scatchard curve for (a) Phb–HSA_FR and (b) Phb–HSA_INC systems. Inserts show the binding isotherms; λ_ex 295 nm.

Figure 10

Scatchard curve for Ket–HSA_FR and Ket–HSA_INC systems; λ_ex 275 nm.

Figure 11

Scatchard curve for Ket–HSA_FR and Ket–HSA_INC systems; λ_ex 295 nm.