Oxidation enhances human serum albumin thermal stability and changes the routes of amyloid fibril formation

Abstract

Oxidative damages are linked to several aging-related diseases and are among the chemical pathways determining protein degradation. Specifically, interplay of oxidative stress and protein aggregation is recognized to have a link to the loss of cellular function in pathologies like Alzheimer's and Parkinson's diseases. Interaction between protein and reactive oxygen species may indeed induce small changes in protein structure and lead to the inhibition/modification of protein aggregation process, potentially determining the formation of species with different inherent toxicity. Understanding the temperate relationship between these events can be of utmost importance in unraveling the molecular basis of neurodegeneration. In this work, we investigated the effect of hydrogen peroxide oxidation on Human Serum Albumin (HSA) structure, thermal stability and aggregation properties. In the selected conditions, HSA forms fibrillar aggregates, while the oxidized protein undergoes aggregation via new routes involving, in different extents, specific domains of the molecule. Minute variations due to oxidation of single residues affect HSA tertiary structure leading to protein compaction, increased thermal stability, and reduced association propensity.

Figure 1. Spectroscopic Analysis of structural and conformational changes induced in human serum albumin by oxidation.

(A) Difference FTIR absorption spectrum in the region 900–1200 cm⁻¹. Samples were prepared in KBr pellet 5% w/w and signal of the HSA sample were subtracted to the OX-HSA in the same conditions; (B) FTIR spectra in the Amide region (1300–1700 cm⁻¹) of HSA (black line) and OX-HSA (red line), 15 mg/ml in D₂O. Data are normalized at Amide I' peak; (C) Far-UV CD spectrum of HSA (black line) and OX-HSA (red line) 0.5 mg/ml K-phosphate buffer at pH 7.4 at room temperature; (D) Intrinsic fluorescence spectra of HSA (black line) and OX-HSA (red line) 0.5 mg/ml K-phosphate buffer at pH 7.4 at room temperature. The excitation wavelength was 280 nm.

Figure 2. ANS titration curves.

Integrated intensity of ANS emission as a function of molar ratio [ANS]/[protein] for HSA (black) and OX-HSA (red) in the range [ANS]/[protein]  = 0.1–2.8. ANS fluorescence was measured at room temperature using an excitation wavelength λ_exc = 380 nm in HSA and OX-HSA samples 0.5 mg/ml in K-phosphate buffer 0.1 M at pH 7.4

Figure 3. Aggregation propensity for HSA and OX-HSA.

Rayleigh scattering variations as a function of temperature measured during an upward temperature scan at 12°C/h for HSA (black) and OX-HSA (red) samples 0.5 mg/ml in K-phosphate buffer 0.1 M at pH 7.4.

Figure 4. Temperature effect on aggregation kinetics.

Temporal evolution of Rayleigh scattering intensity measured at λ = 280 nm for HSA (A) and OX-HSA (B) 0.5 mg/ml in K-phosphate buffer 0.1 M pH 7.4 during isothermal incubation at 60, 65, 70, 75 and 80°C. The inset shows the superimposed kinetics at 70°C when scaled for the Rayleigh scattering plateau value.

Figure 5. Tertiary structure changes during the aggregation process.

Temporal evolution of intrinsic fluorescence emission band spectral moments for HSA (black dots) and OX-HSA (red dots) samples 0.5 mg/ml in K-phosphate buffer 0.1 M at pH 7.4 during isothermal incubation at 70°C. (A) Zeroth moment (M₀) (dots) and Rayleigh scattering (solid line) as a function of time. (B) First moment as a function of time. Rayleigh scattering and fluorescence spectra were acquired under excitation at λ_exc = 280 nm.

Figure 6. Changes in hydrophobic regions during the aggregation process.

Temporal evolution of ANS fluorescence emission band spectral moments for HSA (black dots) and OX-HSA (red dots) samples 0.5 mg/ml in K-phosphate buffer at pH 7.4 during isothermal incubation at 70°C. (A) Zeroth moment (M₀) (dots) and Rayleigh scattering (solid line) as a function of time (B) First moment as a function of time. Spectra were acquired using [ANS]/[protein] concentration ratio 0.3 in order to single out information on domain III changes. ANS Fluorescence spectra were acquired under excitation at λ_exc = 380 nm. Rayleigh scattering was acquired under excitation at λ_exc = 280 nm.

Figure 7. Secondary structure changes.

(A) Temporal evolution of CD signal measured at 222 for HSA (Black line) and OX-HSA (red line) sample 0.5 mg/ml in phosphate buffer 0.1 M at pH 7.4 incubated at 70°C for 800 minutes. Inset: magnification of the early stages (B) Far-UV CD spectra at room temperature of freshly prepared HSA (solid black line) and OX-HSA (solid red line) sample and of the same samples of HSA (dashed black line) and OX-HSA (dashed red line) after 800 minutes of incubation at 70°C. Inset: magnification of significant differences in the spectra. Solid lines at 207 (red) and 209 (grey) nm are guides for eyes.

Figure 8. Morphologies of HSA and OX-HSA aggregates.

TEM images of HSA (a) and OX-HSA (b) aggregates obtained upon thermal incubation 10 mg/ml protein solution in K-phosphate buffer 0.1 M at pH 7.4 at 70°C for 12 hours.