Molecular Serum Albumin Unmask Nanobio Properties of Molecular Graphenes in Shungite Carbon Nanoparticles

Abstract

Serum albumin is a popular macromolecule for studying the effect of proteins on the colloidal stability of nanoparticle (NP) dispersions, as well as the protein-nanoparticle interaction and protein corona formation. In this work, we analyze the specific conformation-dependent phase, redox, and fatty acid delivery properties of bovine albumin in the presence of shungite carbon (ShC) molecular graphenes stabilized in aqueous dispersions in the form of NPs in order to reveal the features of NP bioactivity. The formation of NP complexes with proteins (protein corona around NP) affects the transport properties of albumin for the delivery of fatty acids. Being acceptors of electrons and ligands, ShC NPs are capable of exhibiting both their own biological activity and significantly affecting conformational and phase transformations in protein systems.

Keywords: fatty acids; metastable clusters; protein corona; redox bioactivity; serum albumin; shungite carbon nanoparticles.

Figure 1

(a) Schematic presentation of protein corona in the dispersion of any nanoparticle (NPs) with proteins of different types (adapted from [1]): 1—hard corona; 2—soft corona; (↔)—exchange of proteins between hard and soft corona. Proteins are divided between hard and soft corona depending on the affinity of the proteins for the NP surface. (b). Corona of bovine serum albumin (BSA) around shungite carbon NP. Insert: long rod indicates exchange of fatty acids (which are not a part of the corona) between BSA and NP. Different colors are provided for ease of observation.

Figure 2

General molecular structure of serum albumin (SA). (a) SA domain structure with Cys-34 located in the IA domain cavity. (b) Potential binding sites of fatty acid in the SA structure. Maleimido-proxyl is a spin label that modifies the SH group of Cys-34. 5-DOXYL stearic acid is a spin probe adsorbed at fatty acid binding sites. Structures (a,b) are adapted from [30]. Different colors are provided for ease of observation.

Figure 3

EPR spectra of 5-doxyl-stearic acid (5DSA) spin-probe in different samples: 1—dispersion of shungite carbon nanoparticles (ShC NPs); 2—dispersion of bovine serum albumin (BSA) with molar ratio 5DSA/BSA ≈ 2; 3—dispersion of BSA with molar ratio 5DSA/BSA ≈ 1; 4—dispersion of BSA + ShC NPs. A, A* and B, B* are spectral components (lines), wide and narrow, respectively, due to the immobilization of 5DSA in specific binding sites (A, A*), the relocation of 5DSA between binding sites (B, B* in spectra 2,3), fast rotation of spin probes in ShC NPs.

Figure 4

Relative intensity of scattered light vs. particle size (stokes radius) for bovine serum albumin (BSA) (a) fatty acid-free BSA (blue solid lines) and (b) fatty acid-containing BSA (red solid lines). Dashed lines present the distributions after shungite carbon nanoparticle (ShC NP) administration to the BSA dispersion. Concentration: BSA 1 g/L, ShC NPs 0.06 g/L (25 °C, pH 6).

Figure 5

Heat absorption capacity of native Human and native bovine serum albumin (BSA) solutions (0.5 g/L, pH 6.5) in the absence and in the presence of shungite carbon nanoparticles (ShC NPs) (black curves). Red and blue curves are the Gaussian components.

Figure 6

Temperature dependence of the integral intensity of EPR spectrum of a spin probe 5DSA bound to bovine serum albumin BSA) in the absence (triangles) and in the presence (squares) of shungite carbon nanoparticles (ShC NPs) in protein solution. Protein concentration is 50 mg/mL; 0.1 M NaCl; pH 6; probe/protein ≈ 1. The concentration of ShC NPs in the aqueous dispersion is 0.01 mg/mL Sections of the curves are approximated by straight lines to show the deviation of the dependencies from linearity.

Figure 7

Hypothetical solubility phase diagram of the protein dispersion in the plane temperature—composition m₂/m₃, where m₂ and m₃ are the protein and salt molar concentrations, respectively. Binodals (blue parabolas) and spinodals (green parabolas) are represented as curves with upper critical solution temperature (UCST) and lower critical solution temperature (LCST). Curves 1–6 characterize the change in protein solubility with increasing (from bottom to top) salt concentration m₃. They reflect the overall effect of “salting out”, “salting in”, and “salting out” as re-entrant phase separations induced by salt [78]. Curve 7 is the solubility of protein oligomers; curve 8 is the solubility of microfibrils. Insert: chemical potentials µ of native (N) and denatured (D) proteins on temperature.

Figure 8

(a) Scheme of a diluted shungite carbon nanoparticle (ShC NP) dispersion. Characteristic sizes of NPs, globules, BSU, and stacks composing globules are below. (b) Aqueous ShC dispersions of initial—0.13 mg/mL (1) and diluted—0.06 mg/mL (2) concentration. (c) Particle size distributions in terms of the relative scattered intensity vs. size (stokes radius) for ShC NPs (DLS) and some characteristics of the dispersion used (pH, ζ-potential).