The Importance of Protein-Protein Interactions on the pH-Induced Conformational Changes of Bovine Serum Albumin: A Small-Angle X-Ray Scattering Study

Abstract

The combined effects of concentration and pH on the conformational states of bovine serum albumin (BSA) are investigated by small-angle x-ray scattering. Serum albumins, at physiological conditions, are found at concentrations of approximately 35-45 mg/mL (42 mg/mL in the case of humans). In this work, BSA at three different concentrations (10, 25, and 50 mg/mL) and pH values (2.0-9.0) have been studied. Data were analyzed by means of the Global Fitting procedure, with the protein form factor calculated from human serum albumin (HSA) crystallographic structure and the interference function described, considering repulsive and attractive interaction potentials within a random phase approximation. Small-angle x-ray scattering data show that BSA maintains its native state from pH 4.0 up to 9.0 at all investigated concentrations. A pH-dependence of the absolute net protein charge is shown and the charge number per BSA is quantified to 10(2), 8(1), 13(2), 20(2), and 26(2) for pH values 4.0, 5.4, 7.0, 8.0, and 9.0, respectively. The attractive potential diminishes as BSA concentration increases. The coexistence of monomers and dimers is observed at 50 mg/mL and pH 5.4, near the BSA isoelectric point. Samples at pH 2.0 show a different behavior, because BSA overall shape changes as a function of concentration. At 10 mg/mL, BSA is partially unfolded and a strong repulsive protein-protein interaction occurs due to the high amount of exposed charge. At 25 and 50 mg/mL, BSA undergoes some re-folding, which likely results in a molten-globule state. This work concludes by confirming that the protein concentration plays an important role on the pH-unfolded BSA state, due to a delicate compromise between interaction forces and crowding effects.

Bacterial killing via a type IV secretion system

“…Buffer scattering curves were collected and subtracted from the sample's scattering curve, taking into account its attenuation. The scattering curves were normalized by the protein concentration to check for interference effects 61 or aggregation 62 over the SAXS curves. Guinier analysis was used to calculate the protein radius of gyration, Rg, and to evaluate the monodispersity of the system.…”

Study on the thermodynamics of bovine serum albumin aqueous solutions: experiments, modeling and molecular simulations.

“…For γ-crystallin and lysozyme solutions Malfois et al (MALFOIS et al, 1996) and Tardieu et al (TARDIEU et al, 1999) used a Yukawa potential within the Hyper-Netted Chain approximation, which can only be solved numerically. Barbosa et al (BARBOSA et al, 2010) described the structure factor of bovine serum albumin through a similar approach with the extension introduced by Narayanan and Liu (NARAYANAN;LIU, 2003).…”

“…However, in Zhang et al (ZHANG et al, 2007) and in Barbosa et al (BARBOSA et al, 2010), the attractive parameter (either the depth from the square well potential or the Yukawa parameter) increases when the protein concentration decreases, even though the salt concentration remains constant.…”

“…We left intentionally ε as a function of η due to the experimental evidence that ε is not constant for proteins in aqueous solutions (ZHANG et al, 2007;BARBOSA et al, 2010). As Z must approach 1 as η approaches 0, the expression for ε must follow the limit:…”

“…Previous works (BARBOSA et al, 2010;ZHANG et al, 2007) showed that the attractive parameter of proteins in solution may depend on protein concentration.…”