FTIR Spectroscopy Study of the Secondary Structure Changes in Human Serum Albumin and Trypsin under Neutral Salts

Abstract

The effect of neutral salts on protein conformation was first analyzed by Hofmeister in 1888, however, even today this phenomenon is not completely understood. To clarify this effect, we studied changes in the secondary structure of two proteins: human serum albumin with predominantly α-helical structure and porcine pancreas β-trypsin with the typical β-structural arrangement in aqueous solutions of neutral salts (KSCN, KCl, (NH4)2SO4). The changes in the secondary structure were studied at 23 °C and 80 °C by using the second derivative deconvolution method of the IR spectra. Our results demonstrated that the ability of the salts to stabilize/destabilize these two proteins correlates with the Hofmeister series of ions. At the same time, some exceptions were also observed. The destabilization of the native structures of both α-helical albumin and β-structural trypsin upon interaction with neutral salts leads to the formation of intermolecular β-sheets typical for amyloid fibrils or amorphous aggregates. Thus, our quantitative FTIR-spectroscopy analysis allowed us to further clarify the mechanisms and complexity of the neutral salt actions on protein structures which may lead to strategies preventing unwelcome misfolding of proteins.

Uranyl-Binding Proteins on Silica Nanoparticles for Repeatable Capture of Uranyl Ions

“…On the other hand, proteins with a helical bundle structure require a higher threshold of ionic strength (typically 100−500 mM) to have a defined structure and they tend to aggregate due to unstable structure when ionic strength is low. 25,27,29 Once the ionic strength is above the threshold, the protein has increased stability, as we have observed for SUP at 300 mM NaCl concentration. Due to these to two competing factors, we have observed a significant aggregation at a salt concentration of 100 mM when the salt-out effect is dominated, but have seen a decreased aggregation when salt concentration is further increased to 300 mM under which the helix-bund structure stabilizes the protein.…”

“…Transformed cells were grown in terrific broth (TB) 13 (Ampicillin 100 μg/mL) at 37 °C, 250 rpm for 4 h, protein expression was induced by adding 1 mM isopropyl β-D-1thiogalactopyranoside (IPTG) and incubated at 20 °C, 250 rpm for 16 h. Harvested cells by centrifugation at 10,000 rpm, 10 min at 4 °C were resuspended in lysis buffer (10 mM Tris-HCl pH 7.5, 500 mM NaCl, 1 mM PMSF, 2% Triton-X 100) and lysed using a probe sonicator (QsonicaQ125) for 5 min (30 s on 30 s off, amplitude 90%). Soluble proteins were recovered by centrifugation at 10,000 rpm, 10 min at 4 °C, and purified using metal affinity chromatography (GE Healthcare) by step elution (10,25,50, and 100% of Elution buffer) from 0 to 500 mM imidazole in 10 mM Tris-HCl buffer pH 7.4, 500 mM NaCl. After purification, the engineered proteins were desalted to 10 mM N-(2-hydroxyethyl)piperazine-N′-ethanesulfonic acid (HEPES) pH 7.4 using a G25 Sephadex desalting column (GE Healthcare).…”

Various Simulated Body Fluids Lead to Significant Differences in Collagen Tissue Engineering Scaffolds

“…The proteins in the blood plasma, as well as the amino group in the TRIS contained in the SBF, were capable of spectrally interfering with the amide I band. Human serum albumin, the most abundant protein in plasma, embodies predominantly an α-helical structure and, upon interaction with neutral salts, may lead to the formation of intermolecular β-sheets [ 34 ]. Both these secondary structures are able to influence the deconvoluted band areas in amide I.…”

From Enzyme Stability to Enzymatic Bioelectrode Stabilization Processes

“…While they can be considered as eco-friendly solvents, many proteins are inherently inactive in ILs, requiring the addition of water for activity recovery. This suggests ILs could affect the internal water shell [61,62].…”

“…Changes in the secondary structure of two proteins with helical and beta structural arrangement, respectively, were followed by Fourier Transform Infra-Red spectroscopy (FTIR) in the presence of various salts. It was shown that the stabilization effect of the salt follows the Hofmeister series of ions, although some exceptions were observed with formation of intermolecular β-sheets typical of amorphous aggregates [62]. Glucose oxidase (GOx) stability was studied using microcalorimetry in the presence of various salts [63].…”