Salts employed in hydrophobic interaction chromatography can change protein structure – insights from protein‐ligand interaction thermodynamics, circular dichroism spectroscopy and small angle X‐ray scattering

Abstract

Key requirements of protein purification by hydrophobic interaction chromatography (HIC) are preservation of the tertiary/quaternary structure, maintenance of biological function, and separation of the correctly folded protein from its unfolded forms or aggregates. This study examines the relationship between the HIC retention behavior of hen egg white lysozyme (HEWL) in high concentrations of several kosmotropic salts and its conformation, assessed by circular dichroism (CD) spectroscopy. Further, the physicochemical properties of HEWL in the presence of high concentrations of ammonium sulfate, sodium chloride and magnesium chloride were investigated by small angle X-ray scattering (SAXS) at different temperatures. Radii of gyration were extrapolated from Guinier approximations and the indirect transform program GNOM with protein-protein interaction and contrast variation taken into account. A bead model simulation provided information on protein structural changes using ab initio reconstruction with GASBOR. These results correlated to the secondary structure content obtained from CD spectroscopy of HEWL. These changes in SAXS and CD data were consistent with heat capacity ΔCp -values obtained from van't Hoff plot analyses of the retention data. Collectively, these insights enable informed decisions to be made on the choice of chromatographic conditions, leading to improved separation selectivity and opportunities for innovative column-assisted protein refolding methods.

On-line determination by small angle X-ray scattering of the shape of hen egg white lysozyme immediately following elution from a hydrophobic interaction chromatography column

“…14.0 ± 0.2 15.4 ± 0.6 I(0) (cm −1 ) 0.0054 0.0054 Rear R g (Å) 14.3 ± 0.8 14.8 ± 0.5 I(0) (cm −1 ) 0.0045 0.0045 In contrast, in the absence of the hydrophobic surface of the HIC resin, a different process is expected to occur during the thermal expansion of HEWL in bulk solution under the ex situ conditions with the β-domain becoming less stabilised during thermal unfolding. 18 Moreover, the higher R g value calculated from the ex situ measurements at the lower temperature than that determined by the in situ HIC−SAXS method (Table 1) is concordant with other reported observations 16 and is partly due to the effect of electron density contrast variation between the solution and the protein which arises under the ex situ condition.…”

“…Comparison of the R g values obtained at different temperatures was performed for protein HIC-SAXS data obtained with the same I(0) in order to avoid the effect of contrast variation (due to the dependency of the R g value on the I(0)). 16 The reliability of this observation was indicated by using the Auto R g command in PRIMUS with 95 and 81% quality (obtained at the middle of the peaks) at 15 and 35 °C, respectively. Additional information for the HEWL expansion was achieved with the construction of pair distribution functions from the SAXS signals by using the GNOM software which showed the additional probability to find HEWL residues more distant from the scattering centre (elongation) at the higher temperature, as evident from the P(R) versus R plot in Fig.…”

“…I(0) middle /I(0) rear > I(0) front , this can lead to more favourable conformational re-equilibration of the HEWL species present in the front of the peak, resulting in a lower R g value for HEWL. Table 1 Retention factor k, specific heat capacity (ΔC p ), radius of gyration (R g ) and forward scattering (I(0)) values, calculated using PRIMUS, 16 obtained from the ex situ SAXS and in situ HIC−SAXS experiments at the temperatures (T ) of 15…”

“…[12][13][14][15] In a previous study, we described the effect of different kosmotropic salts on the structural behaviour of HEWL in free solutions using SAXS and CD spectroscopic measurements. 16 However, with some salts, for example, 0.5 M (NH 4 ) 2 SO 4 , the ex situ SAXS results for HEWL conformations in free solution at 15 or 35 °C were inconclusive due to interference to the SAXS signals caused by electron density contrast variation between the protein and the solution. As described in this current report, this challenge has been overcome, permitting more accurate correlation of the protein structure to the HIC behaviour to be obtained, through the development of an in-line system that enables HIC to be directly coupled to an in situ SAXS system (HIC-SAXS), whereby SAXS data can be immediately obtained at several time point intervals as the protein elutes from the HIC column.…”

Analisis Termodinamika Molekul Magnesium Sulphate (MgSO4)

“…Garam mandi (61)(62)(63)(64) Kapasitas Kalor (72)(73)(74)(75) Sintesis organik (16; 76-78) Pengering (34)(35)(36)(37) Pertanian (38)(39)(40) Farmasi (18)(19)(20)(21) Elektrolit pada sintesis (85)(86)(87) Koagulan (31)(32)(33) Mobilitas Ion (65)(66)(67) Bentuk dan struktur (11; 46; 80; 81) MgSO4 umumnya dapat dijumpai dalam garam epsomite yakni MgSO4.7H2O. Di alam senyawa ini terdapat dalam bentuk mineral sulfat.…”