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Denaturation of proteins by surfactants studied by the Taylor dispersion analysis.

ABSTRACT: We showed that the Taylor Dispersion Analysis (TDA) is a fast and easy to use method for the study of denaturation proteins. We applied TDA to study denaturation of β-lactoglobulin, transferrin, and human insulin by anionic surfactant sodium dodecyl sulfate (SDS). A series of measurements at constant protein concentration (for transferrin was 1.9 x 10-5 M, for β- lactoglobulin was 7.6 x 10-5 M, and for insulin was 1.2 x 10-4 M) and varying SDS concentrations were carried out in the phosphate-buffered saline (PBS). The structural changes were analyzed based on the diffusion coefficients of the complexes formed at various surfactant concentrations. The concentration of surfactant was varied in the range from 1.2 x 10-4 M to 8.7 x 10-2 M. We determined the minimum concentration of the surfactant necessary to change the native conformation of the proteins. The minimal concentration of SDS for β-lactoglobulin and transferrin was 4.3 x 10-4 M and for insulin 2.3 x 10-4 M. To evaluate the TDA as a novel method for studying denaturation of proteins we also applied other methods i.e. electronic circular dichroism (ECD) and dynamic light scattering (DLS) to study the same phenomenon. The results obtained using these methods were in agreement with the results from TDA.

SUBMITTER: Jelinska A

PROVIDER: S-EPMC5398553 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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Denaturation of proteins by surfactants studied by the Taylor dispersion analysis.

Jelińska Aldona A Zagożdżon Anna A Górecki Marcin M Wisniewska Agnieszka A Frelek Jadwiga J Holyst Robert R

PloS one 20170420 4

We showed that the Taylor Dispersion Analysis (TDA) is a fast and easy to use method for the study of denaturation proteins. We applied TDA to study denaturation of β-lactoglobulin, transferrin, and human insulin by anionic surfactant sodium dodecyl sulfate (SDS). A series of measurements at constant protein concentration (for transferrin was 1.9 x 10-5 M, for β- lactoglobulin was 7.6 x 10-5 M, and for insulin was 1.2 x 10-4 M) and varying SDS concentrations were carried out in the phosphate-buf ...[more]

PMID: 28426809

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Project description:Hydrodynamic radius (RH) is a descriptive metric of protein structure with the potential to impact drug development, disease diagnosis, and other important research areas of molecular biology. Common instrumental methods for molecular size characterization are disadvantageous due to high sample consumption, measurements made in non-physiological conditions, and/or inaccurate size determinations. Capillary Taylor dispersion analysis (TDA) is a molecular sizing method that utilizes nL sample volumes and achieves absolute size determination without calibration or comparison to standards. One key drawback of TDA is that it reports the concentration-weighted average RH, which may be limiting in the analysis of complex sample mixtures. Here, we describe the development of a 3D printed instrument to integrate capillary electrophoresis (CE) separations online with TDA size characterization. Dual laser-induced fluorescence detectors were developed to enable two-channel detection using a single PMT and fluorescence filter set, achieving detection limits for AlexaFluor 532 of 0.6 ± 0.4 nM and 1.1 ± 0.2 nM for detectors 1 and 2, respectively. Joule heating during CE separations was observed to introduce bias in subsequent TDA measurements. The effects of Joule heating were mitigated by integrating a water circulating sheath flow on the portion of the capillary used for CE. The utility of CE-TDA in bioanalysis was demonstrated by standard-free peak identification in the ficin digestion of IgG1. CE-TDA was further applied to characterizing denaturation dynamics of the Group II heat resistant protein apolipoprotein A-1 (ApoA), in which RH was observed to increase from 2.3 ± 0.2 nm at 20 °C to 5.2 ± 0.5 nm while heated at at 90 °C, then returned to a quasi-native state with RH = 2.9 ± 0.5 nm after cooling to 20 °C. CE-TDA is a powerful analysis mode with potential to impact various domains of bioanalysis. The instrument developed in this work offers a low barrier to entry for researchers interested in adopting CE-TDA.

Project description:This article describes the use of capillary electrophoresis (CE) to examine the influence of different cations (C(+); C(+) = Na(+) and tetra-n-alkylammonium, NR(4)(+), where R = Me, Et, Pr, and Bu) on the rates of denaturation of bovine carbonic anhydrase II (BCA) in the presence of anionic surfactant dodecylsulfate (DS(-)). An analysis of the denaturation of BCA in solutions of Na(+)DS(-) and NR(4)(+)DS(-) (in Tris-Gly buffer) indicated that the rates of formation of complexes of denatured BCA with DS(-) (BCA(D)-DS(-)(n,sat)) are indistinguishable and independent of the cation below the critical micellar concentration (cmc) and independent of the total concentration of DS(-) above the cmc. At concentrations of C(+)DS(-) above the cmc, BCA denatured at rates that depended on the cation; the rates decreased by a factor >10(4) in the order of Na(+) ≈ NMe(4)(+) > NEt(4)(+) > NPr(4)(+) > NBu(4)(+), which is the same order as the values of the cmc (which decrease from 4.0 mM for Na(+)DS(-) to 0.9 mM for NBu(4)(+)DS(-) in Tris-Gly buffer). The relationship between the cmc values and the rates of formation of BCA(D)-DS(-)(n,sat()) suggested that the kinetics of denaturation of BCA involve the association of this protein with monomeric DS(-) rather than with micelles of (C(+)DS(-))(n). A less-detailed survey of seven other proteins (α-lactalbumin, β-lactoglobulin A, β-lactoglobulin B, carboxypeptidase B, creatine phosphokinase, myoglobin, and ubiquitin) showed that the difference between Na(+)DS(-) and NR(4)(+)DS(-) observed with BCA was not general. Instead, the influence of NR(4)(+) on the association of DS(-) with these proteins depended on the protein. The selection of the cation contributed to the properties (including the composition, electrophoretic mobility, and partitioning behavior in aqueous two-phase systems) of aggregates of denatured protein and DS(-). These results suggest that the variation in the behavior of NR(4)(+)DS(-) with changes in R may be exploited in methods used to analyze and separate mixtures of proteins.

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Denaturation of proteins by surfactants studied by the Taylor dispersion analysis.

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Denaturation of proteins by surfactants studied by the Taylor dispersion analysis.

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