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The effect of fatty acid binding in the acid isomerizations of albumin investigated with a continuous acidification method

Del Giudice, Alessandra; Galantini, Luciano; Dicko, Cedric LU and Pavel, Nicolae V. (2018) In Colloids and Surfaces B: Biointerfaces 168. p.109-116
Abstract

The protein Human Serum Albumin (HSA) is known to undergo conformational transitions towards partially unfolded forms triggered by acidification below pH 4.5. The extent of Fatty Acids (FA) binding has been thought to have an impact on the conformational equilibrium between the native and acid forms and to be a possible explanation for the observation of more than one band in early electrophoretic migration experiments at pH 4. We compared the acid-induced unfolding processes of commercial FA-free HSA, commercial “fatted” HSA and FA-HSA complexes, prepared at FA:HSA molar ratios between 1 and 6 by simple mixing and equilibration. We used a method for continuous acidification based on the hydrolysis of glucono-δ-lactone from pH 7 to pH... (More)

The protein Human Serum Albumin (HSA) is known to undergo conformational transitions towards partially unfolded forms triggered by acidification below pH 4.5. The extent of Fatty Acids (FA) binding has been thought to have an impact on the conformational equilibrium between the native and acid forms and to be a possible explanation for the observation of more than one band in early electrophoretic migration experiments at pH 4. We compared the acid-induced unfolding processes of commercial FA-free HSA, commercial “fatted” HSA and FA-HSA complexes, prepared at FA:HSA molar ratios between 1 and 6 by simple mixing and equilibration. We used a method for continuous acidification based on the hydrolysis of glucono-δ-lactone from pH 7 to pH 2.5, and followed the average protein changes by the blue shift of the intrinsic fluorescence emission and by performing a small angle X-ray scattering analysis on selected samples. The method also allowed for continuous monitoring of the increase of turbidity and laser light scattering of the protein samples related to the release of the insoluble ligands with acidification. Our results showed that the presence of FA interacting with albumin, an aspect often neglected in biophysical studies, affects the conformational response of the protein to acidification, and slightly shifts the loss of the native shape from pH 4.2 to pH 3.6. This effect increased with the FA:HSA molar ratio so that with three molar equivalents a saturation was reached, in agreement with the number of high-affinity binding sites reported for the FA. These findings confirm that a non-uniform level of ligand binding in an albumin sample can be an explanation for the early-observed conformational heterogeneity at pH 4.

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author
organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
Acid unfolding, Fatty acid binding, Fluorescence, Human serum albumin, SAXS
in
Colloids and Surfaces B: Biointerfaces
volume
168
pages
109 - 116
publisher
Elsevier
external identifiers
  • scopus:85044537268
ISSN
0927-7765
DOI
10.1016/j.colsurfb.2018.03.038
language
English
LU publication?
yes
id
d7de1414-d575-44af-84d3-b6ccb1c2bc22
date added to LUP
2018-04-12 14:26:20
date last changed
2019-01-14 13:32:25
@article{d7de1414-d575-44af-84d3-b6ccb1c2bc22,
  abstract     = {<p>The protein Human Serum Albumin (HSA) is known to undergo conformational transitions towards partially unfolded forms triggered by acidification below pH 4.5. The extent of Fatty Acids (FA) binding has been thought to have an impact on the conformational equilibrium between the native and acid forms and to be a possible explanation for the observation of more than one band in early electrophoretic migration experiments at pH 4. We compared the acid-induced unfolding processes of commercial FA-free HSA, commercial “fatted” HSA and FA-HSA complexes, prepared at FA:HSA molar ratios between 1 and 6 by simple mixing and equilibration. We used a method for continuous acidification based on the hydrolysis of glucono-δ-lactone from pH 7 to pH 2.5, and followed the average protein changes by the blue shift of the intrinsic fluorescence emission and by performing a small angle X-ray scattering analysis on selected samples. The method also allowed for continuous monitoring of the increase of turbidity and laser light scattering of the protein samples related to the release of the insoluble ligands with acidification. Our results showed that the presence of FA interacting with albumin, an aspect often neglected in biophysical studies, affects the conformational response of the protein to acidification, and slightly shifts the loss of the native shape from pH 4.2 to pH 3.6. This effect increased with the FA:HSA molar ratio so that with three molar equivalents a saturation was reached, in agreement with the number of high-affinity binding sites reported for the FA. These findings confirm that a non-uniform level of ligand binding in an albumin sample can be an explanation for the early-observed conformational heterogeneity at pH 4.</p>},
  author       = {Del Giudice, Alessandra and Galantini, Luciano and Dicko, Cedric and Pavel, Nicolae V.},
  issn         = {0927-7765},
  keyword      = {Acid unfolding,Fatty acid binding,Fluorescence,Human serum albumin,SAXS},
  language     = {eng},
  month        = {01},
  pages        = {109--116},
  publisher    = {Elsevier},
  series       = {Colloids and Surfaces B: Biointerfaces},
  title        = {The effect of fatty acid binding in the acid isomerizations of albumin investigated with a continuous acidification method},
  url          = {http://dx.doi.org/10.1016/j.colsurfb.2018.03.038},
  volume       = {168},
  year         = {2018},
}