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Effect of Heat Treatment on Bovine β Lactoglobulin A, B and C. Explored Using Thiol Availability and Fluorescence.

Manderson, Gavin LU ; Hardman, Michael J and Creamer, Lawrence K (1999) In Journal of Agricultural and Food Chemistry 47(9). p.3617-3627
Abstract
Dilute solutions of -lactoglobulin (-Lg) A, B, and C were heated at temperatures between about 40 and 94 C for 10 min, cooled, and analyzed using Trp fluorescence and extrinsic fluorescence spectra of the probe 1,8-anilinonaphthalene sulfonate (ANS). Thiol availabilities using 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) were determined using a separate set of samples. The normalized ANS fluorescence emission intensity and the thiol availability results showed a 1:1 relationship with the loss of nativelike but not SDS-monomeric protein, as determined by PAGE analysis. The normalized Trp emission intensity results did not show a comparable 1:1 relationship with the loss of nativelike protein, indicating that the Trp intensity arose from... (More)
Dilute solutions of -lactoglobulin (-Lg) A, B, and C were heated at temperatures between about 40 and 94 C for 10 min, cooled, and analyzed using Trp fluorescence and extrinsic fluorescence spectra of the probe 1,8-anilinonaphthalene sulfonate (ANS). Thiol availabilities using 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) were determined using a separate set of samples. The normalized ANS fluorescence emission intensity and the thiol availability results showed a 1:1 relationship with the loss of nativelike but not SDS-monomeric protein, as determined by PAGE analysis. The normalized Trp emission intensity results did not show a comparable 1:1 relationship with the loss of nativelike protein, indicating that the Trp intensity arose from consequential disulfide bond reorganization and not the initial unfolding reaction. The results were also analyzed in terms of two-state models, and the midpoint temperatures (Tmid) for the proteins were generally -Lg C > -Lg A > -Lg B, and the slopes at the midpoint temperatures for the A variant were generally less than those for the B and C variants indicating that -Lg A may denature by a different mechanism from that of -Lg B or -Lg C. The Tmid parameters derived from the ANS fluorescence intensity results were similar to those for thiol availability and both were lower than the Tmid values for Trp emission intensity showing that creation of an ANS binding site on a -Lg molecule was linked to the irreversible exposure of a thiol group and the loss of native -Lg but preceded the decrease in Trp61 fluorescence quenching. These results for the differences between the behavior of the A and B or the C variants involved the creation of a destabilizing cavity by the Val118Ala (A B) substitution and the changed charge distribution within the CD loop caused by the Asp64Gly (A B) substitution. (Less)
Please use this url to cite or link to this publication:
author
publishing date
type
Contribution to journal
publication status
published
subject
keywords
disulfide-linked aggregates, DTNB, aggregate formation, tryptophan fluorescence, Thermal denaturation, ANS fluorescence, -lactoglobulin variants
in
Journal of Agricultural and Food Chemistry
volume
47
issue
9
pages
3617 - 3627
publisher
The American Chemical Society
external identifiers
  • scopus:0032839589
ISSN
0021-8561
DOI
10.1021/jf990591g
language
English
LU publication?
no
id
c6f403e9-5e43-43a5-b487-b45101a3f04f (old id 1115668)
date added to LUP
2008-07-08 11:19:55
date last changed
2017-04-23 03:32:12
@article{c6f403e9-5e43-43a5-b487-b45101a3f04f,
  abstract     = {Dilute solutions of -lactoglobulin (-Lg) A, B, and C were heated at temperatures between about 40 and 94 C for 10 min, cooled, and analyzed using Trp fluorescence and extrinsic fluorescence spectra of the probe 1,8-anilinonaphthalene sulfonate (ANS). Thiol availabilities using 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) were determined using a separate set of samples. The normalized ANS fluorescence emission intensity and the thiol availability results showed a 1:1 relationship with the loss of nativelike but not SDS-monomeric protein, as determined by PAGE analysis. The normalized Trp emission intensity results did not show a comparable 1:1 relationship with the loss of nativelike protein, indicating that the Trp intensity arose from consequential disulfide bond reorganization and not the initial unfolding reaction. The results were also analyzed in terms of two-state models, and the midpoint temperatures (Tmid) for the proteins were generally -Lg C > -Lg A > -Lg B, and the slopes at the midpoint temperatures for the A variant were generally less than those for the B and C variants indicating that -Lg A may denature by a different mechanism from that of -Lg B or -Lg C. The Tmid parameters derived from the ANS fluorescence intensity results were similar to those for thiol availability and both were lower than the Tmid values for Trp emission intensity showing that creation of an ANS binding site on a -Lg molecule was linked to the irreversible exposure of a thiol group and the loss of native -Lg but preceded the decrease in Trp61 fluorescence quenching. These results for the differences between the behavior of the A and B or the C variants involved the creation of a destabilizing cavity by the Val118Ala (A B) substitution and the changed charge distribution within the CD loop caused by the Asp64Gly (A B) substitution.},
  author       = {Manderson, Gavin and Hardman, Michael J and Creamer, Lawrence K},
  issn         = {0021-8561},
  keyword      = {disulfide-linked aggregates,DTNB,aggregate formation,tryptophan fluorescence,Thermal denaturation,ANS fluorescence,-lactoglobulin variants},
  language     = {eng},
  number       = {9},
  pages        = {3617--3627},
  publisher    = {The American Chemical Society},
  series       = {Journal of Agricultural and Food Chemistry},
  title        = {Effect of Heat Treatment on Bovine β Lactoglobulin A, B and C. Explored Using Thiol Availability and Fluorescence.},
  url          = {http://dx.doi.org/10.1021/jf990591g},
  volume       = {47},
  year         = {1999},
}