Effect of Heat Treatment on Bovine β Lactoglobulin A, B and C. Explored Using Thiol Availability and Fluorescence.
(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:
https://lup.lub.lu.se/record/1115668
- author
- Manderson, Gavin LU ; Hardman, Michael J and Creamer, Lawrence K
- publishing date
- 1999
- 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 (ACS)
- external identifiers
-
- scopus:0032839589
- ISSN
- 0021-8561
- DOI
- 10.1021/jf990591g
- language
- English
- LU publication?
- no
- additional info
- The information about affiliations in this record was updated in December 2015. The record was previously connected to the following departments: Connective Tissue Biology (013230151)
- id
- c6f403e9-5e43-43a5-b487-b45101a3f04f (old id 1115668)
- date added to LUP
- 2016-04-01 11:56:07
- date last changed
- 2022-02-18 07:30:26
@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}},
keywords = {{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 (ACS)}},
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}},
doi = {{10.1021/jf990591g}},
volume = {{47}},
year = {{1999}},
}