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case1225734704.pdf (3.15 MB)
ETD Abstract Container
Abstract Header
Characterization of Protein Modification by Products of Lipid Peroxidation
Author Info
Zhu, Xiaochun
Permalink:
http://rave.ohiolink.edu/etdc/view?acc_num=case1225734704
Abstract Details
Year and Degree
2009, Doctor of Philosophy, Case Western Reserve University, Chemistry.
Abstract
In this dissertation, we show that the apparent 4-oxo-2-nonenal (ONE)-Lys Michael adducts (
m
+154) actually represent the isomeric 4-ketoamides. The same type of 4-ketoamide on Lys residues was formed by 9,12-dioxo-10(E)-dodecenoic acid (KODA). By incubation of
β
-lactoglobulin (
β
-LG) with 4-hydroxy-2-nonenal (HNE) and ONE, HNE was found to form Cys/His/Lys/
N
-terminal amine Michael and Lys/
N
-terminal amine Schiff base adducts by mass spectrometry. ONE–Lys 4-ketoamide, ONE–Lys pyrrolinone and Cys/His–ONE–Lys/
N
-terminal amine pyrrole cross-links were detected after both 10 min and 24 h incubations. ONE–Lys Schiff base and ONE–His/Lys/Cys Michael adducts were observed at 10 min. When the ONE concentration is less than stoichiometric with
β
-LG, the predominant protein modification detected is the Cys–ONE–Lys pyrrole cross-link. The cross-linking between Glutathione (GSH) or
β
-alanylhistidine (carnosine) and proteins by ONE was investigated by SDS–PAGE and mass spectrometry. Under the reaction conditions we used, virtually every lysine of
β
-LG was found to be cross-linked to GSH to some degree. Using cytochrome c and ribonuclease A (RNase), we showed that ONE becomes more protein-reactive in the presence of GSH. His and Lys surrogates PHis and PLys were synthesized, both of which have detection limits of 30 fmol with a signal-to-noise ratio of 5:1 by MALDI-TOF-MS. By incubation of PHis or PLys with HNE, ONE, alkenals or a mixture of linoleic acid (
d
0
:d
5
= 1:1), Fe(II) and ascorbic acid, besides the previously reported known adducts, several novel adducts were detected by MALDI-TOF-MS and LC-ESI-MS. When the
β
-LG was incubated with LA or 17,17,18,18,18-
d
5
-(9Z,12Z)-octadeca-9,12-dienoic acid (d5-LA), Fe(II), and ascorbic acid, a novel 2-octenoic acid–His Michael adduct was discovered by LC-ESI-MS. In addition, many of the adducts found represent those previously observed when treating protein with the pure electrophilic modifier. The same modifications by the “mirror-image” carboxy terminus of LA were also identified. 2(E), 4(E)-Decadienal (DDE) was found to modify different proteins through different ways.
β
-LG is intensively cross-linked by DDE but cytochrome c and RNase are resistant to the DDE cross-linking. DDE majorly form Lys Schiff base adducts with cytochrome c and RNase. However, in addition to the these adducts, DDE forms Cys Michael and Lys pyridinium adducts with
β
-LG.
Committee
Lawrence M. Sayre (Advisor)
Vernon E. Anderson (Advisor)
Michael G. Zagorski (Committee Chair)
Mary D. Barkley (Committee Member)
Gregory P. Tochtrop (Committee Member)
Pages
317 p.
Subject Headings
Chemistry
Keywords
Lipid Peroxidation
;
4-hydroxy-2-nonenal
;
4-oxo-2-nonenal
;
Linoleic acid
;
2
;
4-decadienal
;
Glutathione
;
Carnosine
;
Mass Spectrometry
;
LC-ESI-MS
;
MALDI-TOF-MS
;
HPLC
Recommended Citations
Refworks
EndNote
RIS
Mendeley
Citations
Zhu, X. (2009).
Characterization of Protein Modification by Products of Lipid Peroxidation
[Doctoral dissertation, Case Western Reserve University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=case1225734704
APA Style (7th edition)
Zhu, Xiaochun.
Characterization of Protein Modification by Products of Lipid Peroxidation.
2009. Case Western Reserve University, Doctoral dissertation.
OhioLINK Electronic Theses and Dissertations Center
, http://rave.ohiolink.edu/etdc/view?acc_num=case1225734704.
MLA Style (8th edition)
Zhu, Xiaochun. "Characterization of Protein Modification by Products of Lipid Peroxidation." Doctoral dissertation, Case Western Reserve University, 2009. http://rave.ohiolink.edu/etdc/view?acc_num=case1225734704
Chicago Manual of Style (17th edition)
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Document number:
case1225734704
Download Count:
1,359
Copyright Info
© 2008, all rights reserved.
This open access ETD is published by Case Western Reserve University School of Graduate Studies and OhioLINK.