Induction of redox instability of bovine myoglobin by adduction with 4-hydroxy-2-nonenal.
The redox stability of myoglobin (Mb) is compromised by many factors, including lipid oxidation and its products. 4-Hydroxy-2-nonenal (HNE) is an alpha,beta-unsaturated aldehyde derived from the oxidation of omega-6 polyunsaturated fatty acids and is highly reactive and cytotoxic. Our objective was to study potential binding of HNE to Mb and determine how it affects redox stability. OxyMb (0.15 mM) was incubated with HNE (1 mM) at 4, 25, and 37 degrees C at pH 7.4 or 5.6. Samples were analyzed for MetMb formation and by Western blot analyses, LC-MS, LC-MS-MS, circular dichroism (CD), and differential scanning calorimetry (DSC). MetMb formation increased with increasing temperature and was greater at pH 5.6 than at pH 7.4 (P < 0.05). At 37 degrees C, HNE accelerated oxidation at pH 7.4 but not at pH 5.6 (P < 0.05). At both 25 and 4 degrees C, HNE accelerated oxidation at pH 7.4 and 5.6 (P < 0.05). LC-MS revealed the covalent binding of HNE to Mb at both pH values via Michael addition, while Western blot analysis indicated that HNE was bound to histidine (HIS) residues. LC-MS-MS identified six histidine residues of Mb that were readily adducted by HNE, including the proximal (HIS 93) and distal (HIS 64) histidine associated with the heme group. Secondary structure differences between control Mb and Mb incubated with HNE were not detected by CD. However, DSC revealed a decreased T(m) for Mb reacted with HNE at pH 7.4, indicating Mb tertiary structure was altered in a manner consistent with destabilization. These results suggest that HNE accelerates bovine skeletal muscle OxyMb oxidation in vitro by covalent modification at histidine residues.
Alderton
A L
AL
Department of Animal Sciences, University of Kentucky, Lexington, Kentucky 40546-0215, USA.
Faustman
C
C
Liebler
D C
DC
Hill
D W
DW
D000447:Aldehydes / Q000737:chemistry*; D000818:Animals; D002152:Calorimetry, Differential Scanning; D002417:Cattle; D002853:Chromatography, Liquid; D013058:Mass Spectrometry; D009211:Myoglobin / Q000737:chemistry*; D010084:Oxidation-Reduction; D017434:Protein Structure, Tertiary; D013696:Temperature
D000447:Aldehydes; D009211:Myoglobin; C027576:4-hydroxy-2-nonenal