Abstract
BackgroundMyoglobin (Mb) is a sarcoplasmic heme protein primarily responsible for meat color and its chemistry is species specific. 4-hydroxy-2-nonenal (HNE) is a cytotoxic lipid derived aldehyde detected in meat and was reported to covalently adduct with nucleophilic histidine residues of Mb and predispose it to greater oxidation. However, no literature is available on characterization of lipid oxidation induced oxidation of Indian water buffalo (Bubalus bubalis) and goat (Capra hircus) myoglobins.MethodsPresent study characterize the Mb extracted from water buffalo and goat cardiac muscles using two-dimensional gel electrophoresis (2DE), OFFGEL electrophoresis and mass spectrometry (MS). Purified buffalo and goat bright red oxymyoglobin were reacted with HNE in-vitro at physiological pH (7.4) and temperature (37 °C) conditions and the formation of oxidised brown metmyoglobin was measured. The Mb-HNE adducts were detected using MALDI-TOF MS, whereas specific sites of adduction was determined using ESI-QTOF MS/MS.ResultsPurified buffalo and goat Mb samples revealed a molecular mass of 17,043.6 and 16,899.9 Daltons, respectively. The 2DE analysis exhibited 65 (sarcoplasmic protein extract) and 6 (pure Mb) differentially expressed (P < 0.05) protein spots between buffalo and goat samples. OFFGEL electrophoresis revealed an isoelectric point of 6.77 and 7.35 respectively, for buffalo and goat Mb’s. In-vitro incubation of HNE with bright red buffalo and goat oxymyoglobin’s at pH 7.4 and 37 °C resulted in pronounced (P < 0.05) oxidation and formation of brown metmyoglobin. MALDI-TOF MS analysis of Mb-HNE reaction mix revealed covalent binding (via Michael addition) of 3 and 5 molecules of HNE with buffalo and goat Oxy-Mb’s, respectively. ESI-QTOF MS/MS identified seven and nine histidine (HIS) residues of Mb that were readily adducted by HNE in buffalo and goat, respectively.ConclusionThe study demonstrated better redox stability of buffalo Mb than goat Mb. Our findings confirm the hypothesis that relative effect of HNE was greater for Mb’s with 12 ± 1 HIS residues than Mb’s with 9 HIS residues and helps meat processors in developing species-specific processing strategies to reduce the color variability.
Highlights
Myoglobin (Mb) is a sarcoplasmic heme protein primarily responsible for meat color and its chemistry is species specific. 4-hydroxy-2-nonenal (HNE) is a cytotoxic lipid derived aldehyde detected in meat and was reported to covalently adduct with nucleophilic histidine residues of Mb and predispose it to greater oxidation
Primary structure of goat Mb was determined by Suman et al (2009) who reported that goat Mb shared 98.7% similarity with sheep Mb and the distal (64) and proximal (93) histidines responsible for coordinating the heme group and reversible binding of oxygen are conserved in goat Mb, similar to other meat-producing livestock [7]
The extraction and purification of Mb from buffalo and goat is minimally investigated and to our knowledge only two papers are available in the literature for buffalo and goat Mb extraction and characterization [5, 7]
Summary
Myoglobin (Mb) is a sarcoplasmic heme protein primarily responsible for meat color and its chemistry is species specific. 4-hydroxy-2-nonenal (HNE) is a cytotoxic lipid derived aldehyde detected in meat and was reported to covalently adduct with nucleophilic histidine residues of Mb and predispose it to greater oxidation. Dosi et al (2006) have studied the primary structure of buffalo Mb using a combined approach of Edman degradation and MALD-TOF mass spectrometry and found a difference of three amino acids out of 153 compared to beef Mb [5]. These authors have studied the stability, autoxidation and percent metmyoglobin formation in beef and buffalo Mb’s and reported identical results between them. Another important livestock species, goat was reported to share 98.7% sequence similarity with sheep than with buffalo which has got 95.4% sequence similarity. Primary structure of goat Mb was determined by Suman et al (2009) who reported that goat Mb shared 98.7% similarity with sheep Mb and the distal (64) and proximal (93) histidines responsible for coordinating the heme group and reversible binding of oxygen are conserved in goat Mb, similar to other meat-producing livestock [7]
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