BACKGROUND 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. However, no literature is available on characterization of lipid oxidation induced oxidation of Indian water buffalo (Bubalus bubalis) and goat (Capra hircus) myoglobins. METHODS Present 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. RESULTS Purified 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. CONCLUSION The 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.

中文翻译：

---

基于蛋白质组学的方法，用于表征水牛 (Bubalus bubalis) 和山羊 (Capra hircus) 肉肌红蛋白的 4-羟基-2-壬烯醛诱导氧化。

背景技术肌红蛋白(Mb)是一种肌浆血红素蛋白，主要决定肉的颜色，其化学性质具有物种特异性。4-羟基-2-壬烯醛 (HNE) 是一种在肉中检测到的细胞毒性脂质衍生醛，据报道可与 Mb 的亲核组氨酸残基共价加合物，并使其易于发生更大的氧化。然而，还没有关于印度水牛（Bubalus bubalis）和山羊（Capra hircus）肌红蛋白的脂质氧化诱导氧化特征的文献。方法 本研究使用二维凝胶电泳 (2DE)、OFFGEL 电泳和质谱 (MS) 表征从水牛和山羊心肌中提取的 Mb。纯化的水牛和山羊鲜红色氧合肌红蛋白在生理 pH (7.4) 和温度 (37 °C) 条件下与 HNE 体外反应，并测量氧化棕色高铁肌红蛋白的形成。使用 MALDI-TOF MS 检测 Mb-HNE 加合物，而使用 ESI-QTOF MS/MS 确定加合物的具体位点。结果 纯化的水牛和山羊 Mb 样品的分子量分别为 17,043.6 和 16,899.9 道尔顿。2DE 分析显示水牛和山羊样品之间有 65 个（肌浆蛋白提取物）和 6 个（纯 Mb）差异表达（P < 0.05）蛋白点。OFFGEL 电泳显示水牛和山羊 Mb 的等电点分别为 6.77 和 7.35。HNE 与亮红色水牛和山羊氧合肌红蛋白在 pH 7.4 和 37 °C 下进行体外培养，导致显着（P < 0.05）氧化并形成棕色高铁肌红蛋白。Mb-HNE 反应混合物的 MALDI-TOF MS 分析显示，3 个和 5 个 HNE 分子分别与水牛和山羊 Oxy-Mb 共价结合（通过迈克尔加成）。ESI-QTOF MS/MS 分别鉴定出 Mb 的 7 个和 9 个组氨酸 (HIS) 残基，这些残基在水牛和山羊中分别容易被 HNE 加合。结论 研究表明水牛 Mb 的氧化还原稳定性优于山羊 Mb。我们的研究结果证实了这样的假设：HNE 对具有 12 ± 1 个 HIS 残留物的 Mb 的相对影响大于具有 9 个 HIS 残留物的 Mb，并有助于肉类加工商制定物种特异性加工策略以减少颜色变异。