热诱导卵白蛋白-白藜芦醇共价复合物的结构、功能及潜在致敏性

Structure, function and potential allergenicity of heat stressed ovalbumin-resveratrol covalent conjugate

中文关键词:  热诱导卵白蛋白  白藜芦醇  共价复合物  结合位点  功能性质  潜在致敏性

英文关键词:heat stressed ovalbumin  resveratrol  covalent conjugate  binding site  functional property  potential allergenicity

中文摘要:

      目的 研究热诱导卵白蛋白(Heat stressed ovalbumin, HOVA)-白藜芦醇(Resveratrol, RES)共价复合物的结构、功能及潜在致敏性。方法 首先将卵白蛋白(Ovalbumin, OVA)在323 K条件下加热15 min,然后在碱性条件下与RES反应制备HOVA-RES共价复合物,利用十二烷基硫酸钠聚丙烯酰胺凝胶电泳测定HOVA-RES共价复合物的分子量,通过液相色谱-串联质谱法鉴定RES的结合位点,采用内源性荧光光谱、圆二色光谱、紫外吸收光谱解析RES引起的蛋白质构象变化,最后检测HOVA-RES共价复合物的抗氧化性、乳化性、热稳定性及潜在致敏性。结果 碱性条件下HOVA与RES可以共价形式交联,结合位点为Lys187和Lys264。低比例RES复合时,HOVA螺旋结构增加,无规则卷曲减少,而高比例RES复合时,蛋白结构逐渐往无序方向发展。RES的共价修饰会影响蛋白质的功能特性,其中2,2-联苯基-1-苦基肼基（2,2-diphenyl-1-picrylhydrazyl, DPPH）和2,2-联氮-二（3-乙基-苯并噻唑-6-磺酸）二铵盐（2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid), ABTS）清除率提升了约2倍,乳化活性由45.14 m2/g增加到107.88 m2/g,变性峰值温度上升了9.66 ℃。此外,随着RES复合比例的增加,HOVA-RES共价复合物的免疫球蛋白E（immunoglobulin E, IgE）结合能力呈现先下降后上升再下降的趋势，适量RES的共价复合能够显著降低OVA的潜在致敏性。结论 HOVA-RES共价复合可以改变OVA的构象,改善其功能特性,并影响其潜在致敏性,这为OVA的开发利用提供了理论依据。

英文摘要:

      Objective To study the structure, function and potential allergenicity of the covalent conjugate of heat stressed ovalbumin (HOVA) and resveratrol (RES). Methods Ovalbumin (OVA) was heated at 323 K for 15 min and then conjugated with RES to obtain the HOVA-RES covalent conjugate under alkaline condition. The molecular weight of HOVA-RES covalent conjugate was measured using sodium dodecyl sulfate polyacrylamide gel electrophoresis, the binding sites of RES were identified by liquid chromatography-tandem mass spectrometry, then the conformational variation induced by RES were analyzed by endogenous fluorescence spectra, CD spectra and UV absorption spectra. Finally, the antioxidant activity, emulsifying activity, thermal stability and potential allergenicity of HOVA-RES covalent conjugate were tested. Results Under alkaline condition, HOVA and RES could be covalently conjugated, the binding sites were Lys187 and Lys264. When a low proportion of RES was combined, the content of helix structure of HOVA was rose, the random coil content was lessened. While when a high proportion of RES was combined, the protein structure gradually developed in a disordered direction. The covalent modification of RES could affect the functional properties of protein, which displayed the clearance rate of 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) were increased by about 2 times, the emulsifying activity was rose from 45.14 m2/g to 107.88 m2/g, and the denaturation temperature was declined by 9.66 ℃. In addition, with the increase of RES proportion, the immunoglobulin E (IgE) binding ability of HOVA-RES covalent conjugate showed a trend of first declining, then rising and then declining. Proper covalent modification of RES could significantly reduce the potential allergenicity of OVA. Conclusion HOVA-RES covalent conjugating can alter the conformation of OVA, improve its functional properties, and affecte its potential allergenicity, which provides a theoretical reference for the development and utilization of OVA.