大豆巯基肽的共价色谱法制备及其MALDI-TOF-MS分析  

Preparation of soy thiol-containing peptides by covalent chromatography method and its MALDI-TOF-MS analysis

作　　者：丁秀臻[1] 孔祥珍[1] 陈业明[1] 张彩猛[1] 华欲飞[1] 

机构地区：[1]江南大学食品学院,食品科学与工程国家重点实验室,江苏无锡214122

出　　处：《中国油脂》2016年第12期31-36,共6页China Oils and Fats

基　　金：国家自然科学基金(31201380);江苏省自然科学基金(BK2011151)

摘　　要：建立了一种从复杂的大豆球蛋白酶解物中提取巯基肽的高选择性方法。首先,对大豆球蛋白酶解物中的二硫键用1,4-二硫苏糖醇进行还原,对还原剂的用量进行了考察和优化,提高了酶解物中的巯基肽含量。然后用Thiopropyl-Sephrose 6B共价色谱制备还原酶解物中的巯基肽,对共价色谱的吸附、解吸条件进行了考察和优化。研究表明:二硫键还原最佳1,4-二硫苏糖醇用量为20 mmol/L,还原之后酶解物中的巯基含量从1.8μmol/g提高到了113.8μmol/g。共价色谱的最佳条件为还原酶解物的上样量为酶解物中巯基含量占凝胶活性位点的80%,键合时间30 min;解吸时1,4-二硫苏糖醇浓度20 mmol/L,解吸时间20 min。此外,C18柱可以有效地将巯基肽中的1,4-二硫苏糖醇和2-巯基吡啶去除。MALDI-TOF-MS分析表明制备的巯基肽中可检测到45个肽段,其中36个肽段含有巯基,表明Thiolpropyl-Sepharose 6B共价色谱对巯基肽有很高的选择性。A highly selective method to extract thiol - containing peptides from complicated soy glycinin hydrolysates was established. Firstly, the disulfide bonds in soy glycinin hydrolysates were reduced by 1, 4 - dithiothreitol. The dosage of reduction agent was optimized and investigated and the content of thiol - containing peptides in the hydrolysates was improved. Then, the thiol - containing peptides in reduced hydrolysates were prepared by Thiopropyl - Sephrose 6B covalent chromatography, and the adsorption and desorption conditions of the covalent chromatography were optimized and investigated. The results showed that the optimal dosage of 1,4 - dithiothreitol for disulfide bond reduction was 20 mmol/L. After reduction,the content of sulfhydryl groups in hydrolysates increased from 1.8 is,mol/g to 113.8 μmol/g. The optimal covalent chromatography conditions were obtained as follows：the content of sulfhydryl groups 80% of the gel active sites as the optimal loading amount of the thiol - containing peptides, adsorption time 30 min, 1,4 - dithiothreitol concentration for desorption 20 mmol/L, desorption time 20 min. In addition, 1,4 -dithiothreitol and 2 -mereaptopyri- dine were removed from thiol -containing peptides successfully by C18 column. MALDI - TOF - MS showed that 45 peptide fragments were detected in thiol - containing peptides and 36 peptide fragments of them contained sulfhydryl groups, which indicated that Thiolpropyl - Sepharose 6B covalent chromatography showed high selectivity toward thiol -containing peptides.

关 键 词：大豆球蛋白酶解物 巯基肽 还原 Thiolpropyl—Sepharose 6B MALDI—TOF—MS 

分 类 号：O657.63[理学—分析化学] TS201.2[理学—化学]