高效液相色谱分离纯化血管紧张素转换酶活性抑制肽

Isolation and Purification of Angiotensin Converting Enzyme Inhibitory Peptides by High Performance Liquid Chromatography

采用分步高效液相色谱法首次从菠菜核酮糖双磷酸羧化酶(英文缩写为Rubisco)的胃蛋白酶 胰酶复合酶水解产物中分离纯化了血管紧张素转换酶(ACE)活性抑制肽。水解产物经ODS柱分离得到活性组分,这些活性组分再依次经过氨基柱(PhA)、氰基柱(CN)和硝基苯乙基柱(NPE)4种色谱柱分离后,得到4种高度纯化的、具有抑制ACE活性的多肽。经蛋白质序列自动分析仪测定,4种多肽的结构分别为MRWRD,MRW,IAYKPAG和LRIP VA。采用固相多肽合成法分别合成了这4种肽,并采用测定ACE活性抑制率的方法评价其活性大小,结果其抑制ACE活性50%的浓度值(IC50值)分别为2 1,0 6,4 2和0 38μmol/L。通过对照菠菜Rubisco的氨基酸序列,确认IAYKPAG来自于菠菜Rubisco的小亚基,而其余3种多肽则来自于菠菜Rubisco的大亚基。该法具有分离效率高、快速简单、干扰小、重现性好和不易丢失水解蛋白质中活性成分的优点。

Angiotensin Iconverting enzyme (ACE) plays a key physiological role in the regulation of blood pressure. Recently, many studies have focused on various ACEinhibitory peptides derived from casein, fish muscle, and gelatin. It is well known that ribulose bisphosphate carboxylase/oxygenase (Rubisco), which catalyzes the primary step in photosynthetic CO2 fixation, is the most abundant protein on the earth. In this study, inhibitory peptides for ACE were isolated from the pepsinpancreatin digest of spinach Rubisco by high performance liquid chromatography (HPLC). Separated with four different HPLC columns of ODS, PhA, CN and NPE in a successive order, four peptides were obtained. The amino acid sequences of these purified peptides determined by a protein sequencer were MRWRD, MRW, IAYKPAG and LRIPVA, respectively. These peptides were synthesized by a solid peptide synthesizer and their ACE inhibitory activities were determined. The concentrations of the individual peptide to inhibit 50% of ACE activity (IC50) were 21, 06, 42 and 038 μmol/L, respectively. Among the four peptides, only IAYKPAG is derived from the small subunit of spinach Rubisco, and the other three peptides (LRIPVA, MRW and MRWRD) are derived from the large subunit of spinach Rubisco. This method is considered to be rapid and easy to implement, with little matrix interference and good reproducibility and mass recovery from the column. 