猪皮胶原ACE抑制肽的纯化与鉴定

Purification and Identification of ACE Inhibitory Peptides from Porcine Skin Collagen

采用超声协同稀碱对猪皮实施预处理后,酶解30 min制得的高血管紧张素转化酶(Angiotensin converting enzyme,ACE)抑制活性水解液为原料,采用Sephadex G-15、半制备高效液相色谱分离纯化其中胶原ACE抑制肽并对其序列进行鉴定。结果表明,Sephadex G-15分离胶原ACE抑制肽的最佳分离条件为:样品浓度100 mg/m L;上样量2 m L;流速2 m L/min;洗脱剂为蒸馏水;层析柱为1 m×10 mm的层析柱。在该分离条件下,混合肽被分成AP-I、AP-II两个组分,IC50值分别为19.50、1.01 mg/m L。对抑制活性较强的AP-II进一步采用半制备高效液相色谱进行分离,得到8个组分峰,其中峰2、峰5和峰6的IC50值最小,分别为0.73、0.44和0.4 mg/m L。结合IC50值的大小及半制备收集到样品的量,对峰2和峰6采用LC-MS/MS进行序列分析。结果显示峰2的多肽序列为QGPPGPAGPR(P为Hyp),峰6的序列为AGPPGPPGPA,这两个序列位于胶原蛋白α1链中526?535、730?739位。

After being pretreated by ultrasound combined with dilute alkali,porcine skin underwent enzymatic hydrolysis for 30 min to produce hydrolysate with ACE inhibitory activity.This hydrolysate was used as raw material,and the collagen ACE inhibitory peptides were separated and purified using Sephadex G-15 semi-preparative high performance liquid chromatography.The results showed that the optimum separation conditions were as follows：sample concentration of 100 mg/m L;injection volume of 2 m L;flow rate of 2 m L/min;distilled water as the eluent;and a 1 m × 10 mm chromatography column.Under these separation conditions,the mixed peptides were divided into two fractions,AP-I and AP-II.The half maximal inhibitory concentration（IC50） values were 19.50 mg/m L and 1.01 mg/m L,for AP-I and AP-II,respectively.Fraction AP-II,with relatively strong ACE inhibitory activity,was further separated by semi-preparative high performance liquid chromatography,and eight peaks were obtained.The IC50 values of peaks two,five,and six were the lowest（0.44 mg/m L,0.4 mg/m L,and 0.73 mg/m L,respectively）.Based on the IC50 value and the weight of the samples collected from semi-preparative high performance liquid chromatographic separation,liquid chromatography tandem mass spectrometry sequence analysis was conducted for peaks two and six.The results showed that the peptide sequences of peaks two and six were QGPPGPAGPR and AGPPGPPGPA,respectively.Additionally,these sequences occupied amino acid positions 526-535 and 730-739 of the collagen α1 chain,respectively.