三斑海马蛋白ACE抑制肽的制备及其二级结构的研究

Preparation and the Secondary Structure of Angiotensin I-Converting Enzyme(ACE)Inhibitory Peptide from the Seahorse Protein

以三斑海马为原料,经碱性蛋白酶酶解,获得富含血管紧张素转化酶(angiotensin I-converting enzyme,ACE)抑制肽的酶解液。酶解液经透析、Sephadex G-25凝胶过滤色谱和反相高效液相色谱得到进一步分离纯化。结果表明,透析产物的IC50值为(0.44±0.26)mg/mL,相比酶解液(0.81±0.12)mg/mL,其ACE抑制活性更强。透析产物经Sephadex G-25凝胶柱分离纯化后,得到3种组分,其中组分F2的ACE抑制活性最强,IC50值为(0.11±0.08)mg/mL。F2经反相高效液相色谱进一步分离后,得到6个具有ACE抑制活性的组分峰,其中组分F2-4的ACE抑制活性最强,IC50值为(0.005 7±0.000 9)mg/mL。经过3步分离纯化后,成功从三斑海马蛋白中分离得到一种活性较强ACE抑制肽:ProAla-Gly-Pro-Arg-Gly-Pro-Ala(PAGPRGPA),多肽分子量为721.39 Da。圆二色谱分析多肽二级结构表明其主要含无规则卷曲。因此,从三斑海马蛋白中分离得到的多肽可能成为营养保健品和抗高血压药品及相关疾病的一种有益成分,且对海马蛋白资源的开发利用具有重要意义。

In this study,three-spot seahorse was hydrolyzed by alcalase to obtain the hydrolysate containing angiotensin I-converting enzyme(ACE)inhibitory peptides. The enzymatic hydrolysate was further separated and purified by dialysis,Sephadex G-25 gel filtration chromatography and reversed-phase high performance liquid chromatography(RP-HPLC). The results showed that the IC50 value of the dialysis product was(0.44 ±0.26)mg/mL,which was stronger than the enzymatic hydrolysate(0.81 ± 0.12)mg/mL. The dialysis product was separated by Sephadex G-25,three fractions were obtained. Among them,F2 had the strongest ACE inhibitory activity with the IC50 value of(0.11±0.08)mg/mL. F2 was further fractionated by RP-HPLC to obtain six fractions with ACE inhibitory activity. Among them,F2-4 showed strong ACE inhibitory activity with IC50 value of(0.005 7 ± 0.000 9) mg/mL. After three-step purification procedure,the experiment successfully isolated an active ACE inhibitory peptide from the three-spot seahorse protein:Pro-Ala-Gly-Pro-Arg-GlyPro-Ala(PAGPRGPA),the molecular weight of the peptide was 721.39 Da. The secondary structure of the purified peptide was analyzed by circular dichroism to indicate that it mainly contained random coil. Therefore,the peptide isolated from the three-spot seahorse protein might be a beneficial ingredient of nutraceuticals and antihypertensive drugs and related diseases,and was of great significance for the development and utilization of seahorse protein resources.