栉孔扇贝(Chlamys farreri)中抗氧化肽的分离纯化及性质研究

ISOLATION AND CHARACTERIZATION OF ANTIOXIDATIVE PEPTIDE FROM SCALLOP CHLAMYS FARRERI

以栉孔扇贝内脏团为原料,对其中抗氧化肽的制备、纯化及性质进行了研究。首先采用硫酸铵沉淀、DEAE-Sephadex A-50阴离子交换层析的方法,进行了扇贝提取蛋白的制备,其次采用海洋蛋白酶YS-80水解、SephadexG-25凝胶层析的方法,进行扇贝抗氧化肽的制备,获得多肽粗品(海洋肽,PCF),并进一步采用CM Sepharose阳离子交换层析和反相高效液相色谱的方法,对扇贝抗氧化肽进行了纯化,结果得到组分PCF-3A,反相高效液相显示为单一峰。采用茚三酮反应、Sephadex G-15凝胶层析和AccQ.Tag氨基酸分析柱,进行了PCF-3A的理化性质研究。结果表明,茚三酮反应呈阳性;分子量约为794D;由7种氨基酸组成,分别为天冬氨酸、甘氨酸、苏氨酸、丙氨酸、缬氨酸、赖氨酸和脯氨酸。采用邻苯三酚自氧化体系和Fenton反应体系对PCF-3A的抗氧化活性进行了研究,发现该肽可有效清除羟自由基和超氧阴离子,半抑制浓度(IC50)分别为0.39mg/ml和2.85mg/ml,具有明显的抗氧化、抗衰老作用。

The preparation, purification and prime characterization of antioxidative peptide isolated from marine proteins, scallop visceral mass, were studied. Proteins of scallop were extracted in 0.9% NaCl at 4℃, then centrifuged, and the precipitate was discarded. The supernatant was fractionated with ammonium sulfate. The precipitate between 60% and 90% saturation was collected and loaded into a DEAE-Sephadex A-50 column. The column was equilibrated with Tris- HCl buffer（5mmol/L, pH 7.5 ）and eluted with a linear gradient of this buffer containing 0.2mol/L NaCl. The peak monitored at 280nm absorbance comprising mostly of activity was collected. Antioxidative proteins were hydrolyzed with low-temperature marine protease YS-80 at 30℃ （pH 9.0）for 24h, then centrifuged and the precipitate was discarded. And PCF was prepared by taking off macro molecular proteins on a Sephadex G-25 column. The column was equilibrated with redistilled water. The peak monitored at 280nm absorbance comprising peptides was collected. Antioxidative peptide was purified in the first step by means of CM Sepharose FF cationic-exchange chromatography. The column was equilibrated with 2mmol/L phosphate-citric buffer （pH 3.0） and eluted with a linear gradient of this buffer containing 1.0mol/L NaC1. The third peak monitored at 280nm absorbance comprising mostly of activity was collected. In the second step, the peptide was purified by a reversed phase high performance liquid chromatography（HPLC） , the peak eluted at 20.919min has most activities and named as PCF-3A. The major physical and chemical characteristics of PCF-3A were studied. HPLC on C18 was used to check the homogeneity of the isolated product. After the purification with two column chromatographies, the separated product was verified to be homogeneous. The molecular weight was measured by size exclusion chromatography on a Sephadex G-15 column. The column was equilibrated with a 0.02mol/L Tris-HCl buffer containing 0.15moL/L NaCl at pH 7.2. The sample was applied to the column and eluted at a flow rate of 1.0ml/ min. PCF-3A possesses a molecular weight of 794D. The purified PCF-3A was exhibited positive in biuret color reaction. Column of AccQ. Tag was used to analysis the amino acids of PCF-3A. The peptide was hydrolyzed with 6mol/L HC1 at 110℃ for 22h in an sealed tube. Amino acid analysis showed that PCF-3A was composed of D, G, T, A, P, V and K. It is suggested that eight amino acid residues were composed of PCF-3A according to the molecular weight. The scavenging activity of PCF-3A on hydroxyl free radical was studied by Fenton reaction. And scavenging superoxide anion activity of PCF-3A was measured by self-oxidation method of pyrogallol. PCF-3A has distinctive effect antioxidation, the IC5o value for hydroxyl free radical and superoxide anion radical scavenging activities were 0.39 and 2. 85mg/ml respectively. Anti the activity of scavenging hydroxyl free radical is much greater than mannitol.