Purification And Identification Of Pig Skin Collagen Ace Inhibitory Peptide
Keywords
Collagen Angiotensin Converting Enzyme (Ace) Inhibitory Peptide Isolation And Purification Structural Characterization
Abstract
After pretreatment of pig skin with ultrasound and dilute alkali, the high angiotensin converting enzyme (ACE) inhibitory activity hydrolyzate obtained by enzymatic hydrolysis for 30 minutes was used as raw material. Sephadex G-15 and semi-prepared high-efficiency liquid were used. The collagen ACE inhibitory peptide was separated and purified by phase chromatography and its sequence was identified. The results show that the optimal separation conditions for Sephadex G-15 to separate collagen ACE inhibitory peptides are: sample concentration 100 mg/mL; sample loading volume 2 mL; flow rate 2 mL/min; eluent is distilled water; chromatography column is 1 m ¡Á10 mm chromatography column. Under this separation condition, the mixed peptide was divided into two components, AP-I and AP-II, with IC50 values of 19.50 and 1.01 mg/mL respectively. AP-II with strong inhibitory activity was further separated by semi-preparative high performance liquid chromatography, and 8 component peaks were obtained, among which the IC50 values of peak 2, peak 5 and peak 6 were the smallest, which were 0.73, 0.44 and 0.4 mg/ mL. Based on the size of the IC50 value and the amount of sample collected in the semi-preparation, LC-MS/MS was used for sequence analysis of peak 2 and peak 6. The results show that the polypeptide sequence of peak 2 is QGPPGPAGPR (P is Hyp), and the sequence of peak 6 is AGPPGPPGPA. These two sequences are located at positions 526-535 and 730-739 in the collagen ¦Á1 chain. 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/mL; injection volume of 2 mL; flow rate of 2 mL/min; distilled water as the eluent; and a 1 m ¡Á 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/mL and 1.01 mg/mL, 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/mL, 0.4 mg/mL, and 0.73 mg/mL, 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 ¦Á1 chain , respectively.
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Original research was done by Zhang Ying
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