| In this paper, high-performance liquid chromatography (HPLC) and Visible spectrophotometricy (VSP) methods were developed and compared to assay inhibitory activity of angiotensin I-converting enzyme (ACE) in vitro. Grass carp proteins were hydrolyzed by alcalase to prepare ACE inhibitory activity peptides, these peptides were purified by ultrafiltration, macroporous adsorption resin and RP-HPLC. Amino acid sequences of the single peptide with high ACE inhibitory activity were identified by PPSQ-31A/33A Automated Protein/Peptide Sequencers. Furthermore, relationship between structure and ACE inhibitory activity of tripeptides were studied by quantitative structure activity relationship (QSAR) model.Chromatography conditions of HPLC method were determined as follows: ODS C18 chromatography column; diode array detector, detection at 228 nm; flow rate of 0.8 mL/min; solvent-(A) with 0.05% TFA in water and solvent-(B) with 0.05% TFA in acetonitrile; 20μL of sample volume; 25℃of column temperature; elution with a gradient of 10-60% B in 10 min and another 60-10% B in 2 min. Chromogenic reaction conditions of VSP method were sure as follows:2.5 of qunolin/BSC ratio(V/V); 30℃of reaction temperature; 30 min of reaction time in dark; 3.7 mL of ethanol; 30 min of further incubation time in dark. Comparisons of two methods showed that the lowest detectable limit of HPLC method is much smaller than VSP method, and HPLC method had better linearity, higher precision, repeatability and stability. Furthermore, IC50 values of captopril, soybean peptides, Grass Carp peptides were 0.00206±0.00005,192±4.53 and 153±4.29μg/mL by VSP method with 1.07,1.07,1.18 and 1.44 fold by HPLC method, and K, value of captopril was 7.09 nM by VSP method with 1.44 fold by HPLC method.To prepare ACE inhibitory activity peptides, grass carp proteins were hydrolyzed by alcalase on the conditions of pH 9.0,50℃of temperature and 48 AU/kg of enzyme/substrate ratio and stoped at DH of 17.25%. Its IC50 valve and peptide content were 0.872mg/mL and 65.48%. Grass carp peptides was purified by ultrafiltration, macroporous adsorption resin and RP-HPLC and a single peptide with high ACE inhibitory activity (IC50 valve of 0.00534±0.00003 mg/mL) was prepared, its amino acid sequence was VAP determined by utomated Protein /Peptide Sequencers. The results of activity stability test showed that VAP were not hydrolyzed by pepsin, chymotrypsin and ACE. And the result of inhibitory kinetics study showed that VAP is a competitive ACE inhibitor isolated from grass carp protein, and its Ki value is 3.34 nM.76 different tripeptides were collected from previously published works to build QSAR model. In this model, hydrophilicity, steric properties or side chain bulk/molecular size and electronic properties of each individual amine acid and hydrophilicity of tripeptide were designed as ten abscissas(X), log IC50 valve was designed as ordinate(Y). The model could explain 42.55% of Y-variance and 80.16 % of X- variance with the predictive ability of 60.3% for tripeptides and its equation was as following:Y=3.4639+0.0084X1-0.6281X2-0.0025X3+0.8119X4+0.5677X5-0.0107 X6+0.6139X7+0.5338X8-0.0228X9+1.0223X10QSAR studies on tripeptide suggest that side chain bulk of the amine acids at carbon terminus is most significant effect factor on ACE inhibitory activity of tripeptide;low electronic properties, high side chain bulk and hydrophilicity of the amine acids at amino terminus, such as Phe, Met, Ile, Leu, et al, is positively related to ACE inhibitory activity; amine acids such as Lys, Pro, Met, et al with high side chain bulk, low electronic properties and hydrophilicity were preferred for carbon amino terminus and middle position. |
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