Isolation, purification and characterization of proteins with bioactive peptides exhibiting anti-hypertensive activity from winged bean (Psophocarpus tetragonolobus (L.) DC)
2011
Torio, M.A.O.
Total soluble proteins of winged bean var. Gloria, was extracted using a buffer mixture of Tris-HCl and phosphate buffer. The major storage protein was then purified using ammonium sulfate fractionation hydrophobic interaction chromatography and gel filtration chromatography. The estimated molecular weight of the major storage protein was 19175.6 Da on SDS-PAGE [sodium dodecyl sulfate-poly-acrylamide gel electrophoresis]. The protein concentration was 4.10 mg/ml. In Silico analysis was done to determine if the protein sequence of the major storage protein in winged bean contains peptides exhibiting ACE [Angiotensin-I-Converting Enzyme] inhibitory activity and anti-hypertensive activity. Many peptide sequences were found for WBA-1 [winged bean albumin-1] and WBA, in particular VK [Val-Lys], MY [Met-Tyr] and FQP [Phe-Gln-Pro]. The partially purified protein was digested using themolysin. The ACE inhibitory activity of the partially purified protein and the digested protein at an hour and 24 hours digestion time with thermolysin to protein r/v ratio of 8:1 was determined. the ACE activity was 1.34 x 10 sup-3 U. The digests were found to inhibit the ACE at one hour and 24 hour digestion time the percent inhibitions 88.70 and 92.47. The ACE inhibitory activities were 1.19 x 10 sup-3 U and 1.01 x 10 sup-3 U. The undigested protein also inhibited the ACE having 37.24 percent inhibition. Other low molecular weight proteins or denatured proteins may have caused this. Although the other proteins were digested, it was assumed that the peptides came from the major storage protein having the highest percent concentration (17.37%) from densitometric analysis. Reverse Phase High Performance Liquid Chromatography (HPLC) was done to themolytic hydrolysates to further separate and purify the bioactive peptides. Three randomly selected (with highest peaks) fractions were again assayed for ACE inhibitory activity. Results showed that the inhibitory activities obtained were 1.4492 x 10 sup-3 U for fraction 4, 1.692 x 10 sup-3 U for fraction 8, and 4.872 x 10 sup-3 U for fraction 9. The percent inhibition of the three fractions 4, 8 and 9 were 57.965, 64.38%, and 80.75%, respectively. Fraction 9 has the highest inhibitory activity and percent inhibition and this was further subjected to Thin Layer Chromatography. From the results, the possible sequence of the bioactive peptide fragment was VK for it has the closest values to the standard amino acid RF values.
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