Preliminary investigation on the DNA and crude protein of Pigeon pea (Cajanus cajan) and winged bean (Psophocarpus tetragonolobus)- crops containing bioactive peptides exhibiting angiotensin-converting enzyme (ACE) inhibitory activity

Date

2011

Degree

Bachelor of Science in Chemistry

College

College of Arts and Sciences (CAS)

Adviser/Committee Chair

Mary Ann O. Torio

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Abstract

The search for bioactive peptides with antihypertensive activity in pigeon pea and winged bean was done by In silico analysis using GenBank database. The proteins, legumin in pigeon pea and albumin-1 in winged bean, were found to contain bioactive peptides which exhibit anti-hypertensive or ACE inhibitory activity. The nucleotide

sequence corresponding to the proteins found to contain bioactive peptides with anti- hypertensive activity were downloaded and used as a basis for designing a primer. The

designed primers were then used for polymerase chain reaction (PCR) amplification. Genomic DNA of pigeon pea and winged bean was extracted using the method of Cheung et al. – Datta et al. – Dellaporta et al. as modified by Angeles et al. (2005) and viewed using agarose gel electrophoresis with GelRed. The extracted genomic DNAs were used as template for PCR using the designed primers specific for the crop. The forward and reverse primers are 5’-TCCTCTTCTCCTTGATGACGC-3’ and 5’-TGGACTCAGGCACAATAACCC-3’ for pigeon pea and 5’-ACTCCACCACAACCAAGG-3’ and 5’- GTAAGACACCACAAGCC-3’ for winged bean. Results of PCR showed one major band for each crop as revealed by the bands in agarose gel electrophoresis. The bands correspond to products with 600 bp and 100 bp for pigeon pea and winged bean, respectively. The PCR products corresponding to these results were sent for DNA sequencing. DNA analysis revealed the presence of bioactive peptides after translating the DNA sequence into amino acid sequence using Expasy Translate tool. Confirmation of the identity of the fragments amplified was done using BLAST search and BioEdit pairwise alignment. Some fragments of the PCR product of pigeon pea were found in legumin and other genes of the crop. The PCR product of winged bean, however, did not match with albumin-1 but some fragments were also found in other genes of the crop. The ACE inhibitory peptides found in the PCR product of pigeon pea are RF, FP, LF, IP, RP, VP, RA, AA, FR, IF, GI, GA, GL, KG, GS, GV, GE, LG, SF, EI, EV, LQ, and PT while in winged bean the peptides are RL, VW, VK, VG, IG, GD, TG, DG, KL. Protein analysis was also done. The crude protein of each crop was isolated using 0.01M sodium borate at pH 8.2 for pigeon pea and Tris-HCl phosphate buffer saline (0.1 M Tris-HCl buffer, pH 8.3, with 35 mM potassium phosphate buffer and 0.4 M NaCl) for winged bean. The crude proteins were then subjected to Bradford assay to determine its concentration. Pigeon pea and winged bean were found to contain 11.76 mg/mL and 13.40 mg/mL crude protein, respectively. The crude proteins were also digested using pepsin as enzyme, then subjected to SDS-PAGE to confirm digestion and spectrophotometric assay to determine percent inhibition of ACE activity using the method of Cheung and Cushman (1979). ACE assay revealed that pigeon pea and winged bean exhibit 81.63 and 74.44 percent inhibition, respectively. This confirms that pigeon pea and winged bean have bioactive peptides exhibiting ACE-inhibitory activity.

Language

English

LC Subject

Pigeon pea, Winged bean, Proteins--Analysis

Call Number

LG 993.5 2011 C4 /M36

Document Type

Thesis

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