Preliminary investigation on the DNA and protein of winged bean (Psophocarpus tetrafonolobus) and lima bean (Phaseolus lunatus) with bioactive peptide exhibiting Angiotensin Converting Enzyme (ACE) inhibitory activity

Date

5-2011

Degree

Bachelor of Science in Chemistry

College

College of Arts and Sciences (CAS)

Adviser/Committee Chair

Mary Ann O Torio

Co-adviser

Roberta N. Garcia

Committee Member

Mark Rickard N. Angelia

Abstract

The presence of ACE inhibitors in lima bean (Phaseolus tuna.) and winged bean (Psophocarpus tetragonolobus) were investigated using DNA and protein analysis. In silico analysis showed that phaseolin of lima bean and winged bean albumin-1 of winged bean contain bioactive peptides exhibiting Angiotensin Converting Enzyme (ACE) activity in their respective amino acid sequence, which are LLP and VK, respectively. The genomic DNA of the samples was isolated using Cheung et al.- Datta et al.- Dellaporta et al. as modified by Angeles et al. (2005) and the modified Doyle and Doyle (1990) extraction method. The gene coding for the bioactive peptide of the samples was amplified via polymerase chain reaction (PCR) using the isolated genomic DNA as the template and a designed pair of primers. Forward and reverse primers for lima bean are (5'-ATGTCCAAACCCGAAACCCTC-3') and (5*-TGGGCTATTGAAGGAGGCATC-3'), respectively. The forward and reverse primer for winged bean, on the other hand, are (5'-TGGICTGTGGTGGATTTC-3') and (5'-GTAAGACACCACAAGCCT-3'), respectively. The amplified products were sent to Macrogen, Korea and the PCR product of lima bean was found to contain the target nucleotide sequence coding for the antihypertensive peptide. LLP. based from the pairwise alignment with the original DNA sequence, thereby, confirming that lima bean is a potential natural remedy for hypertension. The PCR product of winged bean, on the other hand, does not contain the target bioactive peptide. Instead, another bioactive peptide. LA. was found between the primer sequences of the PCR product, which is also located in the original amino acid sequence of winged bean albumin-1. The crude protein of both samples was isolated using ascorbic acid buffer (0.5M NaCI, 0.25M ascorbic acid, pH 2.4) for lima bean and Tris-HCI phosphate buffer saline (PBS) (0.1 M Tris-HCI buffer. pH 8.3 with 35 mM potassium phosphate buffer and 0.4 M NaCI) for winged bean and was found to have an estimated protein concentration of 14.3mg/m1 and 17.1mg/m1 for lima bean and winged bean, respectively. The crude isolates were subjected to pepsin digestion and characterized using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Spectrophotometric assay for ACE inhibitory activity by Cheung and Cushman (1979) revealed that lima bean and winged bean have a percentage ACE inhibition of 61.4% and 76.5%, respectively. The percentage inhibition obtained further substantiates the presence of antihypertensive peptides in both samples.

Language

English

Location

UPLB Main Library Special Collections Section (USCS)

Call Number

LG 993.5 2011 C4 M34

Document Type

Thesis

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