Screening of food legumes for methionine rich proteins using an in-vitro labeling technique involving biotin-avidin interactions
2007
Castillo-Isarel, K., Philippines Univ. Los Baños, College, Laguna (Philippines). Inst. of Chemistry | Laurena, A. C., Philippines Univ. Los Baños, College, Laguna (Philippines). Inst. of Plant Breeding
Some Philippine food legumes were screened for the presence of methionine-rich proteins(MRPs) in their cotyledons using a non-radioactive in-vitro labeling method which was developed for the detection and semi-quantitative analysis of MRPs. The key reaction is the specific alkylation of the thioether moiety of methionine residues in polypeptides by biotinylated, iodoacetate at pH 2 via a SsubN2 mechanism. Visualization of positive results is facilitated by the strong biotin-avidin interaction where avidin is tagged with horseradish peroxidase, and the development of a colored product with 4-chloronaphthol (Opti-4CN) as substrate. Screening of the legume samples was carried out using dot blots of the albumin preparations on PVDF membrane. The methionine contents (met index) of the albumin were estimated based on the relative intensities of the spots on the blots. Samples found to contain putatively significant levels of methionine were cowpea (paayap), stringbean (pole sitao), hyacinth bean (batao). The non-radioactive protocol was also applied in tissue printing of fresh legume seeds at different stages of maturity on nitrocellulose membrane. This was to determine the localization of MRPs in the seeds, their expression at different stages of growth and development and clues on their biological functions. Results showed that MRP expressions vary from one species to another, but generally are optimally expressed in mature seeds and are localized in the hilum areas of the seeds. These suggest possible roles of MRPs in the seed maturation and deposition of nutrient reserves.
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