Non-radioactive detection of methionine-rich proteins in the cotyledons of some Philippine food legumes

Methionine is a limiting amino acid in legumes. Its detection in proteins by conventional radioactive method, which takes 14 days after electrophoresis and electroblotting, has paved the way for the identification and the cloning of the gene encoding the protein in soybean. A non-radioactive in vitro labeling method for the detection and semi-quantitative analysis of methionine-rich proteins (MRP) in the dot blot and SDS-PAGE [ sodium dodecyl sulfate-poly-acrylamide gel electrophoresis] blots of albumin preparations of some Philippine food legume cotyledons was developed based on the reaction established for the conventional protocol. The key reaction is the specific alkylation of the thioether moiety of methionine residues in polypeptides via an SN2 mechanism. Detection of the MRP is facilitated by a biotin-avidin tagging reaction, with biotinylated iodoacetate as alkylating reagent at Ph2, interacting specifically with avidin linked to horseradish peroxidase, and developing a purple quinone product in the presence of the pseudosubstrate, 4-chloronaphthol (Opti-4CN). Blocking with BSA prevents non-specific interactions. The optimized non-radioactive MRP protocol was used to detect putative MRPs in the albumin extracted from cotyledons of some Philippine food legumes on dot blots and electrophoretic blots. The approximate methionine content (Met index) of the crude albumins was estimated based on the relative intensities of the spots on the blots quantified by Quantiscan, a computer software. The intensity of spots/bands is correlated with methionine. Samples found to contain putatively significant levels of methionine were cowpea (Vigna unguiculata L. (Walp.)), pole sitao (Vigna sesquipedalis L.) and batao (Dolichos lablab L. HY III) with met indices of 2.6, 2.3 and 2.1 respectively, while corn prolamine, the standard with 6.9. A 30kD MRT band was identified from two accessions of batao (Dolichos lablab L. HY 97 and HY 57).