NON-RADIOACTIVE IN-VITRO LABELING TECHNIQUE FOR DETECTION OF METHIONINE-RlCH PROTEINS IN PHILIPPINE FOOD LEGUMES

摘要

A method icas developed for a non-radioactive in-vitro labeling for the detection of and semi-quantitative analysis for any of the methionine-rich proteins (MKPs) in dot blots of albumin preparations from cotyledons of some Philippine food legume. Thekey reaction is the specific alkvlalion of the thioether moiety of met hi on uie residues in polypeptides by biotmylated iodoacetate at pll 2 via an S2 mechanism. Visualization of positive results is facilitated by the strong biotin-avidin interactionwhere avidin is tagged with horseradish peroxidase;and the development of a colored product with 4-chloronaphthol (Opti-4CN) as substrate. Experimental parameters;which include blocking time;concentration of reagents;and incubation time;were optimized. Ariahsis icas dramatically shortened in time;with a comparable sensitivity to the established radioactive method. The optimized non-radioactive protocol ivas used to detect putative MRPs in the albumin extracted from the cotyledons. Samples found to contain putatively significant levels of methionine were con-pea (Vigna unguiculata subsp. unguiculata);pole sitao (V'igna iinguiciilata subsp. sesquipedalis);and batao (Dolichos lablab syn. Lablab purpureus).