CHANGES IN KUNITZ TRYPSIN INHIBITOR DURING GERMINATION OF SOYBEANS: AN IMMUNOELECTROPHORESIS ASSAY SYSTEM

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Abstract
A rocket immunoelectrophoresis system was developed for assay of the major trypsin inhibitor of soybean seeds, the Kunitz soybean trypsin inhibitor (STI). Antibodies specific for purified Kunitz inhibitor were produced by subcutaneous injection of the purified inhibitor in rabbits. Electrophoresis of samples containing STI into agarose gels containing antisera to STI resulted in the formation of precipitin bands (rockets) whose height was linearly related to the amount of STI in the sample in the range of 35–175 nanograms. The smallest amount of STI which can be detected is approximately 5–10 nanograms. For many purposes, this assay system represents a substantial improvement over enzyme assays because only a single protein is recognized rather than all proteins with trypsin inhibitory activity. Trypsin‐STI complexes are also recognized in the immunoelectrophoresis assay, suggesting that at least two of the antigenic determinants of STI are accessible to antibodies when the inhibitor is complexed with enzyme. The Kunitz trypsin inhibitor concentration of soybeans (Glycine max L. var. Steele) decreased by 13% on a dry weight basis during the first 9 days of germination. Enzymatic assay of total trypsin inhibitor activity showed similar decreases during germination. Since rapid decreases in Kunitz inhibitor content do not take place during germination it seems improbable that the Kunitz inhibitor functions as a storage protein in the seed.