| Summary: | 碩士 === 國立臺灣大學 === 食品科技研究所 === 92 === Soybean (Glycine max Merrill) is highly nutritive due to its high protein and lipid contents. Some bioactive compounds such as isoflavones and phytoestrogen were reported to help scavenge free radicals and alleviate feminine menopause syndrome, respectively, and furthermore, prevent cancer. Activation of enzymes during germination is relevant to hydrolysis of nutritive components as well as to the plant growth. Proteomic researches on the activity changes of some prominent enzymes, such as lipoxidase, peroxidase, pectinesterase (PE) and trypsin inhibitor, appeared to be of great interest during germination. Therefore, two-dimensional electrophoresis (2-DE) and
immunoblotting techniques were tried to investigate the quantitative changes of enzymes during germination.
First, four rabbits were immunized with one of above four enzymes to prepare polyclonal antibodies. Subsequently, soybeans germinated 1/3, 1, 3, 5, and 7 days were homogenized and the obtained protein portions were applied on a SDS-PAGE and a 2-D electrophoresis. Finally, Western blotting analysis was conducted to compare the color developed.
Results showed that ELISA value increase with the increasing time of boosting, and the antiserum titer against PE, lipoxidase, trypsin inhibitor, and peroxidase were determined to be 105, 105, 105, and 106, respectively, by an indirect ELISA method.
For mini gel SDS-eletrophoresis, high molecular weight proteins reduced and low-molecular weight proteins increased with the increasing cultivation time of soybeans, suggesting marked protein hydrolysis occurred during germination. Immunoblotting results suggested that PE, peroxidase, and trypsin inhibitor reduced during germination, while lipoxidase remained unchanged.
Changes in the trends of protein distribution assayed by 2-DE during germination were similar to those in mini gel SDS-electrophoresis. Immunoblotting stains revealed the decreasing trypsin inhibitor and increasing peroxides during germination. Decrease in trypsin inhibitor was considered to be favorable for the enhanced protease activity during germination, which was consistent with the phenomenon of sprout growth. However, increase in peroxidase activity during germination facilitated the reduction of fat hydroperoxide, which could be toxic to sprout growth. 2-DE and the subsequent immunoblotting results showed that PE, and lipoxidase both increased during germination. Increase in lipoxidase was favorable for the plant anti-microbial actions during germination to avoid the possible attack. Combination of 2-DE with immunoblotting stains provided convenient techniques for the detection of proteins in quantitative changes during germination.
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