Amino Acids

, Volume 43, Issue 1, pp 379–388 | Cite as

Proteomic characterization of Kunitz trypsin inhibitor variants, Tia and Tib, in soybean [Glycine max (L.) Merrill]

  • K. J. Lee
  • J.-B. Kim
  • B.-K. Ha
  • S. H. Kim
  • S.-Y. Kang
  • B.-M. LeeEmail author
  • D. S. KimEmail author
Original Article


The soybean Kunitz trypsin inhibitor (KTi) has several polymorphic variants. Of these, Tia and Tib, which differ by nine amino acids, are the two main types. In this study, differences in KTi proteome between Tia and Tib were investigated using three soybean cultivars and three mutant lines. Two cultivars, Baekwoon (BW) and Paldal (PD), and one mutant line, SW115-24, were Tia type, whereas one soybean cultivar, Suwon115 (SW115), and two mutant lines, BW-7-2 and PD-5-10, were Tib type. Protein from the six soybean lines was extracted and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), non-denaturing polyacrylamide gel electrophoresis (non-denaturing PAGE), and two-dimensional polyacrylamide gel electrophoresis (2-DE). By SDS-PAGE, there was no difference between soybean cultivars and mutant lines, except for SW115-24. Western blot analysis revealed that, in comparison with Tia, Tib type accumulated relatively low amounts of KTi. By non-denaturing PAGE, the three soybean lines of Tib type were characterized by slower mobility than the three soybean lines of Tia type. Zymography detected eight distinct zones of trypsin inhibitory activity among which Tia and Tib lacked the fifth and sixth zone, respectively. By two-dimensional native polyacrylamide gel electrophoresis (2-DN), the spots related to trypsin inhibitory activity showed different mobilities, whereas only one KTi (21.5 kDa) spot was resolved by 2-DE. By two-dimensional zymography (2-DZ), Tib showed a broader activity zone (pI 4–7) in comparison with Tia (pI 4–5). The results indicate that the genotypes with a different type of KTi present different proteomic profiles and trypsin inhibitory activities.


Kunitz trypsin inhibitor Mutation Proteome Soybean 



Two-dimensional polyacrylamide gel electrophoresis


Two-dimensional native polyacrylamide gel electrophoresis


Two-dimensional zymography


Coomassie brilliant blue R-250


Isoelectric focusing


Immobilized pH gradient


Isoelectric point


Sodium dodecyl sulfate-polyacrylamide gel electrophoresis



This research was supported by the Technology Development Program for Agriculture and Forestry, the Ministry for Food, Agriculture, Forestry, and Fisheries, the Korea Atomic Energy Research Institute (KAERI) and the Ministry of Education, Science and Technology (MEST), Korea.


  1. Birk Y (1961) Purification and some properties of a highly activity inhibitor of trypsin and α-chymotrypsin from soybean. Biochim Biophys Acta 54:378–381PubMedCrossRefGoogle Scholar
  2. Bowman DE (1946) Differentiation of soybean antitryptic factors. Proc Soc Exp Biol Med 63:547–550PubMedGoogle Scholar
  3. Choi NS, Yoo KH, Yoon KS, Maeng PJ, Kim SH (2004) Nano-scale proteomics approach using two-dimensional fibrin zymography combined with fluorescent SYPRO ruby dye. Biochem Mol Biol 37:298–303CrossRefGoogle Scholar
  4. Gallagher SR (1995) One-dimensional electrophoresis using non-denaturing conditions. In: Current Protocols in Protein Science. Wiley, New York, pp 10.3.1–10.3.11Google Scholar
  5. Gore A, Obermaier C, Boguth G, Harder A, Scheibe B, Wildgruber R, Weiss W (2000) The current state of two-dimensional electrophoresis with immobilized pH gradients. Electrophoresis 21:1037–1053CrossRefGoogle Scholar
  6. Herbert B (1999) Advances in protein solubilization for two-dimensional electrophoresis. Electrophoresis 20:660–663PubMedCrossRefGoogle Scholar
  7. Hymowitz T (1973) Electrophoretic analysis of SBTI-A2 in the USDA soybean germplasm collection. Crop Sci 13:420–421CrossRefGoogle Scholar
  8. Hymowitz T, Kaizuma N (1981) Soybean seed protein electrophoresis profiles from 15 Asian countries or regions; hypotheses on paths of dissemination of soybean from China. Econ Bot 35:10–23Google Scholar
  9. Kaizuma N, Oikawa K, Miura M (1980) Consideration on the cause of the differential ti alleles frequency distributions found among some regional populations of soybean (Glycine max (L.) Merrill) land varieties. J Fac Agric Iwate Univ 15:81–96Google Scholar
  10. Krishnan HB (2001a) Characterization of a soybean [Glycine max (L.) Merr.] mutant with reduced levels of Kunitz trypsin inhibitor. Plant Sci 160:979–986PubMedCrossRefGoogle Scholar
  11. Kim DS, Lee KJ, Kim J-B, Kim SH, Song JY, Seo YW, Lee B-M, Kang S-Y (2010) Identification of Kunitzt trypsin inhibitor mutations using SNAP markers in soybean mutant lines. Theor Appl Genet 121:751–760PubMedCrossRefGoogle Scholar
  12. Kim SH, Choi NS, Lee WY (1998) Fibrin zymography: a direct analysis of fibrinolytic enzyme on gel. Anal Biochem 263:115–116PubMedCrossRefGoogle Scholar
  13. Kim SH, Hara S, Hase S, Ikenaka T, Tode H, Kitamura K, Kaizuma N (1985) Comparative study on amino acid sequence of Kunitz-type soybean trypsin inhibitors, Tia, Tib, and Tic. J Biochem 19:435–448Google Scholar
  14. Krishnan HB, Jiang G, Krishnan AH, Wiebold WJ (2000) Seed storage protein composition of non-nodulating soybean [Glycine max (L.) Merr.] and its influence on protein quality. Plant Sci 157:191–199PubMedCrossRefGoogle Scholar
  15. Krishnan HB (2001b) Characterization of a soybean [Glycine max (L.) Merr.] mutant with reduced levels of Kunitz trypsin inhibitor. Plant Sci 160:979–986PubMedCrossRefGoogle Scholar
  16. Kunitz M (1945) Crystallization of a trypsin inhibitor from soybean. Science 101:668–669PubMedCrossRefGoogle Scholar
  17. Lajolo FM, Genovese MI (2002) Nutritional significance of lectins and enzyme inhibitors from legumes. J Agric Food Chem 50:6592–6598PubMedCrossRefGoogle Scholar
  18. Li FS (1993) Studies on the ecological and geographical distribution of the Chinese resources of wild soybean (G. Soja). Sci Agri Sin 26:47–55Google Scholar
  19. Natarajan SS, Xu C, Bae H, Caperna TJ, Garrett WM (2006) Characterization of storage proteins in wild (Glycine soja) and cultivated (Glycine max) soybean seeds using proteomic analysis. J Agric Food Chem 54:3114–3120PubMedCrossRefGoogle Scholar
  20. Martz F, Wilczynska M, Kleczkowski LA (2002) Oligomerization status, with the monomer as active species, defines catalytic efficiency of UDP-glucose pyrophosphorylase. Biochem J 367:295–300PubMedCrossRefGoogle Scholar
  21. Orf JH, Hymowitz T (1979) Inheritance of the absence of the Kunitz trypsin inhibitor in seed protein of soybeans. Crop Sci 19:107–109CrossRefGoogle Scholar
  22. Roychaudhuri R, Sarath G, Zeece M, Markwell J (2004) Stability of the allergenic soybean Kuntiz trypsin inhibitor. Biochem Biophys Acta 1699:207–212PubMedGoogle Scholar
  23. Singh LC, Wilson M, Hadley HH (1969) Genetic differences in soybean trypsin inhibitor separated by disc electrophoresis. Crop Sci 9:489–491CrossRefGoogle Scholar
  24. Song SI, Kim CH, Baek SJ, Choi YD (1993) Nucleotide sequences of cDNA encoding the precursors for soybean (Glycine max) trypsin inhibitors (Kunitz type). Plant Physiol 101:1401–1402PubMedCrossRefGoogle Scholar
  25. Wang KJ, Kaizuma N, Takahata Y, Hatakeyama S (1996) Detection of two new variants of soybean Kunitz trypsin inhibitor through electrophoresis. Breed Sci 46:39–44Google Scholar
  26. Wang KJ, Takahata Y, Ito K, Zhao YP, Tsutsumi KI, Kaizuma N (2001) Genetic characterization of a novel soybean Kunitz trypsin inhibitor. Breed Sci 51:185–190CrossRefGoogle Scholar
  27. Wang KJ, Yamashita T, Watanabe M, Takahata Y (2004) Genetic characterization of a novel Tib-derived variant of soybean Kunitz trypsin inhibitor detected in wild soybean (Glycine soja). Genome 47:9–14PubMedCrossRefGoogle Scholar
  28. Wang KJ, Li XH (2005) Tif type of soybean Kunitz trypsin inhibitor exists in wild soybean of northern China. In: Proceedings of the 8th national soybean research conference of China, pp 167–168Google Scholar
  29. Wang KJ, Takahata Y, Kono Y, Kaizuma N (2008) Allelic differentiation of Kunitz trypsin inhibitor in wild soybean (Glycine soja). Theor Appl Genet 117:565–573PubMedCrossRefGoogle Scholar
  30. Weder JKP, Kahley R (2003) Reaction of lentil trypsin-chymotrypsin inhibitors with human and bovine proteinase. J Agric Food Chem 51:8045–8050PubMedCrossRefGoogle Scholar
  31. Xin H, Xie KF, Dong AW, Uan QY, Gu QM (1999) The amino acid sequence determination of a new variant of Kunitz soybean trypsin inhibitor (SBTi-A2). Soyb Genet Newsl. Accessed 24 Mar 1999
  32. Yu M, King J (1988) Surface amino acids as sites of temperature-sensitive folding mutations in the P22 tailspike protein. J Biol Chem 263:1424–1431PubMedGoogle Scholar
  33. Zhao SW, Wang H (1992) A new electrophoretic variant of SBTi-A2 in soybean seed protein. Soyb Genet Newsl 19:22–24Google Scholar

Copyright information

© Springer-Verlag 2011

Authors and Affiliations

  1. 1.Advanced Radiation Technology InstituteKorea Atomic Energy Research InstituteJeongeupRepublic of Korea
  2. 2.Department of Plant BiotechnologyDongguk UniversitySeoulRepublic of Korea

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