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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

Abstract

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.

Keywords

Kunitz trypsin inhibitor Mutation Proteome Soybean 

Abbreviations

2-DE

Two-dimensional polyacrylamide gel electrophoresis

2-DN

Two-dimensional native polyacrylamide gel electrophoresis

2-DZ

Two-dimensional zymography

CBB

Coomassie brilliant blue R-250

IEF

Isoelectric focusing

IPG

Immobilized pH gradient

pI

Isoelectric point

SDS-PAGE

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis

Notes

Acknowledgments

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.

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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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