Isolation and Characterization of a Trypsin Inhibitor and a Lectin from Glycine max cv. Large Black Soybean

  • Ye, Xiu Juan (School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong) ;
  • Ng, Tzi Bun (School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong)
  • Published : 2009.10.31

Abstract

Trypsin inhibitors and lectins are defense proteins produced by many organisms. From Chinese 'Large Black Soybeans', a 60 kDa lectin and a 20 Da trypsin inhibitor (TI) were isolated using chromatography on Q-Sepharose, Mono Q, and Superdex 75. The TI inhibited trypsin and chymotrypsin with an $IC_{50}$ of 5.7 and $5{\mu}M$, respectively. Trypsin inhibitory activity of the TI was stable from pH 3 to 13 and from 0 to $65^{\circ}C$. Hemagglutinating activity of the lectin was stable from pH 2 to 13 and from 0 to $65^{\circ}C$. The TI was inhibited by dithiothreitol, signifying the importance of disulfide bond. The TI and the lectin inhibited HIV-1 reverse transcriptase ($IC_{50}$=44 and $26{\mu}M$), and proliferation of breast cancer cells ($IC_{50}$=42 and $13.5{\mu}M$) and hepatoma cells ($IC_{50}$=96 and $175{\mu}M$). The hemagglutinating activity of the lectin was inhibited most potently by L-arabinose. Neither the lectin nor the TI displayed antifungal activity.

Keywords

References

  1. Deshimaru M, Hanamoto R, Kusano C, Yoshimi S, Terada S. Purification and characterization of proteinase inhibitors from wild soja (Glycine soja) seeds. Biosci. Biotech. Bioch. 66: 1897-1903 (2002) https://doi.org/10.1271/bbb.66.1897
  2. Cheung AH, Wong JH, Ng TB. Trypsin-chymotrypsin inhibitors from Vigna mungo seeds. Protein Peptide Lett. 16: 277-284 (2009) https://doi.org/10.2174/092986609787601714
  3. Fernandes AO, Banerji AP. The field bean protease inhibitor can effectively suppress 7,12-dimethylbenz[a]anthracene-induced skin tumorigenesis in mice. Cancer Lett. 104: 219-224 (1996) https://doi.org/10.1016/0304-3835(96)04253-X
  4. Gladysheva IP, Polekhina OV, Karmakova TA, Nemtsova ER, Yakubovskaya RI, Shen WC, Kennedy AR, Larionova NI. Potential of block copolymer- and immuno-conjugates for tumor-targeted delivery of Bowman-Birk soybean proteinase inhibitor. J. Control. Release 74: 303-308 (2001) https://doi.org/10.1016/S0168-3659(01)00337-6
  5. Birk Y. Plant Protease Inhibitors: Significance in Nutrition, Plant Protectior. Springer-Verlag, Berlin-Heidelberg, Germany. pp. 1-126 (2003)
  6. von Hofe E, Newberne PM, Kennedy AR. Inhibition of Nnitrosomethylbenzylamine- induced esophageal neoplasms by the Bowman-Birk protease inhibitor. Carcinogenesis 12: 2147-2150 (1991) https://doi.org/10.1093/carcin/12.11.2147
  7. Kobayashi H, Suzuki M, Kanayama N, Terao T. A soybean Kunitz trypsin inhibitor suppresses ovarian cancer cell invasion by blocking urokinase upregulation. Clin. Exp. Metastasis 21: 159-166 (2004) https://doi.org/10.1023/B:CLIN.0000024751.73174.c2
  8. Ye XY, Ng TB, Rao PF. A Bowman-Birk-type trypsin-chymotrypsin inhibitor from broad beans. Biochem. Bioph. Res. Co. 289: 91-96 (2001) https://doi.org/10.1006/bbrc.2001.5965
  9. Lam SSL, Wang HX, Ng TB. Purification and characterization of novel ribosome inactivating proteins, $\alpha$- and $\beta$-pisavins, from seeds of the garden pea Pisum sativum, Biochem. Bioph. Res. Co. 253: 135-142 (1998) https://doi.org/10.1006/bbrc.1998.9764
  10. Fakhoury AM, Woloshuk CP. Inhibition of growth of Aspergillus flavus and fungal α-amylases by a lectin-like protein from Lablab purpureus. Mol. Plant Microbe In. 14: 955-961 (2001) https://doi.org/10.1094/MPMI.2001.14.8.955
  11. Nasi A, Picariello G, Ferranti P. Proteomic approaches to study stucture functions and toxicity of legume seeds lectins perspectives for the assessment of food quality and safety. J. Proteomics 72: 527- 538 (2009) https://doi.org/10.1016/j.jprot.2009.02.001
  12. Wong JH, Wong CC, Ng TB. Purification and characterization of $\alpha$- specific lectin with mitogenic activity from pinto beans. Biochim. Biophys. Acta 1760: 808-813 (2006) https://doi.org/10.1016/j.bbagen
  13. Le Berre-Anton V, Bompard-Gilles C, Payan E, Rouge P. Characterization and functional properties of the α-amylase inhibitor (alpha -AI) from kidney bean (Phaseolus vulgaris) seeds. Biochim. Biochim. Biophys. Acta 1343: 31-40 (1997) https://doi.org/10.1016/S0167-4838(97)00100-3
  14. Franco-Fraguas L, Pl$\acute{u}$ A, Ferreira F, Massaldi H, Su$\acute{u}$rez N, Batista- Viera F. Preparative purification of soybean agglutinin by affinity chromatography and its immobilization for polysaccharide isolation. J. Chromatogr. B 790: 365-372 (2003) https://doi.org/10.1016/S1570-0232(03)00086-2
  15. Nielsen TB, Reynolds JA. Measurements of molecular weights by gel electrophoresis. Method Enzymol. 48: 3-10 (1978) https://doi.org/10.1016/S0076-6879(78)48003-6
  16. Wang HX, Ng TB. Concurrent isolation of a Kunitz-type trypsin inhibitor with antifungal activity and a novel lectin from Pseudostellaria heterophylla roots. Biochem. Bioph. Res. Co. 342: 349-353 (2006) https://doi.org/10.1016/j.bbrc.2006.01.109
  17. Tsoi AY, Ng TB, Fong WP. Immunomodulatory activity of a chymotrypsin inhibitor from Momordica cochinchinensis seeds. J. Pept. Sci. 12: 605-611 (2006) https://doi.org/10.1002/psc.765
  18. Azarkan M, Dibiani R, Goormaghtigh E, RaussensV, Baeyens- Volant D. The papaya Kunitz-type trypsin inhibitor is a highly stable $\beta$-sheet glycoprotein. Biochim. Biophys. Acta 1764: 1063-1072 (2006) https://doi.org/10.1016/j.bbapap.2006.02.014
  19. Wong JH, Ng TB. Isolation and characterization of a glucose/ mannose-specific lectin with stimulatory effect on nitric oxide production by macrophages from the emperor banana. Int. J. Biochem. Cell B. 38: 234-243 (2006) https://doi.org/10.1016/j.biocel.2005.09.004
  20. Wong JH, Ng TB. Sesquin, a potent defensin-like antifungal peptide from beans with inhibitory activities toward tumor cells and HIV-1 reverse transcriptase. Peptides 26: 1120-1126 (2005) https://doi.org/10.1016/j.peptides.2005.01.003
  21. Cheung AH, Ng TB. Isolation and characterization of a trypsinchymotrypsin inhibitor from the seeds of green lentil (Lens culinaris). Protein Peptide Lett. 14: 859-864 (2007) https://doi.org/10.2174/092986607782110310
  22. Ye XY, Ng TB. Mungin, a novel cyclophilin-like antifungal protein from the mung bean. Biochem. Bioph. Res. Co. 273: 1111-1115 (2000) https://doi.org/10.1006/bbrc.2000.3067
  23. Ho VSM, Ng TB. A Bowman-Birk trypsin inhibitor with antiproliferative activity from Hokkaido black soybeans. J. Peptide Sci. 14: 278-282 (2008) https://doi.org/10.1002/psc.922
  24. de Mejia EG, Bradford T, Hasler C. The anticarcinogenic potential of soybean lectin and lunasin. Nutr. Rev. 61: 239-246 (2003) https://doi.org/10.1301/nr.2003.jul.239-246
  25. Valentiner U, Fabian S, Schumacher U, Leathem AJ. The influence of dietary lectins on the cell proliferation of human breast cancer cell lines in vitro. Anticancer Res. 23: 1197-1206 (2003)