A Trypsin-Chymotrypsin Inhibitor with Antiproliferative Activity from Small Glossy Black Soybeans

Abstract

A trypsin inhibitor with a molecular mass of about 19 kDa was isolated from seeds of Chinese black soybean Glycine max cv. “Small Glossy Black”. It was isolated using a protocol that comprised ion exchange chromatography on Q-Sepharose, SP-Sepharose and DEAE-cellulose. It was adsorbed on all three ion exchangers. It inhibited trypsin with an IC₅₀ of 19 µM and chymotrypsin with an IC₅₀ of 14.3 µM. Its trypsin inhibitory activity was stable in the pH range pH 3 – pH 13 and in the temperature range 0 °C − 60 °C. The trypsin inhibitor was inhibited by dithiothreitol (from 5 to 25 mM) in a dose-dependent manner. It exhibited an N-terminal sequence highly homologous to Kunitz-type trypsin inhibitors. It inhibited HIV-1 reverse transcriptase with an IC₅₀ of 0.16 µM, and suppressed proliferation of MCF-7 breast cancer cells with an IC₅₀ of 4.3 µM and HepG2 hepatoma cells with an IC₅₀ higher than 25 µM. The trypsin inhibitor lacked antifungal activity and mitogenic activity towards mouse splenocytes.

Key words

black soybean - trypsin inhibitor - isolation - antiproliferative activity - Glycine max - Leguminosae

References

• 1 Wong J H, Ng T B. Isolation and characterization of a glucose/mannose/rhammose-specific lectin from the knife bean Canavalia gladiata.  Arch Biochem Biophys. 2005;  439 91-98
• 2 Lam S SL, Wang H X, Ng T B. Purification and characterization of novel ribosome inactivating proteins, alpha- and beta-pisavins, from seeds of the garden pea Pisum sativum.  Biochem Biophys Res Commun. 1998;  253 135-142
• 3 Wong J H, Ng T B. Sesquin, a potent defensin-like antifungal peptide from beans with inhibitory activities toward tumor cells and HIV‐1 reverse transcriptase.  Peptides. 2005;  26 1120-1126
• 4 Le Berre-Anton V, Bompard-Gilles C, Payan E, Rouge P. Characterization and functional properties of the alpha-amylase inhibitor (alpha-AI) from kidney bean seeds.  Biochim Biophys Acta. 1997;  1343 31-40
• 5 Birk Y. Plant protease inhibitors. Berlin, Heidelberg, New York; Springer-Verlag 2003
• 6 Ye X Y, Ng T B, Rao P F. A Bowman-Birk-type trypsin-chymotrypsin inhibitor from broad beans.  Biochem Biophys Res Commun. 2001;  289 91-96
• 7 Conners R, Konarev A V, Forsyth J, Lovegrove A, Marsh J, Joseph-Horne T, Shewry P, Brady R L. An unusual helix-turn-helix protease inhibitory motif in a novel trypsin inhibitor from seeds of Veronica (Veronica hederifolia L.).  J Biol Chem. 2007;  282 27760-27768
• 8 Kowalska J, Pszczoła K, Wilimowska-Pelc A, Lorenc-Kubis I, Zuziak E, Ługowski M, Łegowska A, Kwiatkowska A, Sleszynska M, Lesner A, Walewska A, Zabłotna E, Rolka K, Wilusz T. Trypsin inhibitors from the garden four o'clock (Mirabilis jalapa) and spinach (Spinacia oleracea) seeds: isolation, characterization and chemical synthesis.  Phytochemistry. 2007;  68 1487-1496
• 9 Deshimaru M, Hanamoto R, Kusano C, Yoshimi S, Terada S. Purification and characterization of proteinase inhibitors from wild soja (Glycine soja) seeds.  Biosci Biotechnol Biochem. 2002;  66 1897-1903
• 10 Wong R C, Fong W P, Ng T B. Multiple trypsin inhibitors from Momordica cochinchinensis seeds, the Chinese drug mubiezhi.  Peptides. 2004;  25 163-169
• 11 Oliveira A S, Migliolo L, Aquino R O, Ribeiro J K, Macedo L L, Andrade L B, Bemquerer M P, Santos E A, Kiyota S, de Sales M P. Purification and characterization of a trypsin-papain inhibitor from Pithecelobium dumosum seeds and its in vitro effects towards digestive enzymes from insect pests.  Plant Physiol Biochem. 2007;  45 858-865
• 12 Azarkan M, Dibiani R, Goormaghtigh E, Raussens V, Baeyens-Volant D. The papaya Kunitz-type trypsin inhibitor is a highly stable beta-sheet glycoprotein.  Biochim Biophys Acta. 2006;  1764 1063-1072
• 13 Cheung A H, Ng T B. Isolation and characterization of a trypsin-chymotrypsin inhibitor from the seeds of green lentil (Lens culinaris).  Protein Pept Lett. 2007;  14 859-864
• 14 Troncoso M F, Cerda Zolezzi P, Hellman U, Wolfenstein-Todel C. A novel trypsin inhibitor from Peltophorum dubium seeds, with lectin-like properties, triggers rat lymphoma cell apoptosis.  Arch Biochim Biophys. 2003;  411 93-104
• 15 Ng T B, Lam S K, Fong W P. A homodimeric sporamin-type trypsin inhibitor with antiproliferative, HIV reverse transcriptase-inhibitory and antifungal activities from wampee (Clausena lansium) seeds.  Biol Chem. 2003;  384 289-293
• 16 Macedo M L, de Sa C M, Freire M D, Parra J R. A Kunitz-type inhibitor of coleopteran proteases, isolated from Adenanthera Pavonina L seeds and its effect on Callosobruchus macclatus.  J Agric Food Chem. 2004;  52 2533-2540
• 17 Kennedy A R. The evidence for soybean products as cancer preventive agents.  J Nutr. 1995;  125 S733-S743
• 18 Fernandes A O, Banerji A P. The field bean protease inhibitor can effectively suppress 7, 12-dimethylbenz[a]anthracene-induced skin tumorigenesis in mice.  Cancer Lett. 1996;  104 219-224
• 19 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. 2004;  21 159-166
• 20 Troncoso M F, Biron V A, Longhi S A, Retegui L A, Wolfenskein-Todel C. Peltophorum dubium and soybean Kunitz-type trypsin inhibitors induce human Jurkat cell apoptosis.  Int J Immunopharmacol. 2007;  7 625-635
• 21 von Hofe E, Newberne P M, Kennedy A R. Inhibition of N-nitrosomethylbenzylamine-induced esophageal neoplasms by the Bowman-Birk protease inhibitor.  Carcinogenesis. 1991;  12 2147-2150
• 22 Tsoi A Y, Ng T B, Fong W P. Immunomodulatory activity of a chymotrypsin inhibitor from Momordica cochinchinensis seeds.  J Pept Sci. 2006;  12 605-611
• 23 Zhao M, Naude R J, Muramoto K, Oelofsen W. Purification and characterization of ostrich pancreatic secretory trypsin inhibitor.  Int J Pept Protein Res. 1996;  48 174-181
• 24 Yang L, Resnick M I, Marengo S R. A simple procedure for isolating microgram quantities of biologically active bikunin from human urine.  BJU Int. 2005;  96 647-653
• 25 Kobayashi H, Suzuki M, Hirashima Y, Terao T. The protease inhibitor bikunin, a novel anti-metastatic agent.  Biol Chem. 2003;  284 749-754
• 26 Ho V SM, Ng T B. A Bowman-Birk trypsin inhibitor with antiproliferative activity from Hokkaido black soybeans.  J Pept Sci. 2008;  14 278-282
• 27 Ye X Y, Wang H X, Ng T B. Sativin; a novel antifungal miraculin like protein isolated from legumes of the sugar snap Pisum sativum var. macro carpon.  Life Sci. 2000;  67 775-781
• 28 Nielsen T B, Reynolds J A. Measurements of molecular weights by gel electrophoresis.  Methods Enzymol. 1978;  48 3-10
• 29 Zhang X, Wang H X, Ng T B. Isolation and characterization of a novel trypsin inhibitor from fresh lily bulbs.  Planta Med. 2008;  74 546-550
• 30 Wang H X, Ng T B. Concurrent isolation of a Kunitz-type trypsin inhibitor woth antifungal activity and a nivel lectin from Pseudostellaria heterophylla roots.  Biochem Biophys Res Commun. 2006;  342 349-353
• 31 Bottcher M, Grosse F. HIV‐1 protease inhibits its homologous reverse transcriptase by protein-protein interaction.  Nucleic Acids Res. 1997;  25 1709-1714
• 32 Wang H, Ng T B. 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 Biol. 2006;  38 224-243