Journal of Protein Chemistry

, Volume 15, Issue 2, pp 177–184 | Cite as

Trypsin inhibitors from ridged gourd (Luffa acutangula Linn.) seeds: Purification, properties, and amino acid sequences

  • Umesh C. Haldar
  • Sanat K. Saha
  • Ronald C. Beavis
  • Nirmal K. Sinha


Two trypsin inhibitors, LA-1 and LA-2, have been isolated from ridged gourd (Luffa acutangula Linn.) seeds and purified to homogeneity by gel filtration followed by ion-exchange chromatography. The isoelectric point is atpH 4.55 for LA-1 and atpH 5.85 for LA-2. The Stokes radius of each inhibitor is 11.4 å. The fluorescence emission spectrum of each inhibitor is similar to that of the free tyrosine. The biomolecular rate constant of acrylamide quenching is 1.0×109 M−1 sec−1 for LA-1 and 0.8 × 109 M−1 sec−1 for LA-2 and that of K2HPO4 quenching is 1.6×1011 M−1 sec−1 for LA-1 and 1.2×1011M−1 sec−1 for LA-2. Analysis of the circular dichroic spectra yields 40%α-helix and 60%Β-turn for La-1 and 45%α-helix and 55%Β-turn for LA-2. Inhibitors LA-1 and LA-2 consist of 28 and 29 amino acid residues, respectively. They lack threonine, alanine, valine, and tryptophan. Both inhibitors strongly inhibit trypsin by forming enzymeinhibitor complexes at a molar ratio of unity. A chemical modification study suggests the involvement of arginine of LA-1 and lysine of LA-2 in their reactive sites. The inhibitors are very similar in their amino acid sequences, and show sequence homology with other squash family inhibitors.

Key words

Trypsin inhibitor physicochemical properties amino acid sequence sequence homology 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Birks, J. (1970).Photophysics of Aromatic Molecules, Wiley-Interscience, New York, pp. 433–447.Google Scholar
  2. Chase, T., Jr., and Shaw, E. (1967).Biochem. Biophys. Res. Commun. 29, 508–514.Google Scholar
  3. Ellman, G. L. (1959).Arch. Biochem. Biophys. 82, 70–77.PubMedGoogle Scholar
  4. Erlanger, B. F., Kokowsky, N., and Cohen, W. (1961).Arch. Biochem. Biophys. 95, 271–278.PubMedGoogle Scholar
  5. Favel, A., Mattras, H., Coletti-Previero, M. A. Zwilling, R., Robinson, E. A., and Castro, B. (1989).Int. J. Peptide Protein Res. 33, 202–208.Google Scholar
  6. Habeeb, A. F. S. A. (1966).Anal. Biochem. 14, 328–336.PubMedGoogle Scholar
  7. Hara, S., Makino, J., and Ikenaka, T. (1989).J. Biochem. 105, 88–92.PubMedGoogle Scholar
  8. Haynes, R., and Feeney, R. E. (1968).Biochemistry 7, 2879–2885.PubMedGoogle Scholar
  9. Hider, R. C., Drake, A. F., Morrison, I. E. G. Kupryszewski, G., and Wilusz, T. (1987).Int. J. Peptide Protein Res. 30, 397–403.Google Scholar
  10. Hirs, C. H. W. (1967).Meth. Enzymol. 11, 197–199.Google Scholar
  11. Hojima, Y., Pierce, V. J., and Pisano, J. J. (1982).Biochemistry 21, 3741–3746.PubMedGoogle Scholar
  12. Hummel, B. C. W. (1959).Can. J. Biochem. Physiol. 37, 1393–1399.PubMedGoogle Scholar
  13. Laskowski, M., Jr., and Kato, I. (1980).Annu. Rev. Biochem. 49, 593–626.PubMedGoogle Scholar
  14. Liu, T. Y., and Chang, Y. H. (1971).J. Biol. Chem. 246, 2842–2848.PubMedGoogle Scholar
  15. Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951).J. Biol. Chem. 193, 265–275.PubMedGoogle Scholar
  16. Miura, S., and Funatsu, G. (1995).Biosci. Biotech. Biochem. 59, 469–473.Google Scholar
  17. Ozawa, K., and Laskowski, M., Jr. (1966).J. Biol. Chem. 241, 3955–3961.PubMedGoogle Scholar
  18. Patthy, L., and Smith, E. L. (1975).J. Biol. Chem. 250, 557–564.PubMedGoogle Scholar
  19. Reisfeld, R. E., Lewis, U. J., and Williams, D. E. (1962).Nature 195, 281–283.PubMedGoogle Scholar
  20. Richardson, M. (1977).Phytochemistry 16, 159–169.Google Scholar
  21. Ryan, C. A. (1981).The Biochemistry of Plants, Academic Press, New York, pp. 351–370.Google Scholar
  22. Seigel, L. M., and Monty, K. J. (1966).Biochim. Biophys. Acta 112, 346–362.PubMedGoogle Scholar
  23. Swank, R. T., and Munkres, K. D. (1971).Anal. Biochem. 39, 462–477.PubMedGoogle Scholar
  24. Vesterberg, O., and Svenson, H. (1966).Acta Chem. Scand. 20, 820–834.PubMedGoogle Scholar
  25. Wieczorek, M., Otlewski, J., Cook, J., Parks, K., Leluk, J., Willimowska-Pelc, A., Polanowski, A., Wilusz, T., and Laskowski, M., Jr. (1985).Biochem. Biophys. Res. Commun. 126, 646–652.PubMedGoogle Scholar
  26. Yang, J. T., Chuen-Shang, and Hugo, M. M. (1986).Meth. Enzymol. 130, 208–215.PubMedGoogle Scholar

Copyright information

© Plenum Publishing Corporation 1996

Authors and Affiliations

  • Umesh C. Haldar
    • 1
  • Sanat K. Saha
    • 1
  • Ronald C. Beavis
    • 2
  • Nirmal K. Sinha
    • 1
  1. 1.Department of ChemistryBose InstituteCalcuttaIndia
  2. 2.Department of Pharmacology and Chemistry, Skirball InstituteNew York University Medical CenterNew York

Personalised recommendations