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Purification and Characterization of Hieronymain III. Comparison with Other Proteases Previously Isolated from Bromelia hieronymi Mez

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Abstract

A new proteolytic enzyme, named hieronymain III, has been purified by ion-exchange chromatography from unripe fruits of Bromelia hieronymi Mez. The new peptidase belongs to the cysteine catalytic type, as well as hieronymain I and II, the other two peptidases previously isolated from this species. Hieronymain III showed optimum alkaline pH range (8.6–9.3) and the molecular mass (MALDI-TOF) was 23713 Da. The N-terminal sequence (AVPQSIDWRRYGAVTTSRNQG) exhibited a higher percentage identity with hieronymain II (93%) than with hieronymain I (71%). The three peptidases showed notable differences on synthetic substrates degradation: whereas hieronymain III was the only one able to hidrolyze Z-Arg-Arg-p-nitroanilide, hieronymain I and II could degrade Z-Phe-Arg-p-nitroanilide; on the other hand, PFLNA was only split by hieronymain I. Finally, the three proteases showed different preferences on N-α-CBZ-p-nitrophenyl aminoacid ester substrates. From a biotechnological point of view, cleavage specificity differences are significant enough to use these enzymes as potential tools in that area.

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Abbreviations

AMPSO:

3-[(1, 1-dimethyl-2-hydroxyethyl)amino]-2-hydroxy-propanesulfonic acid

BLAST:

Basic local alignment search tool

CAPS:

3-(ciclohexylamino)-1-propanesulfonic acid

CBZ:

Carbobenzoxy

DMSO:

Dimethyl sulfoxide

DTT:

Dithiothreitol

E-64:

trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane

EDTA:

Ethylendiaminetetraacetic acid

IEF:

Isoelectric focusing

FPLC:

Fast protein liquid chromatographay

MALDI-MS TOF:

Matrix-assisted laser desorption/ionization mass spectrometry time of flying

MES:

2-(N-morpholino)ethanesulfonic acid

MOPS:

3-(N-morpholino)propanesulfonic acid

PFLNA:

p-Glu-Phe-Leu-p-nitro-anilide

Q-Sepharose HP:

Quaternary sepharose high performance

SDS-PAGE:

Sodium dodecyl sulfate polyacrylamide gel electrophoresis

SP-Sepharose HP:

Sulphopropyl sepharose high performance

TAPS:

N-tris (hydroxymethyl) methyl-3-aminopropanesulfonic acid

TFA:

Trifluoroacetic acid

Z:

Benzyloxycarbonyl

References

  1. Barrett AJ, Rawlings ND, Woessner JF (2004) In: Handbook of proteolytic enzymes, 2nd edn. Academic Press, London, UK, pp 1051–1204

    Google Scholar 

  2. Murachi T, Yamazaki M (1970) Biochemistry 9:1935–1938

    Article  CAS  Google Scholar 

  3. Rowan AD, Buttle DJ (1994) Method Enzymol 244:555–568

    Article  CAS  Google Scholar 

  4. Vallés D, Furtado S, Cantera AMB (2007) Enzyme Microb Tech 40:409–413

    Article  CAS  Google Scholar 

  5. Pardo MF, López LMI, Caffini NO, Natalucci CL (2001) Biol Chem Hoppe Seyler 382:871–874

    Article  CAS  Google Scholar 

  6. Cabral H, Leopoldino AM, Tajara EH, Greene LJ, Faça VM, Mateus RP, Ceron CR, Judice WAS, Julianod L, Bonilla-Rodriguez GO (2006) Protein Pept Lett 13:83–89

    Article  CAS  Google Scholar 

  7. Bruno MA, Pardo MF, Caffini NO, López LMI (2003) J Protein Chem 22:127–134

    Article  CAS  Google Scholar 

  8. Bruno MA, Trejo SA, Avilés FX, Caffini NO, López LMI (2006) Protein J 25:224–231

    Article  CAS  Google Scholar 

  9. Abreu Payrol J, Obregón WD, Natalucci CL, Caffini NO (2005) Fitoterapia 6:540–548

    Article  CAS  Google Scholar 

  10. Abreu Payrol J, Obregón WD, Trejo SA, Caffini NO (2007) Protein J 27:88–96

    Article  CAS  Google Scholar 

  11. López LMI, Sequeiros C, Natalucci CL, Brullo A, Maras B, Barra D, Caffini NO (2000) Protein Expr Purif 18:133–140

    Article  CAS  Google Scholar 

  12. López LMI, Sequeiros C, Trejo SA, Pardo MF, Caffini NO, Natalucci CL (2001) Biol Chem Hoppe-Seyler 382:875–878

    Article  Google Scholar 

  13. Harrach T, Eckert K, Schulze-Forster K, Nuck R, Grunow D, Maurer HR (1995) J Protein Chem 14:41–52

    Article  CAS  Google Scholar 

  14. Napper AD, Bennett SP, Borowski M, Holdridge MB, Leonard MJ, Rogers EE, Duan Y, Laursen RA, Reinhold B, Shames SL (1994) Biochem J 301:727–735

    CAS  Google Scholar 

  15. Rowan AD, Buttle DJ, Barrett AJ (1988) Archiv Biochem Biophys 267:262–270

    Article  CAS  Google Scholar 

  16. Ota S, Muta E, Katanita Y, Okamoto Y (1985) J Biochem 98:219–228

    CAS  Google Scholar 

  17. Rowan AD, Buttle DJ, Barrett AJ (1990) Biochem J 266:869–875

    CAS  Google Scholar 

  18. Silverstein RM (1974) Anal Biochem 62:478–484

    Article  CAS  Google Scholar 

  19. Carter CE, Marriage H, Goodenough PW (2000) Biochemistry 39:11005–11013

    Article  CAS  Google Scholar 

  20. Lee KL, Albee KL, Bernasconi RJ, Edmunds T (1997) Biochem J 327:199–202

    CAS  Google Scholar 

  21. Wiederanders B (2003) Acta Biochim Pol 50:691–713

    CAS  Google Scholar 

  22. Grudkowska M, Zagdańska B (2004) Acta Biochim Pol 51:609–624

    CAS  Google Scholar 

  23. Walsh G (2002) Proteins Biochemistry and Biotechnology. John Wiley and Sons Ltd, West Sussex, England, p 420

    Google Scholar 

  24. Haileselassie SS, Lee BH, Gibbs BF (1999) J Dairy Sci 82:1612–1617

    Article  CAS  Google Scholar 

  25. Silva SV, Malcata FX (2005) Int Dairy J 15:1–15

    Article  CAS  Google Scholar 

  26. Perpetuo EA, Juliano L, Lebrun I (2003) J Prot Chem 22:601–606

    Article  CAS  Google Scholar 

  27. Torres-Llanez M, Vallejo-Cordova B, González-Córdova A (2005) Arch Latinoam Nutr 55:111–117

    Google Scholar 

  28. Bruno MA, Pardo MF, Caffini NO, López LMI (2002) Acta Farm Bonaerense 21:51–56

    CAS  Google Scholar 

  29. Natalucci CL, Priolo NS, Buttazzoni MS, Caffini NO (1985) Acta Farm Bonaerense 4:93–98

    CAS  Google Scholar 

  30. Anderson JW (1968) Phytochemistry 7:1973–1988

    Article  CAS  Google Scholar 

  31. Bradford MB (1976) Anal Biochem 72:248–254

    Article  CAS  Google Scholar 

  32. Natalucci CL, Brullo A, López LMI, Hilal RM, Caffini NO (1996) J Food Biochem 19:443–454

    Article  CAS  Google Scholar 

  33. Salvesen GS, Nagase H (2001) In: Beynon R, Bond JS (eds) Proteolytic Enzymes, 2nd edn. Oxford University Press, Oxford, pp 116–119

  34. Filippova IY, Lysogorskaya EN, Oksenoit ES, Rudenskaya GN, Stepanov VM (1984) Anal Biochem 143:293–297

    Article  CAS  Google Scholar 

  35. Laemmli UK (1970) Nature 227:680–685

    Article  CAS  Google Scholar 

  36. Barrett AJ, Kirschke H (1981) Method Enzymol 80:535–561

    Article  CAS  Google Scholar 

  37. Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ (1997) Nucleic Acids Res 25:3389–3402

    Article  CAS  Google Scholar 

  38. Rawlings ND, Barrett AJ (1994) Method Enzymol 244:461–486

    Article  CAS  Google Scholar 

  39. Ritonja A, Buttle DJ, Rawlings ND, Turk V, Barrett AJ (1989) FEBS Lett 258:109–112

    Article  CAS  Google Scholar 

  40. Muta E, Okamoto Y, Ota S (1994) DDBJ, Accession D38531, (Direct Submission)

  41. Taylor MA, Al-Sheikh M, Revell DF, Sumner IG, Connerton IF (1999) Plant Sci 145:41–47

    Article  CAS  Google Scholar 

  42. Kyndt T, Van Damme EJ, Van Beeumen J, Gheysen G (2007) FEBS J 274:451–462

    Article  CAS  Google Scholar 

  43. Ohyanagi H, Tanaka T, Sakai H, Shigemoto Y, Yamaguchi K, Habara T, Fujii Y, Antonio BA, Nagamura Y, Imanishi T, Ikeo K, Itoh T, Gojobori T, Sasak T (2006) Nucleic Acids Res 34:D741–D744

    Article  CAS  Google Scholar 

  44. Walreavens V, Jaziri M, van Beeumen J, Schnek AG, Kleinschmidt T, Looze Y (1993) Biol Chem 374:501–506

    CAS  Google Scholar 

  45. Ling JQ, Kojima T, Shiraiwa M, Takahara H (2003) Biochim Biophys Acta 1627:129–139

    CAS  Google Scholar 

  46. Funk V, Kositsup B, Zhao C, Beers EP (2002) Plant Physiol 128:84–94

    Article  CAS  Google Scholar 

  47. Sakuta C, Oda A, Konishi M, Yamakawa S, Kamada H, Satoh S (2001) Biosci Biotechnol Biochem 65:2243–2248

    Article  CAS  Google Scholar 

  48. Jones ML, Chaffin GS, Eason JR, Clark DG (2005) J Exp Bot 56:2733–2744

    Article  CAS  Google Scholar 

Download references

Acknowledgments

N. O. Caffini is member of the CICPBA Researcher Career; L. M. I. López is member of the CONICET Researcher Career; M. A. Bruno is UNLP fellow. The present work was supported by grants from ANPCyT (PICT 38088), CONICET (PIP 5931), CYTED (Project IV.22), CICPBA (Res. 527/06), and University of La Plata, Argentina (Project X-445). Authors wish thank to Elisa F. Villagra and Adrián Ojeda for very helpful technical assistance.

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Authors and Affiliations

  1. Laboratorio de Investigación de Proteínas Vegetales (LIPROVE), Departamento de Ciencias Biológicas, Facultad de Ciencias Exactas, Universidad Nacional de La Plata, C.C. 711, La Plata, B1900AVW, Argentina

    Mariela A. Bruno, Néstor O. Caffini & Laura M. I. López

  2. Institut de Biotecnología i de Biomedicina, Universidad Autònoma de Barcelona, Campus Universitari, Bellaterra, Cerdanyola del Vallès, Barcelona, 08193, Spain

    Sebastián A. Trejo

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  1. Mariela A. Bruno
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  2. Sebastián A. Trejo
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  3. Néstor O. Caffini
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  4. Laura M. I. López
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Correspondence to Mariela A. Bruno.

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Bruno, M.A., Trejo, S.A., Caffini, N.O. et al. Purification and Characterization of Hieronymain III. Comparison with Other Proteases Previously Isolated from Bromelia hieronymi Mez. Protein J 27, 426–433 (2008). https://doi.org/10.1007/s10930-008-9152-1

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