Identification and characterization of proteases from skin mucus of tambacu, a Neotropical hybrid fish

  • C. M. C. Salles
  • P. Gagliano
  • S. A. T. Leitão
  • J. B. Salles
  • H. L. M. Guedes
  • V. P. F. Cassano
  • S. Giovanni De-Simone
Article

Abstract

Skin secretions of fishes constitute a rich source of proteins with a broad spectrum of antimicrobial properties. We report here the characterization of proteases from skin mucus of tambacu, an economically important Neotropical hybrid fish. The effects of pH on the proteolytic activities of the mucus acting on various substracts – hemoglobin, casein, bovine serum albumin (BSA) and ovalbumin (OVA) – were tested. Optimal pH values for protease activity on hemoglobin were 4.5 and 8.5, on casein, 8.5, on BSA, 5.0 and 7.5, and on ovalbumin, 4.5 and 6.5. The proteolytic activity was inhibited on all of these substrates in the presence of specific inhibitors: caseinolytic activity was inhibited by inhibitors of serine and metalloproteases; hemoglobinolytic activity was inhibited by serine, aspartic and metalloproteases inhibitors; albuminolytic activity on BSA was inhibited by serine and aspartic proteases inhibitors, and on ovalbumin, by cysteine and aspartic proteases inhibitors. Gelatin zymography revealed that the skin mucus of tambacu consisted primarily of serine and metalloproteases. Hemoglobin zymography showed one proteolytic band inhibited by EDTA, whereas casein zymography showed two proteases inhibited by serine proteases inhibitors. We were able to identify all classes of proteases in the mucus from the skin of tambacu. These, and these results suggest that the proteolytic activities of the skin mucus of fish may play an important role in the defense against microorganisms and ectoparasites.

Keywords

Enzymatic specificity Protease Skin mucus Tambacu 

Abbreviations

ACA

6-Amino-caproic acid

CEPTA/IBAMA

Centro de Pesquisas de Peixes Neotropicais

E-64

l-trans-3-Carboxyoxiran-2-carbonyl-l-leucylagmatine

EDTA

Ethylenediaminetetraacetic acid

pHMB

p-Hydroxy-mercurybenzoate

PMSF

Phenylmethylsulfonyl fluoride

TLCK

N-Tosyl-lysine chloromethyl ketone

TPCK

N-Tosyl-l-phenylalanine chloromethyl ketone

Notes

Acknowledgements

We are grateful to Dr. Jayme Bastos (Biochemistry Department, UERJ-University of the State of Rio de Janeiro, Brazil) for the donation of tambacu from Morro Grande Farm. This work was supported by CNPq, Faperj and Oswaldo Cruz Foundation – Fiocruz.

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Copyright information

© Springer Science+Business Media, Inc. 2007

Authors and Affiliations

  • C. M. C. Salles
    • 1
  • P. Gagliano
    • 2
  • S. A. T. Leitão
    • 4
  • J. B. Salles
    • 3
  • H. L. M. Guedes
    • 2
  • V. P. F. Cassano
    • 4
  • S. Giovanni De-Simone
    • 2
  1. 1.Laboratório de Química e Função de Proteínas e PeptídeosCentro de Biociências e Biotecnologia/Universidade Estadual do Norte FluminenseCampos dos GoytacazesBrazil
  2. 2.Laboratório de Bioquímica de Proteínas e PeptídeosDBBM/IOC/FIOCRUZManguinhosBrazil
  3. 3.Laboratório de BiotecnologiaCBB/UENFCampos dos GoytacazesBrazil
  4. 4.Laboratório de Bioquímica ToxicológicaIBRAG/UERJRio de JaneiroBrazil

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