Trypsins from yellowfin tuna (Thunnus albacores) spleen: purification and characterization

Comp Biochem Physiol B Biochem Mol Biol. 2006 May;144(1):47-56. doi: 10.1016/j.cbpb.2006.01.006. Epub 2006 Feb 24.

Abstract

Two anionic trypsins (A and B) were purified to homogeneity from yellowfin tuna (Thunnus albacores) spleen by a series of column chromatographies including Sephacryl S-200, Sephadex G-50 and DEAE-cellulose. Purity was increased to 70.6- and 91.5-fold with approximately 2.8% and 15.6% yield for trypsin A and B, respectively. The apparent molecular weight of both trypsins was estimated to be 24 kDa by size exclusion chromatography and SDS-PAGE. Both trypsin A and B appeared as a single band on native-PAGE. Trypsin A and B exhibited the maximal activity at 55 and 65 degrees C, respectively, and had the same optimal pH at 8.5 using TAME as a substrate. Both trypsins were stable to heat treatment up to 50 degrees C and in the pH range of 6.0 to 11.0. Both trypsin A and B were stabilized by calcium ion. The activities were inhibited effectively by soybean trypsin inhibitor, TLCK and partially inhibited by EDTA, but were not inhibited by E-64, N-ethylmaleimide, iodoacetic acid, TPCK and pepstatin A. Activity of both trypsins continuously decreased with increasing NaCl concentration (0-30%). Apparent Km and Kcat of trypsin A and B for TAME were 0.2-0.33 mM and 66.7-80 S(-1), respectively. The N-terminal amino acid sequences of trypsin A, IVGGYECQAHSQPHQVSLNA, and trypsin B, IVGGYECQAHSQPPQVSLNA, indicated the high homology between both enzymes.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acids / analysis
  • Animals
  • Chromatography, DEAE-Cellulose
  • Electrophoresis, Polyacrylamide Gel
  • Hydrogen-Ion Concentration
  • Molecular Weight
  • Peptide Fragments / chemistry
  • Sequence Homology
  • Spleen / enzymology*
  • Trypsin / classification
  • Trypsin / isolation & purification*
  • Trypsin / metabolism
  • Trypsin Inhibitors / pharmacology
  • Tuna*

Substances

  • Amino Acids
  • Peptide Fragments
  • Trypsin Inhibitors
  • Trypsin