Ostrich trypsinogen: Purification, kinetic properties and characterization of the pancreatic enzyme

Mark D. Bodley, Ryno J. Naudé, Willem Oelofsen, A. Pátthy

Research output: Contribution to journalArticle

6 Citations (Scopus)

Abstract

Trypsinogen is a serine protease zymogen (EC. 3.4.21.4) which has proved to be of key significance in a family of about 20 structurally and functionally related pancreatic digestive enzymes. This study was an endeavour to isolate, purify and characterize a stable form of ostrich trypsinogen, which has thus far not yet been accomplished. Trypsinogen (anionic) was isolated and purified by alkaline extraction of pancreatic acetone powder, followed by Toyopearl DEAE 650M, hydroxylapatite and LBTI-Sepharose affinity chromatography. The enzyme was chemically, physically and kinetically characterized, using amidase and esterase activity and spectrofluorometric determinations. Effects of CaCl2 and pH, among others, were examined. Purification of homogeneous anionic ostrich trypsinogen was achieved. Immunochemical analysis and spectrofluorometric reaction with sulphonyl-Ala-Ala-Pro-Arg-7-amino-4-methylcoumarin indicated trypsin-free ostrich trypsinogen, with an average Mr of 23,016 and a pI of 4.93. N-terminal sequence data revealed an unique activation peptide sequence, VPGDADDDK. Certain concentrations of Ca2+ enhanced trypsinogen activation, whilst others appeared to have the opposite effect. The kcat Km values obtained at different pHs, using Nα-benzoyl-dl-arginine-p-nitroanilide, p-toluenesulphonyl-arginine-methylester and p-toluenesulphonyl-lysine-methylester, followed the pH profile activity trend closely, with maximum catalytic activity at about pH 8 for both ostrich and bovine activated trypsinogen. Ostrich trypsin has significantly higher amidase activity than bovine trypsin, while esterase activities of the two enzymes have an inverse ratio. Kinetic pKa values were 7.2 and 7.4 for ostrich and bovine activated trypsinogens, respectively. The existence of ostrich trypsinogen in a now homogeneous stable form, free of autocatalytic inducing impurities, together with its characterization scenario will hopefully make a significant contribution to the field of comparative biochemistry. This study also confirms that ostrich trypsinogen is closely related to its serine protease counterparts.

Original languageEnglish
Pages (from-to)719-728
Number of pages10
JournalInternational Journal of Biochemistry and Cell Biology
Volume27
Issue number7
Publication statusPublished - Jul 1995

Fingerprint

Struthioniformes
Trypsinogen
Purification
Kinetics
Enzymes
amidase
Trypsin
Serine Proteases
Esterases
Arginine
Chemical activation
Affinity chromatography
Agarose Chromatography
Biochemistry
Enzyme Precursors
Durapatite
Acetone
Affinity Chromatography
Powders
Sepharose

Keywords

  • Activation
  • Immunochemical
  • Ostrich trypsinogen
  • Purification

ASJC Scopus subject areas

  • Cell Biology
  • Biochemistry

Cite this

Ostrich trypsinogen : Purification, kinetic properties and characterization of the pancreatic enzyme. / Bodley, Mark D.; Naudé, Ryno J.; Oelofsen, Willem; Pátthy, A.

In: International Journal of Biochemistry and Cell Biology, Vol. 27, No. 7, 07.1995, p. 719-728.

Research output: Contribution to journalArticle

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AU - Pátthy, A.

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