Biochemical characterization of a cysteine proteinase from Bauhinia forficata leaves and its kininogenase activity

In this work, the proteinase activity detect in the acetone precipitate (80%, v/v) of B. forficata leaves, trivially known as cow paw, and popularly used in folk medicine for treatment of diabetes mellitus, was purified by chromatography on Sephadex G-25, Canecystatin-Sepharose, and on Con A-Sepharo...

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Bibliographic Details
Published in:Process biochemistry (1991) 2011-02, Vol.46 (2), p.572-578
Main Authors: Andrade, Sheila S., Silva-Lucca, Rosemeire A., Santana, Lucimeire A., Gouvea, Iuri E., Juliano, Maria A., Carmona, Adriana K., Araújo, Mariana S., Sampaio, Misako U., Oliva, Maria Luiza V.
Format: Article
Language:English
Subjects:
acetone
Bauhinia
Bauhinia forficata
Biochemistry
bradykinin
chromatography
Circular dichroism
cows
Cysteine
Cysteine proteinase
diabetes mellitus
EDTA
EDTA (chelating agent)
energy transfer
enzyme activity
Enzymes
fluorescence
humans
Kininogen
Leaves
Molecular weight
Papain
Plant enzyme
polyacrylamide gel electrophoresis
Protein purification
Proteinase
proteolysis
Substrate specificity
sugarcane
traditional medicine
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Summary:In this work, the proteinase activity detect in the acetone precipitate (80%, v/v) of B. forficata leaves, trivially known as cow paw, and popularly used in folk medicine for treatment of diabetes mellitus, was purified by chromatography on Sephadex G-25, Canecystatin-Sepharose, and on Con A-Sepharose. The molecular weight 30kDa was estimated by SDS-PAGE and zymography, and the N-terminal sequence and CD spectra indicated a relationship with the papain family of cysteine proteinases. Denominated baupain, the enzyme was activated by dithiotreitol and inhibited by E-64 and iodoacetamide, but not by benzamidine, TLCK, TPCK and EDTA. The S2 and S1 substrate specificity of baupain, assayed with two series of fluorescence resonance energy transfer (FRET) peptide substrates derived from Abz-KLRSSK-Q-EDDnp, indicates a preference for Phe and Tyr at P2 position over Leu found in papain. Baupain releases bradykinin from HMWK (human high molecular weight kininogen) though its proteolytic activity is blocked by the sequence motif QVVA of kininogen (Kiapp=1.9×10−8M). Canecystatin, from sugar cane, which also lodges the QVVA sequence, inhibits baupain (Kiapp=0.18×10−9M).
ISSN:1359-5113
1873-3298
DOI:10.1016/j.procbio.2010.10.008