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

Abstract

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 30 kDa 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 (K iapp = 1.9 × 10 −8 M). Canecystatin, from sugar cane, which also lodges the QVVA sequence, inhibits baupain (K iapp = 0.18 × 10 −9 M).

Unfolding Studies of the Cysteine Protease Baupain, a Papain-Like Enzyme from Leaves of Bauhinia forficata: Effect of pH, Guanidine Hydrochloride and Temperature

“…This classification was corroborated by the results of CDPro estimation (44% α-helix and 16% β-sheet) and spectrum sharpness (with the band at 210 nm slightly more intense than that at 222 nm), which is similar to the papain spectrum that shows two bands (208 and 222 nm) in neutral pH [12]. Together with biochemical data obtained previously [1], the current results allow us to classify baupain in the clan CA of the papain family.…”

“…This is similar to the papain spectrum that shows two bands at 222 and 208 nm, characteristic of an α-helix structure [10]. Analysis of secondary structure content using the CDPro software package with the SELCON3, CONTINLL, CDSSTR programs yielded 44% α-helix, 16% β-sheet, 12% β-turn, and 28% unordered structures, with a standard deviation of 2% [1]. Cluster analysis showed that baupain belongs to the α+β tertiary structure class, like other cysteine proteases, for example procerain and bromelain [8,11].…”

“…The results of baupain characterization showed that it is a 23,000 Da single polypeptide chain protein, active at neutral pH, and belonging to the CA clan of the papain family. Our findings also showed that baupain has kininogenase activity [1] and blocks thrombin-induced human platelet aggregation, possibly by unspecific cleavage of protease activated receptors 1 (PAR1) [2].…”

“…Baupain was purified as described by Andrade et al [1]. Briefly, the proteins were precipated with acetone from crude extract (7%) and applied on a size exclusion Sephadex G-25 column followed by canecystatin-Sepharose and Con A Sepharose chromatographies.…”

“…We have previously reported the biochemical characterization of a cysteine protease from Bauhinia forficata leaves, named baupain [1]. Bauhinia forficata leaves extract is used in folk therapy in Brazil as an infusion for diabetes mellitus.…”