The Thiol Proteinases from the Latex ofCarica papayaL. I. Fractionation, Purification and Preliminary Characterization

Abstract

Three thiol proteinases, namely papain, chymopapain and proteinase Ω were purified to homogeneity from the latex of Carica papaya L. During the purification procedure, the thiol function of the cysteinyl residues were protected either as mixed disulfides with cysteamine or 2-thiopyridone or as iS-sulphenylthiosulfate derivative or after blocking with pchloromercuribenzoic acid. In marked contrast with earlier publications, chymopapain also was found to be a monothiol proteinase as papain and proteinase Ω. The active sites of chymopapain and proteinase Ω could not be distinguished from that of papain neither by the analysis of the pH dependence of Κςχ/Κη nor by the examination of the pH dependence of the fluorescence emission spectra. Die Thiol-Proteinasen aus dem Milchsaft vonCarica papaya L. I. Fraktionierung, Reinigung und vorl ufige Charakterisierung Zusammenfassung: Aus dem Milchsaft des Melonenbaumes (Carica papaya L.) wurden die drei Thiol-Proteinasen Papain, Chymopapain und Proteinase Ω isoliert und bis zur Einheitlichkeit gereinigt. W hrend der Aufarbeitung wurde die ThiolFunktion der Cysteinreste gesch tzt, und zwar durch Umwandlung in gemischte Disulfide mittels Cysteamin, 2-Thiopyridon, S-Sulphenylthiosulfat-Derivate oder durch Umsetzung mit p-Chloromercuribenzoes ure. Im Gegensatz zu fr heren Ver ffentlichungen erwies sich Chymopapain ebenfalls als Monothiol-Proteinase wie Papain und Proteinase Ω. Die aktiven Zentren von Chymopapain und Proteinase Ω konnten weder durch die pHAbh ngigkeit der kau/Km -Werte noch durch ihre Fluoreszenz-Emissionsspektren vom aktiven Zentrum des Papains unterschieden werden.

Molecular Cloning and Functional Expression of cDNA Encoding a Cysteine Proteinase Inhibitor, Cystatin, from Job's Tears (Coix lacryma-jobiL. var.Ma-yuenStapf)

“…The method for assaying proteinase activity 23) was modied as follows. The sample to be analyzed (0.2 ml) was incubated at 379 C for 15 min with 0.2 ml of papain solution (0.1 mg W ml) and 1.4 ml of an activation buŠer that contained 100 mM phosphate in addition to 2.5 mM dithiothreithol and 10 mM EDTA (pH 6.8).…”

Purification and In Situ Immobilization of Papain with Aqueous Two-Phase System

“…The release of p -nitroaniline was determined spectrophotometrically at 410 nm using a ε410 = 8800 M −1 cm −1 . x was chosen so that ΔA 410 never exceeded 1.0 after 15 min [26], [27]. One unit of activity (nkat) is the amount of proteinase (free, immobilized or CLEAs) that hydrolyses one nmol of substrate per second under the abovementioned conditions.…”

Purification and preliminary characterization of latex proteases of Vasconcellea candicans (A. Gray) A. DC (Mito)

“…Estos resultados concuerdan con otras investigaciones donde al purificar papaína de papaya encontraron diferentes bandas de proteínas como 27 kDa, 30 kDa, 33 kDa (Kyndt et al, 2005), papaína 23 kDa y quimopapaína 27 kDa (Arnon, 1970), caricain 24 kDa (Dubois et al, 1988), CC-IV 28 kDa (Gravina et al, 1994), CC23 23 kDa (Pereira et al, 2001), CCIb y CMS1MS2 de 22,99 kDa (Walraevens et al, 1999;Gomes et al, 2008). La microheterogeneidad observada durante las etapas de purificación indica que hay múltiples isoformas de las enzimas proteolíticas en el látex de V. candicans, lo cual también fue reportado para V. heilbornii (Badillo, 2000) y para V. cundinamarcensis (Walraevens et al, 1999).…”