Crystalline Papain.
Creators: name: Kimmel, Joe R
Publisher: name: University of Utah
Year: 2012
Subjects: Biochemistry
Recrystallization

description: Papain, a proteolytic enzyme found in the latex of the green fruit of the tropical papaw, Carica papaya, has been known for any years.  It has a long and illustrious history and figured prominently in the development of the concept of sulfhydryl enzymes.  The latex of the green fruit when dried has many commercial uses because of its proteolytic activity.  Therefore, dried papaya latex is readily available. In the past it has not been possible to prepare crystalline papain with dried latex as the starting material, but the crystalline enzyme had been prepared from fresh papaya latex.  This dissertation describes a method whereby crystalline papain can be prepared in good yield from certain sources of commercial dried papaya latex. Crystalline papain was found to require both a metal-chelating agent (e.g. Versene) and a reducing agent (e.g. cysteine) for maximal activation.  Specific ion effects on papain activity were noted in the absence of Versene.  The optimum pH for hydrolysis of alpha-benzoyl-L-argininamide by papain extends from pH 5.0 to 7.5.  Like the crystalline proteinase from pancreas, papain has esterase action on tosyl-L-arginine methyl ester.  The pH optimum for ester hydrolysis is similar to that for the hydrolysis of the amide substrate.  Papain is inhibited by iodoacetamide and p-chloromercuribenzoate but not by diisoprophylfluorophosphate.  Crystalline papain has a broad specificity since it hydrolyzes a variety of acyl and un-substituted amino acids amides. The most sensitive substrate yet found is alpha-benzoyl-L-argininamide.  Amides of N-substituted aromatic amino acids are hydrolyzed very slowly.  Free dipeptides are resistant but tri-peptides are hydrolyzed slowly.  Carbobenzoxy-L-isoglutamine is split but strong product inhibition is observed.  Carbobenzoxy-L-glutamic acid acts as an inhibitor for all types of substrates studied.  This indicates that the same active center on the enzyme is responsible for the broad action of the enzyme.  Crystalline papain is about three times as active on urea-denatured hemoglobin as crystalline trypsin.  A crystalline mercury derivative of papain has been prepared.  This derivative contains 1 gram atom of mercury per two moles of papain.  The molecular weight of papain calculated from the mercury content of mercuripapain is 21,700.  Mercuripapain is inactive but is fully activated in the presence of cysteine and Versene. The amino acid composition of crystalline papain has been determined.  The protein has a high content of tyrosine and glycine, 1 histidine residue per mole and no methionine.  The molecular weight calculated from the amino acid composition of 20,300.  The N-terminal amino acid sequence of papain is isoleucylprolylglutamic acid.  There is one N-terminal residue per 23,900 grams of papain indicating that the molecule is a single peptide chain.  On the basis of the amino acid composition this chain is 182 amino acid residues long.  Since sulfhydryl groups appear to be essential for papain action, some of the amino acid sequences containing cysteine were identified.  This was done with per-formate-oxidized   papain.  The sequences indentified were serylcystein acid, aspartylcysteic acid, valylcysteic acid, and possible glycylprolycysteic acid.  Physical studies with crystalline papain indicate that the material is in a high state of purity.  Mono-dispersity is the ultracentrifuge and the electrophoresis apparatus are observed only at pH 4.  At this pH the sedimentation constant (s20,w) is 2.42 Sveberg units; the diffusion constant is 10.27 X 10[-7] sq. cm per second.  The partial specific volume (V) was calculated from the amino acid composition and was found to be 0.724.  These values were used to calculate the molecular weight and yielded the figure 20,700.  Electrophoresis of papain indicated an isoelectric point at pH 8.75.  The mobility curve has a broad zone between pH 3.9 and 6.0 over which the mobility remains constant.  Comparison of this curve with that of insulin and lysozyme suggests that most of the free carboxyl groups of papain and lysozyme are those of aspartyl residues and that all, or nearly all, of the glutamyl residues are in the amide form.  Oxidized papain exists in aqueous solution as a mixture of monomeric and dimeric forms.  The relative amounts being a function of the protein concentration.  At pH 8, mercuripapain contains a mixture of monomers and a heavy aggregate apparently containing 6 moles of papain and 3 moles o mercury.  The relative amounts of the two forms are dependent upon the protein concentration, the aggregate dissociating to monomers on dilution.  Ordinary papain appears to contain trace metal impurities which also cause aggregation at pH 8.  All of at the physical and chemical evidence accumulated indicated that crystalline papain is essentially homogeneous.  There is no data to suggest any gross contamination with protein impurities.  Furthermore, these same studies all give values for the molecular weight that are in good agreement with one another.  Therefore, it is possible to characterize papain as a protein consisting of 182 amino acid residues arranged in a single peptide chain.  This protein has a molecular weight of about 20.500.  It hydrolyzes many peptide bonds and one or more free thiol groups on the protein molecule are necessary for this action.