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Enzyme Manual: Papain

I.U.B.: 3.4.22.2
C.A.S.: 9001-73-4

Enzymatic Reaction (image will open in a new window)

Papain is a cysteine hydrolase that is stable and active under a wide range of conditions. It is very stable even at elevated temperatures (Cohen et al. 1986). The latex of Carica papaya is a rich source of four cysteine endopeptidases including papain, chymopapain, glycyl endopeptidase, and caricain. The proteins are synthesized as inactive precursors that become active within two minutes of the plant being wounded and the latex expelled. Papain is a minor constituent, but has been more widely studied because it is more easily purified (Azarkan 2003).

History:

In 1873 G.C. Roy first investigated the action of papain in an article published in the Calcutta Medical Journal entitled “The Solvent Action of Papaya Juice on Nitrogenous Articles of Food”. Papain was first named in the late nineteenth century by Wurtz and Bouchut who partially purified the product from the sap of papaya (Menard and Storer 1998). When named, it was simply recognized as a proteolytically active constituent in the latex of tropical papaya fruit (Wurtz and Bouchut 1879).

Throughout the mid-1950s and 1960s, purification and separation techniques improved greatly and pure papain was isolated. The study of papain allowed for great advances in understanding enzymes as proteins. In 1968, papain was the second enzyme to be crystallized and its structure determined by x-ray methods. Papain was the first cysteine protease to have its structure identified (Drenth et al. 1968).

In the 1980s, the geometry of the active site was reviewed and the three-dimensional structure was determined to a 1.65 Angstrom resolution (Kamphuis et al. 1984). The precursors and inhibitors of papain were extensively studied into the 1990s (Vernet et al. 1991).

Today’s research aims to further understand the specificity (Portaro et al. 2000) and the structural perturbations brought about by inhibitors, low pH, metal ions, and fluorinated alcohols (Alphey and Hunter 2006, Huet et al. 2006, Kaul et al. 2002, Naeem et al. 2004).

Specificity:

Papain has fairly broad specificity; it has endopeptidase, amidase, and esterase activities. The active site consists of seven subsites (S1-S4 and S1’-S3’) that can each accommodate one amino acid residue of a substrate (P1-P4 and P1’-P3’) (Schechter and Berger 1967). Specificity is controlled by the S2 subsite, a hydrophobic pocket that accommodates the P2 side chain of the substrate. Papain exhibits specific substrate preferences primarily for bulky hydrophobic or aromatic residues at this subsite (Kimmel and Smith 1954). Outside of the S2 subsite preferences, there is a lack of clearly defined residue selectivity within the active site.

Molecular Characteristics:

The mature forms of all papaya proteinases are between 212 and 218 amino acids, and exhibit a strong degree of homology (Azarkan 2003). X-ray structure analysis has shown that they adopt identical three-dimensional folds (Pickersgill et al. 1991, O’Hara et al. 1995, and Maes et al.1996).

Papain is synthesized as a zymogen with a 133 amino acid N-terminal region that is not part of the active enzyme (Cohen et al. 1986). The papain precursor gene, prepropapain, has been cloned and expressed either in parts or as a whole (Cohen et al. 1986 and Choudhury et al.2009). 

Composition:

Papain is a single-chained polypeptide with three disulfide bridges and a sulfhydryl group necessary for the activity of the enzyme. Papain is expressed as an inactive precursor, prepropapain. The formation of active papain requires several cleavage steps including an initial cleavage of the 18 amino acid preregion (the signal sequence), followed by further cleavage of the glycosylated 114 amino acid proregion (Vernet et al. 1995). This proregion serves as an intrinsic inhibitor and folding template. For further detail see Revell et al. 1993, Taylor et al. 1992, and Cohen et al. 1990. 

Protein Accession Number: P00784

CATH Classification (v. 3.2.0): 

  • Class: Alpha Beta
  • Architecture: Alpha-Beta Complex
  • Topology: Cathepsin B; Chain A

Molecular Weight: 

  • 23.4 kDa (Theoretical)

Optimal pH: 6.0-7.0

Isoelectric Point:

  • 8.88 (Theoretical)

Extinction Coefficient: 

  • 53,610 cm-1 M-1 (Theoretical)
  • E1%,280 = 22.88 (Theoretical)

Active Site Residues:

  • Cysteine (C158)
  • Histidine (H292)
  • Asparagine (N308)

Activators:

  • Cysteine
  • Sulfide and sulfite
  • Heavy metal chelating agents like EDTA
  • N-bromosuccinimide
(White and White 1997) 

Inhibitors:

  • PMSF
  • TLCK, TPCK
  • alph2-macroglobulin
  • Hg2+ and other heavy metals
  • AEBSF
  • Antipain
  • cystatin
  • E-64
  • Leupeptin
  • Sulfhydryl binding agents
  • Carbonyl reagents
  • Alkylating agents

         (White and White 1997)

 Applications:

  • Cell isolation where it is more gentle than other proteases (i.e.: cortical neurons, retina, and smooth muscle)
  • Protein structural studies, peptide mapping
  • Red cell surface modification for antibody screening or identification
  • Fab preparation from IgG and IgM antibodies
  • Solubilization of integral membrane proteins
  • Production of glycopeptides from purified proteoglycans
  • Enzymatic wound debridement

Assay

Method: A titrimetric determinatin of the acid produced during the hydrolysis of benzoyl-L-arginine ethyl ester (BAEE). One unit will hydrolyze one micromole of benzoyl-L-arginine ethyl ester per minute at 25°C and pH 6.2 under the specified conditions.

Reagents

  • Enzyme diluent (Activation buffer): Prepare fresh daily by mixing the following:

     

    0.01 M EDTA 10 ml
    0.06 M Mercaptoethanol 0.1 ml
    0.05 M Cysteine⋅HCl 10 ml
    Reagent grade water 70 ml
  • Substrate solution: Prepare fresh daily by mixing the following:

     

    0.058 M BAEE 15.0 ml
    0.01 M EDTA 0.8 ml
    0.05 M Cysteine⋅HCl 0.8 ml
  • Adjust pH to 6.2 and dilute to a final volume of 21 ml with reagent grade water.
  • Titrant: 0.01-0.02 N NaOH, standardized

Enzyme

Activate enzyme by dissolving in enzyme diluent to a concentration of 0.05-0.1 mg/ml. Under these conditions activation is complete within 30 minutes.

Determination of protein concentration

equation

Procedure

The reaction can be measured with either an automatic titrator or a laboratory pH meter. The titration vessel should be maintained at 25°C.

Pipette the following into the titration vessel at 25°C:

Substrate solution 5.0 ml
3.0 M NaCl 5.0 ml
Reagent grade water 5.0 ml

At zero time add 0.1 ml of appropriately diluted enzyme and adjust the pH to 6.2. Record the amount of standardized NaOH added per minute to maintain the pH at 6.2 after a constant rate is achieved.

Calculation

calculation

Technical note: Mercuripapain must be activated before use. Mercury is removed from the enzyme in activation buffer. After 30 minutes in this solution, the enzyme is completely activated and the mercury has been chelated. The mercuripapain suspension contains no free mercury. The product has been extensively dialyzed prior to packaging.

References

• Akahane, K., and Umeyama, H.: Binding Specificity of Papain and Cathepsin B , Enzyme 36, 141, 1986
 
• Alecio, M., Dann, M., and Lowe, G.: The Specificity of the S1 Subsite of Papain , Biochem J 141, 495, 1974
 
• Allen, G., and Lowe, G.: Investigation of the Active Site of Papain with Fluorescent Probes , Biochem J 133, 679, 1973
 
• Arnon, R.: Papain , Methods in Enzymology Vol. 19, G. Perlmann and L. Lorand, Academic Press, NY, 226, 1970
 
• Asboth, B., Stokum E., Khan, I., and Polgar, L.: Mechanism of Action of Cysteine Proteinases. Oxyanion Binding Site Is Not Essential in the Hydrolysis of Specific Substrates , Biochemistry 24, 606, 1985
 
• Azarkan, M., El Moussaoui A., van Wuytswinkel, D., Dehon, G., and Looze, Y.: Fractionation and Purification of the Enzymes Stored in the Latex of Carica papaya , J Chromatogr B Analyt Technol Biomed Life Sci 790, 229, 2003
 
• Balls, A., and Lineweaver, H.: Isolation and Properties of Crystalline Papain , J Biol Chem 130, 669, 1939
 
• Barrett, A., and Buttle, D.: Names and Numbers of Papaya Proteinases , Biochem J 228, 527, 1985
 
• Blumberg, S., Schechter, I., and Berger, A.: The Purification of Papain by Affinity Chromatography , Eur J Biochem 15, 97, 1970
 
• Brisson, J., Carey, P., and Storer, A.: Benzoylamidoacetonitrile Is Bound as a Thiomidate in the Active Site of Papain , J Biol Chem 261, 9087, 1986
 
• Brocklehurst, K., and Kierstan, M.: Propapain and Its Conversion to Papain: A New Type of Zymogen Activation Mechanism Involving Intramolecular Thiol-Disulfide Interchange , Nature New Biol 242, 167, 1973
 
• Brocklehurst, K., Kierstan, M., and Little, G.: The Reaction of Papain with Ellman's Reagent (5,5'-Dithiobis-2-nitrobenzoate Dianion) , Biochem J 128, 811, 1972
 
• Brubacher, L., and Bender, M.: The Preparation and Properties of Trans-Cinnamoyl-Papain , J Am Chem Soc 88, 5871, 1966
 
• Brubacher, L., and Bender, M.: The Reaction of Trans-Cinnamoyl-Papain with a Series of Polyglycinamides of Varying Chain Length , Biochem Biophys Res Commun 27, 176, 1967
 
• Campbell, P., and Kaiser, E.: The Reaction of A Cyclic Sulfonate Ester with the Sulfhydryl Proteolytic Enzyme Papain , Bioorg Chem 1, 432, 1971
 
• Carotti, A., Smith, R., Wong, S., Hansch, C., Blaney, J., and Langridge, R.: Papain Hydrolysis of X-Phenyl-N-methane-sulfonyl Glycinates: A Quantitative Structure-Activity Relationship and Molecular Graphics Analysis , Arch Biochem Biophys 229, 112, 1984
 
• Choudhury, D., Roy, S., Chakrabarti, C., Biswas, S., and Dattagupta, J.: Production and Recovery of Recombinant Propapain with High Yield , Phytochem 70, 465-72, 2009
 
• Clagett, J., and Englehard, W.: Immunosuppressive Activity of the 3-alpha-1 Fraction of Papain , J Appl Bacteriol 93, 1493, 1967
 
• Cohen, L., Coghlan, V., and Dihel, L.: Cloning and Sequencing of Papain-Encoding cDNA , Gene 48, 219, 1986
 
• Cohen, L., Fluharty, C., and Dihel, L.: Synthesis of Papain in Escherichia coli , Gene 88, 263, 1990
 
• deJersey, J.: On the Specificity of Papain , Biochemistry 9, 1761, 1970
 
• Dekeyser, P., Smedt, S., Demeester, J., and Lauwers, A.: Fractioning and Purification of the Thiol Protinases from Papaya Latex , J Chromatogr B Biomed Appl 656, 203, 1994
 
• Drenth, J., Jansonius, J., Koekoek, R., Swen, H., and Wolters, B.: Structure of Papain , Nature 218, 929, 1968
 
• Finkbeiner, S., and Stevens, C.F.: Applications of Quantitative Measurements for Assessing Glutamate Neurotoxicity , Proc Natl Acad Sci U S A 85, 4071, 1988
 
• Fujimoto, S., Kanazawa, H., Ishimitsu, S., and Ohara, A.: On the Mechanism of Inactivation of Papain by Hydroxylamine , Chem Pharm Bull (Tokyo) 38, 546, 1990
 
• Glick, B., and Brubacher, L.: Evidence for Nonproductive Binding Subsites Within the Active Site of Papain , Can J Biochem 52, 877, 1974
 
• Hall, P., Anderson, C., and Crawford, G.: Acridine Dyes as Effectors in Papain Catalysis , Arch Biochem Biophys 153, 162, 1972
 
• Hill, R., and Smith, E.: Biochim Biophys Acta 19, 376, 1956
 
• Hinkle, P., and Kirsch, J.: Evidence for Conformational Restrictions within the Active Site of Acyl Papains Which Influence the Rates of Hydrolysis , Biochemistry 9, 4633, 1970
 
• Hinkle, P., and Kirsch, J.: Evidence for Conformational Restrictions within the Active Site of Acyl Papains Which Influence the Rates of Hydrolysis. Correction , Biochemistry 10, 1510, 1971
 
• Hinkle, P., and Kirsch, J.: Kinetics and Thermodynamics of the Reactions of Acyl-Papains. Effect of pH, Temperature, Solvents, Ionic Strength and Added Nucleophiles , Biochemistry 10, 3700, 1971
 
• Huettner, J., and Baughman, R.: Primary Culture of Identified Neurons From the Visual Cortex of Postnatal Rats , J Neurosci 6, 3044, 1986
 
• Jori, G., Gennari, G., Toniolo, C., and Scoffone, E.: Probing the Topography of Proteins in Solution by Photosensitized Oxidation, The Catalytic Region of Papain , J Mol Biol 59, 151, 1971
 
• Kamphuis, I., Kalk, K., Swarte, M., and Drenth, J.: Structure of Papain Refined at 1.65 A Resolution , J Mol Biol 179, 233, 1984
 
• Kanazawa, H., Fujimoto, S., and Ohara, A.: On the Mechanism of Inactivation of Active Papain by Ascorbic Acid in the Presence of Cupric Ions , Biol Pharm Bull 17, 189, 1994
 
• Kimmel, J., and Smith, E.: Crystalline Papain. I. Preparation, Specificity and Activation , J Biol Chem 207, 515, 1954
 
• Lake, A., and Lowe, G.: The Kinetics of Papain- and Ficin-Catalyzed Hydrolyses in the Presence of Alcohols , Biochem J 101, 402, 1966
 
• Liener, I.: The Sulfhydryl Proteases , Food Related Enzymes, Adv. in Chem. Series 136, J. Whitaker, Amer. Chem. Soc., Washington, DC, 202, 1974
 
• Light, A., and Smith, E.: Chymotryptic Digest of Papain. I , J Biol Chem 235, 3144, 1960
 
• Light, A., and Smith, E.: Chymotryptic Digest of Papain. II , J Biol Chem 235, 3151, 1960
 
• Light, A., Frater, R., Kimmel, J., and Smith, E.: Current Status of the Structure of Papain: The Linear Sequence, Active Sulfhydryl Group, and the Disulfide Bridges , Proc Natl Acad Sci U S A 52, 1276, 1964
 
• Light, A., Glazer, A., and Smith, E.: Chymotryptic Digest of Papain. III , J Biol Chem 235, 3159, 1960
 
• Lin, W., Armstrong, D., and Gaucher, G.: Formation and Repair of Papain Sulfenic Acid , Can J Biochem 53, 298, 1975
 
• Lindahl, P., Alriksson, E., Jšrnvall, H., and Bjork, I.: Interaction of the Cysteine Proteinase Inhibitor Chicken Cystatin with Papain , Biochemistry 27, 5074, 1988
 
• Lowe, G.: Structural Relationships between Some Plant and Animal Proteases , Nature 212, 1263, 1966
 
• Lowe, G., and Yuthavong, Y.: Kinetic Specificity in Papain-Catalyzed Hydrolyses , Biochem J 124, 107, 1971
 
• Lowe, G., and Yuthavong, Y.: pH-Dependence and Structure-Activity Relationships in the Papain-Catalyzed Hydrolysis of Anilides , Biochem J 124, 117, 1971
 
• Mackenzie, N., Grant, S., Scott, A., and Malthouse, J.: 13C NMR Study of the Sterospecificity of the Thiohemiacetals Formed on Inhibition of Papain by Specific Enantiomeric Aldehyde , Biochemistry 25, 2293, 1986
 
• Maes, D., Bouckaert, J., Poortmans, F., Wyns, L., and Looze, Y.: Structure of Chymopapain at 1.7 A Resolution , Biochemistry 35, 16292, 1996
 
• Martin, B., Feinman, R., and Detwiler, T.: Platelet Stimulation by Thrombin and Other Proteases , Biochemistry 14, 1308, 1975
 
• Matsumoto, K., Murata, M., Sumiya, S., Kitamura, K., and Ishida, T.: Clarification of Substrate Specificity of Papain by Crystal Analyses of Complexes with Covalent-type Inhibitors , Biochim Biophys Acta 1208, 268, 1994
 
• McCluskey, R., and Thomas, L.: The Removal of Cartilage Matrix in vivo by Papain. Identification of Crystalline Papain as the Cause of the Phenomenon , J Exp Med 108, 371, 1958
 
• Miller, W.: Degradation of Poly-a, L-glutamic Acid. I. Degradation of High Molecular Weight PGA by Papain , J Am Chem Soc 83, 259, 1961
 
• Mitchel, R., Chaiden, I., and Smith, E.: The Complete Amino Acid Sequence of Papain. Additions and Corrections , J Biol Chem 245, 3485, 1970
 
• Mole, J., and Horton, H.: Kinetics of Papain-Catalyzed Hydrolysis of a-N-Benzoyl-L-Arginine-p-Nitroanilide , Biochemistry 12, 816, 1973
 
• Morihara, K.: Chemical Nature of the Active Site of Papain. I. Inhibition by Carbonyl Reagents with Active Methylene Groups , J. Biochem. 62, 250, 1967
 
• Moussaoui, A., Nijs, M., Paul, C., Wintjens, R., Vincentelli, J., Azarkan, M., and Looze, Y.: Revisiting the Enzymes Stored in the Laticifers of Carica papaya in the Context of their Possible Participation in the Plant Defense Mechanism , Cell Mol Life Sci 58, 556, 2001
 
• Narinesingh, D., Ngo, T.: Papain Covalently Immobilized on Fractogel Derivative: Preparation, Bioreactor Flow Kinetics, and Stability , Biotechnol Appl Biochem 9, 450, 1987
 
• Neumann, H., Shinitzky, M., and Smith, R.: The Activation of Papain and Ficin by Phosphorothioate , Biochemistry 6, 1421, 1967
 
• Nolan, C., and Smith, E.: Absence of Sialic Acids and Other Carbohydrates in Crystalline Papain , Proc Soc Exp Biol Med 105, 287, 1960
 
• Ozawa, K., Ohnishi, T., and Tanaka, S.: Activation and Inhibition of Papain , J. Biochem. 51, 372, 1962
 
• Pickersgill, T., Rizkallah, P., Harris, G., and Goodenough, P.: Determination of the Structure of Papaya Protease Omega , Acta Crystallogr 47, 766, 1991
 
• Polg‡r, L.: Isolation of Highly Active Papaya Peptidases A and B from Commercial Chymopapain , Biochim Biophys Acta 658, 262, 1981
 
• Potter, J., McCluskey, R., Weissmann, G., and Thomas, L.: The Removal of Cartilage Matrix by Papain , J Exp Med 112, 1173, 1960
 
• Revell, D., Cummings, N., Baker, K., Collins, M., Taylor, M., Sumner, I., Pickersgill, R., Connerton, I., and Goodenough, P.: Nucleotide Sequence and Expression in Escherichia coli of cDNAs Encoding Papaya Proteinase Omega from Carica papaya , Gene 127, 221, 1993
 
• Schack, P., and Kaarsholm, N.: Absence in Papaya Peptidase A Catalyzed Hydrolysis of a pKa~4 Present in Papain-Catalyzed Hydrolyses , Biochemistry 23, 631, 1984
 
• Schechter, I., and Berger, A.: On the Size of the Active Site in Proteases. I. Papain , Biochem Biophys Res Commun 27, 157, 1967
 
• Schneider, M., and Goldstein, G.: Digestion of Articular Cartilage by Papain in Vitro , Proc Soc Exp Biol Med 124, 991, 1967
 
• Schwert, G., Neurath, H., Kaufman, S., and Snoke, J.: The Specific Esterase Activity of Trypsin , J Biol Chem 172, 221, 1948
 
• Shapira, E., and Arnon, R.: The Mechanism of Inhibition of Papain by Its Specific Antibodies , Biochemistry 6, 3951, 1967
 
• Shapira, E., and Arnon, R.: Antibodies to Papain. Resolution on DEAE-Sephadex into Fractions with Different Precipitating Capacities , Biochim Biophys Acta 140, 177, 1967
 
• Shipton, M., Kierstan, M., Malthouse, J., Stuchbury, T., and Brocklehurst, K.: The Case for Assigning a Value of Approximately 4 to pKa1 of Essential Histidine-Cysteine Interactive Systems of Papain, Bromelain and Ficin , F.E.B.S. Lett. 50, 365, 1975
 
• Skalski, M., Lewis, S., Maggio, E., and Shafer, J.: Effects of Proflavine on Papain's Catalytic Activity. A Proflavine-Mediated Decrease in Km , Biochemistry 12, 1884, 1973
 
• Sluyterman, L.: The Activation Reaction of Papain , Biochim Biophys Acta 139, 430, 1967
 
• Sluyterman, L., and DeGraaf, M.: The Effect of Salts Upon the pH Dependence of the Activity of Papain and Succinyl-Papain , Biochim Biophys Acta 258, 554, 1972
 
• Sluyterman, L., and Wijdenes, J.: Cyanuration of Papain Activity and Fluorescence of the Products , Biochim Biophys Acta 263, 329, 1972
 
• Sluyterman, L., and Wijdenes, J.: Benzoylamido-acetonitrile as an Inhibitor of Papain , Biochim Biophys Acta 302, 95, 1973
 
• Smith, D., Maggio, E., and Kenyon, G.: Simple Alkanethiol Groups for Temporary Blocking of Sulfhydryl Groups of Enzymes , Biochemistry 14, 766, 1975
 
• Smith, E., and Kimmel, J.: The Enzymes, 2nd Ed. Vol. 4, P. Boyer, H. Lardy, and K. Myrback, Academic Press, NY, 133, 1960
 
• Smith, E., Kimmel, J., and Brown, P.: J Biol Chem 207, 533, 1954
 
• Smith, L., Garvin, P., Gesler, R., and Jennings, R.: Enzyme Dissolution of the Nucleus Pulposus , Nature 198, 1311, 1963
 
• Storer, A., and Menard, R.: Catalytic Mechanism in Papain Family of Cysteine Pepsidases , Vol. 244, , , 486, 1994
 
• Taylor, M., Pratt, K., Revell, D., Baker, K., Sumner, I., and Goodenough, P.: Active Papain Renatured and Processed from Insoluble Recombinant Propapain Expressed in Escherichia coli , Protein Eng Vol. 5, , , 455, 1992
 
• Ungar-Waron, H., Jaton, J., and Sela, M.: Action of Papain on Normal Rabbit Immunoglobulin M , Biochim Biophys Acta 140, 542, 1967
 
• Van Eyk, H.: Fragmentation of Human Globulin with Papain , Biochim Biophys Acta 127, 241, 1966
 
• Vernet, T., Berti, P., de Montigny, C., Musil, R., Tessier, D., Macnard, R., Magny, M., Storer, A., and Thomas, D: Processing of the Papain Precursor. The Ionization State of a Conserved Amino Acid Motif within the Pro Region Participates in the Regulation of Intramolecular Processing , J Biol Chem 270, 10838, 1995
 
• Weinstein, B.: The Evolution of Papain , Biochem Biophys Res Commun 41, 441, 1970
 
• Westerik, J., and Wolfenden, R.: Aldehydes as Inhibitors of Papain , J Biol Chem 247, 8195, 1972
 
• White, J., and White, D.: Source Book of Enzymes , Source Book of Enzymes, , C.R.C. Press, , 1997
 
• Whitehouse, M., and Leader, J.: Biochemical Properties of Anti-Inflammatory Drugs. IX. Coupling of Oxidative Phosphorylation and Inhibition of a Thiol Enzyme (Papain) by Some Cyclic beta-Diones and Ninhydrin , Biochem Pharmacol 16, 537, 1967
 
• Williams, D., and Whitaker, J.: Kinetics of Papain Catalyzed Hydrolyses of Neutral Substrates , Biochemistry 6, 3711, 1967
 
• Wolthers, B., Dreuth, J., Jansonius, J., Koekoek, R., and Swen, H.: The Three Dimensional Structure of Papain , Structure-Function Relationships of Proteolytic Enzymes, P. Desnuelle, H. Neurath, and M. Ottesen, Academic Press, NY, 272, 1970
 
• Zucker, S., Buttle, D., Nicklin, M., and Barrett, A.: The Proteolytic Activities of Chymopapain, Papain and Papaya Proteinase III , Biochim Biophys Acta 828, 196, 1985
 

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