Data Sheet - Papain

Source:
Latex from the fruit of Carica papaya L.
Systematic name:
Peptidyl peptide hydrolase;
E.C.-No.
3.4.22.2
CAS-No.
9001-73-4

Occurrence

Papain is one of the proteolytic enzymes found in the latex of the leaves and of the green fruit of the papaya tree (Carica papaya L., Caricaceae). In industrial preparations, referred to as refined Papain, Papain EC 3.4.22.2 represents only a minor part when compared to the amount of the other proteinases, papaya proteinase Ω and Chymopapain (EC 3.4.22.6), the latter being by far the most abundant enzyme. Some other hydrolytic enzymes, e.g. papaya lysozyme, are also present in refined Papain though only in minute amounts.

Characteristics

Note: Papain EC 3.4.22.2 contributes less than 10 % to the total proteinase content of industrial preparations of papaya latex, in the following referred to as refined Papain. The characteristics of refined Papain are largely influenced by the two other proteinases, papaya proteinase W and Chymopapain, because these two make up about 15 % and 75 %, respectively, of the total proteinase content of refined Papain. Therefore, the data below consider the respective values for all three proteinases of refined Papain, where appropriate.

Specificity: Papain EC 3.4.22.2 hydrolyzes a wide variety of proteins and peptides. It has also esterase, thiolesterase, transesterase and transamidase activity. It has a preference for bonds involving the carbonyl end of α-NH2 substituted arginine and lysine, and to a lesser extent, histidine, glycine, glutamine and tyrosine (1,2,3). Phenylalanine in a position Phe-X- enhances the susceptibility of a peptide to hydrolysis at the C-terminal end of X (1,2).

Chymopapain and papaya proteinase Ω have substrate specificities close but not identical to that of Papain. The presence of these enzymes in refined Papain therefore extends the range of bonds susceptible to hydrolysis and hence the degree of digestion of protein by refined Papain when compared with Papain EC 3.4.22.2 alone.

Effectors: All three aforementioned proteinases are thiol enzymes, containing in the active centre a highly reactive cysteine which is essential for catalysis. Alkylating reagents such as p-chloro-mercuribenzoate (PCMB), N-ethylmaleimide (NEM), and iodoacetate cause irreversible inactivation. Heavy metals (e.g. Hg2+, Zn2+, Fe2+, Cu2+), air and H2O2 are also inhibitory. The papaya proteinases can on the other hand be activated by the application of mild reducing agents like cysteine, sulfide, cyanide or thioglycolic acid, supplemented by a heavy metal chelator such as EDTA, or by the addition of 2,3-dimercaptopropanol, a compound which combines the functions of both a thiol reagent and a metal binder (1,4).

Catalytic optima: Depending on the nature of the substrate, refined Papain shows maximum activity at pH 4.0-7.0. The optimum temperature is in the range of 60-70 °C.

Stability: Papain EC 3.4.22.2 and papaya proteinase Ω are unstable under acidic conditions. At pH values lower than pH 2.8 rapid and irreversible inactivation occurs even at ambient temperature (1,4). In contrast, Chymopapain is extremely stable at pH 2. Papain EC 3.4.22.2 shows high stability towards urea and organic solvents; exposure to up to 9 M urea, 70% methanol, or 15% dimethylsulfoxide (DMSO) cause no loss of activity (1,4). All three papaya proteinases are particularly resistant to heat.

Solubility: Refined Papain is almost completely soluble in water, insoluble in most organic solvents. Chymopapain exhibits high solubility in saturated NaCl above pH 3 and is more soluble in aquatic solution than Papain EC 3.4.22.2.

Molecular weight: Papain EC 3.4.22.2: approx. 23,400, Chymopapain: approx. 24,000, papaya proteinase Ω: approx. 24,000.

Composition: Papain, Chymopapain and papaya proteinase W consist of single polypeptide chains of 212, 216, and 218 amino acids, respectively. The amino acid sequences, the secondary structures, and the three-dimensional structures of the three papaya proteinases show a lot of significant similarities, suggesting that these enzymes are closely related to each other (5). On the other hand, based on the differences in the amino acid composition and sequence it can be ruled out that the three proteinases arose from each other or from a common ancestor by proteolysis (5).

Isoelectric point: Papain EC 3.4.22.2: 8.7-8,9,
                               Chymopapain: 10.3 - 10.7,
                               papaya proteinase Ω: > 11.0.

Spectral data: Papain EC 3.4.22.2: E278 (1%, 1cm) = 25.0 (6),
                        Chymopapain: E280 (1%, 1cm) = 18.4 - 18.7 at pH 7.0 (6).

Assay according to FIP

(1 FIP unit ≈ 10,000-13,000 USP units)

The method described here is the one given by Lauwers & Scharpé (1).
Note:   The assay reflects the activity of all three proteinases contained in refined Papain.

Principle

Papain hydrolyzes the synthetic substrate N-benzoyl-L-arginine ethyl ester (BAEE). The amount of acid liberated (at pH 7.0 and 25 °C) is measured by titration with sodium hydroxide, recorded as a function of time.

Unit definition
1 FIP unit of Papain is the amount of Enzyme that hydrolyzes under the standard conditions 1 mmole of BAEE per minute.

Reagents

  1. Substrate solution: 0.06 M N-benzoyl-L-arginine ethyl ester (BAEE). Dissolve 514 mg BAEE x HCl in distilled water, adjust to pH 7.0, and add water to a final volume of 25.0 ml.
  2. Activation solution: Dissolve 157.5 mg cysteine.HCl, 63.2 mg EDTA x 2H2O and 1.17 g NaCl in distilled water, adjust to pH 7.0 and add water to a final         volume of 100.0 ml. Prepare fresh daily.
  3. Enzyme solution: Prepare a solution of Papain in distilled water. The solution should contain about 3 FIP units/ml and have a pH of 7.0. This solution is kept in ice ware during the assay.
  4. Enzyme Reference Solution: Dissolve a suitable amount of Papain FIP standard in pure water and adjust to pH 7.0 to obtain a solution containing approximately 3 FIP units/ml. This solution is kept in ice ware during the assay.
  5. Papain FIP Reference Standard is issued by: Centre for Standards FIP, Harelbekestraat 72, B-9000 Ghent, Belgium.
  6. 0.02 N NaOH: Merck 9142.

Apparatus
The reaction vessel is provided with a stirring device (e.g. magnetic stirrer) and is maintained by a thermostat to 25.0 ± 0.1 °C. The vessel is covered with a lid containing holes, one for each of the electrodes, one for the burette tip, one for the nitrogen inlet and one for the introduction of the reactants. A pH meter accurate to 0.01 pH units is necessary. The microburette has a total capacity of 0.5 ml and can deliver accurately 0.001 ml. A stop-watch, giving up to 0.2 seconds.

Procedure

Transfer into the reaction vessel:

flush with N2 and equilibrate to 25.0 °C. Then add

pre-equilibrated to 25.0 °C, start stop-watch and maintain pH constantly at 7.0 by continuous addition of 

Follow the reaction for 5 min.

Calculation

The specific activity of the Enzyme in FIP units/mg is calculated as:

m´ x V x A
m x V´

m       =   mass in milligrams of the substance to be examined
m´      =   mass in milligrams of Papain standard
V       =   volume in ml of 0.02 N NaOH used per min. by the test solution
V´      =   volume in ml of 0.02 N NaOH used per min. by the reference solution
A       =   activity of Papain FIP standard in units/mg

Assay according to current USP

(1 FIP unit ≈ 10,000-13,000 USP units)

Unit definition
One USP Unit of Papain activity is the activity that releases the equivalent of 1 µg of tyrosine from a specified casein substrate under the conditions of the Assay, using the enzymes concentration that liberates 40 µg of tyrosine per ml of test solution.

Reagents

  1. 0.05 M Dibasic sodium Phosphate solution: Dissolve 0.71 g Na2HPO4 (M = 141.96 g/mol) in pure H2O to make 100 ml.
  2. 0.05 M Citric acid solution: Dissolve 1.05 g Citric acid monohydrate (M = 210.14 g/mol) in pure H2O to make 100 ml.
  3. Buffer solution (pH 6.00): Dissolve 1.78 g Na2HPO4 (M = 141.96 g/mol) in 200 ml pure H2O. Give 3.5 g Disodium edetate (M = 372.24 g/mol) and 1.53 g Cysteine x HCl x H2O (M = 175,64 g/mol). Adjust the pH with 1 N NaOH or HCl to pH 6.00 and fill with pure H2O to 250 ml (Prepare fresh daily).
  4. Casein Substrate solution: Give 0.5 g Casein (Hammersten-type or BRP) into 25 ml 0.05 M Na2HPO4 solution. Place in a boiling water bath for 30 minutes with occasional stirring. Cool to room temperature and add 0.05 M Citric acid solution under rapidly stirring to adjust a pH of 6.00. Dilute with pure water to 50 ml. (Prepare fresh daily)
  5. 30% (m/V) Trichloroacetic acid solution: Dissolve 30 g TCA (M = 163.39 g/mol) with pure H2O to make 100 ml.
  6. Preparation of Enzyme solution: Prepare an enzyme solution in that kind that the release of 40 µg Tyrosine in test does not exceed. {400 USP-U/10 ml ≈ 40 USP-u/ml (= 40 µg Tyrosine)} Use the enzyme dilution within 60 Minutes.

Initial weight of Enzyme = [(40 USP-E*10 ml/1.5 ml)*dilution factor*ml Enzyme solution] ÷ declared Activity

Procedure

Label test tubes in duplicate Ta, Tb, S1a, S1b, S2a, S2b, S3a, S3b. For Blank as follow: BTa, BTb, BS1a, BS1b, BS2a, BS2b, BS3a, BS3b Add to the test tubes in the order given:

  1. Give from 0.5 M Phosphat-buffer solution pH 6.00 (C) in S1a, S1b & BS1a, BS1b 1.0 ml, in S2a, S2b & BS2a, BS2b, Ta and Tb 0.5 ml and in S3a, S3b and BS3a, BS3b 0.0 ml;
  2. Give in each tube 5.0 ml of Casein solution (D). Mix by shaking. Place all tubes in a water bath at 40 ± 0.5 °C.
  3. When temperature equilibrium is reached, start the stop watch and at t = 0 add 1.0 ml of Papain Reference Standard dilution into the tubes S1a, S1b, and 1.5 ml of Papain Reference Standard dilution into the tubes S2a, S2b and 2.0 ml of Papain Reference Standard dilution into the tubes S3a, S3b. Also add 1.5 ml of Papain Test dilution into the tubes T1a, T1b. Shake the tubes and let at 40 °C for exactly 60 Minutes. Exactly 60 min after addition of casein solution, taking into account the regular interval adopted, add 3.0 ml of 30% trichloroacetic acid (E) to tubes S1a, S1b, S2a, S2b, S3a, S3b and Ta, Tb. Mix. For 30-40 Minutes allow to coagulate fully the precipitated protein at 40 °C.
  4. For the Blanks add at first 3.0 ml of 30% trichloroacetic acid (E) to the tubes BTa, BTb, BS1a, BS1b, BS2a, BS2b, BS3a, BS3b. Mix by shaking and than give as follows: 1.0 ml of Papain Reference Standard dilution into the tubes BS1a, BS1b, 1.5 ml of Papain Reference Standard dilution into the tubes BS2a, BS2b and 2.0 ml of Papain Reference Standard dilution into the tubes BS3a, BS3b. Also add 1.5 ml of Papain Test dilution into the tubes BT1a, BT1b.
  5. Withdraw all the tubes from the water bath and allow to stand at room temperature for 30 min.
  6. Filter the contents of each tube twice through the same suitable filter paper previously washed with 30% trichloroacetic acid (E), then with water and dried. Measure the absorbance of the clear filtrates at 280 nm against the filtrate of their respective Blanks.

Plot the absorbance reading for S1, S2 and S3 (= E280-correc.) against the enzymes concentration of each corresponding level (mg/ml). By interpolation from this curve, taking into consideration dilution factors, calculation the potency in the weight of Papain taken by the formula:

E280-correc. = E280-Test - E280-Blank
mg / ml = { [ (Initial weight of enzyme / Enzyme volume) / dilution factor] * „ml in Test“} / 10 ml
Factor = 1 / (mg/ ml)
USP-u/mg for Papain sample = Factor * [declared Activity of Reference Standards * (mg/ml)read from the curve]

 

Availability

Standard qualities
Papain BPC 1954 (3000 - 4000 USP units/mg)
Papain 1 x USP ≥  6,000 USP units/mg
Papain 5 x USP ≥ 30,000 USP units/mg
Papain Concentrate ≥ 40,000 USP units/mg
Papain 2.5-3.5 FIP-units/mg
Papain 0.5-2.5 FIP-units/mg

Customized qualities are available upon request.

References

  1. Lauwers, A.; Scharpé, S.: Pharmaceutical Enzymes, drugs and pharmaceutical sciences., Volume 84, Marcel Dekker, Inc., New York-Basel-Hong Kong, 1997.
  2. International Union of Biochemistry. Nomenclature Committee: Enzyme Nomenclature. Academic Press, Inc., London, 1984.
  3. Glazer, A.N., Smith, E.L. in: The Enzymes (P.D. Boyer, ed.) 3rd ed., Vol. III, p. 502. Academic Press, New York, 1971.
  4. Arnon, R. in: Methods of Enzymology (G.E. Perlmann, L. Lorand, eds.) Vol. XIX, p. 226. Academic Press, Inc., Orlando, Florida, 1970.
  5. Dubois, T., Kleinschmidt, T., Schnek, A.G., Looze, Y., Braunitzer, G., Biol. Chem. Hoppe-Seyler 369 (1988) 741.
  6. Merck Index, 11th ed., Merck & Co., Inc., Rahway, USA, 1989.

 

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