Purification of Papain

Papain might be purified by the method of Stepanov and Rudenskaya (66) on a bacitracin-Sepharose column, but this has not been proved since crude papain contains papain, chymopapain, and an aminopepti-dase, which may not be separated by this method.

The Kimmel and Smith method (71) described here is an improved method of that of Balls and Lineweaver (72) and gives crystalline papain. They started with hard particles of commercial latex that had to be ground to a fine powder to permit extraction. Currently, papain is separated from the insoluble latex by filtration and dried at ~ 65°C in a spray drier. The dried material is soluble at 20% and retains

Figure 19 Chromatography of commercial papain preparation on bacitracin-Sepharose. Papain (70 mg) in 7 mL of 0.1 M phosphate buffer, pH 7.0, containing 0.1 M Na2EDTA and 3 mM dithiothreitol was applied to the bacitracin-Sepharose column (1 x 12 cm) equilibrated with the same buffer. The arrow indicates start of elution with 25% isopro-panol in 1 M NaCl. Fraction I contained inert material; Fraction II, papain (activity measured against azocasein). (From Ref. 66.)

Figure 19 Chromatography of commercial papain preparation on bacitracin-Sepharose. Papain (70 mg) in 7 mL of 0.1 M phosphate buffer, pH 7.0, containing 0.1 M Na2EDTA and 3 mM dithiothreitol was applied to the bacitracin-Sepharose column (1 x 12 cm) equilibrated with the same buffer. The arrow indicates start of elution with 25% isopro-panol in 1 M NaCl. Fraction I contained inert material; Fraction II, papain (activity measured against azocasein). (From Ref. 66.)

Figure 20 Separation of serine proteinase and carboxypep-tidase T from Thermoactinomyces sp. Enzyme mixture (17.5 mg) dissolved in 7 mL of 0.1 M ammonium acetate, pH 6.5, containing 1 mM CaCl2, was applied to the bacitracin-Sepharose column (1 x 12 cm). Arrow I indicates start of elution with 20 mM Na2EDTA; arrow II, elution with 25% isopropanol in 1 M NaCl, pH 6.5. Fraction I contained inactive material; Fraction II, carboxypeptidase T (activity measured after reactivation by Ca2+); Fraction III serine thiol-dependent proteinase (active against substrate Z-Ala-Ala-Leu-NHC6H4NO2). (From Ref. 66.)

Figure 20 Separation of serine proteinase and carboxypep-tidase T from Thermoactinomyces sp. Enzyme mixture (17.5 mg) dissolved in 7 mL of 0.1 M ammonium acetate, pH 6.5, containing 1 mM CaCl2, was applied to the bacitracin-Sepharose column (1 x 12 cm). Arrow I indicates start of elution with 20 mM Na2EDTA; arrow II, elution with 25% isopropanol in 1 M NaCl, pH 6.5. Fraction I contained inactive material; Fraction II, carboxypeptidase T (activity measured after reactivation by Ca2+); Fraction III serine thiol-dependent proteinase (active against substrate Z-Ala-Ala-Leu-NHC6H4NO2). (From Ref. 66.)

Figure 21 (Top) Affinity chromatography of crystalline subtilisin on a 4-(4-aminophenylazo)phenylarsonic acid-CH-Sepharose column. See text for experimental details. (Bottom) Purification of subtilisin from a crude preparation. See Top and text for details. (From Ref. 69.)

full activity. In water, it has a pH of — 5.5 (Whitaker, unpublished data).

The soluble papain (1 L) was carefully adjusted to pH 9.0 with - 110 mL of 1 N NaOH which is added slowly with stirring (Fraction 1; Table 4). A fine grayish denatured precipitate formed, which was removed by centrifugation at 2600 rpm for 1 h. The solution should be clear (Fraction 2).

Fraction 2 was cooled to 4°C and brought to 0.4 saturation with solid ammonium sulfate (250 g/L). A white precipitate formed. After 1-2 h at 4°C, the precipitate (Fraction 3) was recovered by centrifugation at 2500 rpm for 1 h. The supernatant fraction was saved for preparation of chymopapain. The precipitate was washed over with 400-500 mL cold (4°C) 50% saturated ammonium sulfate, and the precipitate was separated by centrifugation as above.

Fraction 3 was redissolved in 600 mL of 0.02 M cysteine (pH 7-7.5; freshly prepared), and 60 g of solid sodium chloride was slowly added. Papain was precipitated as a fine white solid. After standing for 1 h at 4°C, it was centrifuged in the cold for 1 h at 2500 rpm. The supernatant liquid was discharged. The precipitate was Fraction 4.

Fraction 4 was suspended in 400 mL of 0.02 M cysteine at pH 6.5 and adjusted to pH 5 with 1 M HCl as needed. At room temperature, the suspension developed a marked crystalline sheen in — 30 min. It was then placed in a cold room (or refrigerator) at 4°C overnight. The light crystals (Fraction 5) were removed by centrifugation at 2000-2500 rpm for 4-5 h. The supernatant liquid may be discarded. After several hours a small, insignificant amount of crystals formed.

Recrystallization was by dissolving Fraction 5 in the minimum amount of distilled water at room temperature and adding very slowly, with stirring 10 mL saturated sodium chloride per 300 mL protein solution. Crystallization began when — 75% of the 10-mL of saturated sodium chloride was added at room temperature. The suspension was then moved to a 4°C room

Table 4 Preparation of Crystalline Papain from Dried Papaya Latexa

Fraction No.b

Volume

Total protein

0 0

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