The purification of papain by affinity chromatography.

A water insoluble derivative of a papain inhibitor was prepared by covalently linking Gly-Gly-Tyr(Bzl)-Arg to an agarose resin. The immobilized inhibitor binds active papain specifically. When papain prepared by the method of Kimmel and Smith was activated and applied to a column of the immobilized inhibitor at moderate ionic strength (20 mM EDTA, pH 4.3), about 50% of the total protein was not bound and was found to be catalytically inactive. The bound enzyme was released with distilled water. It contained one mole of SH per mole of protein. When assayed with α-N-benzoyl-l-arginine ethyl ester (25°, pH 6.0) the purified enzyme had a kcat of 28.5 sec-1 and a Km of 18 mM. The specific activity of the purified papain was about twice that of the original preparation and the Km was unchanged. The enzyme, inactivated by an equimolar amount of mercuric chloride could be fully activated even after prolonged storage.

[1]  B. G. Wolthers,et al.  Binding of competitive (product) inhibitors to papain in the active and inactive state. , 1970, Biochimica et biophysica acta.

[2]  A. Berger,et al.  Mapping the active site of papain with the aid of peptide substrates and inhibitors. , 1970, Philosophical transactions of the Royal Society of London. Series B, Biological sciences.

[3]  J. Kirsch,et al.  The mechanism of the activation of papain. , 1969, Biochemical and biophysical research communications.

[4]  C. Anfinsen,et al.  Selective enzyme purification by affinity chromatography. , 1968, Proceedings of the National Academy of Sciences of the United States of America.

[5]  A. Berger,et al.  On the active site of proteases. 3. Mapping the active site of papain; specific peptide inhibitors of papain. , 1968, Biochemical and biophysical research communications.

[6]  J. Drenth,et al.  Structure of Papain , 1968, Nature.

[7]  A. Burger.  Design of Active-Site-Directed Irreversible Enzyme Inhibitors. , 1967 .

[8]  J. Porath,et al.  Chemical Coupling of Proteins to Agarose , 1967, Nature.

[9]  L. Sluyterman.  The activation reaction of papain. , 1967, Biochimica et biophysica acta.

[10]  J. Porath,et al.  Chemical Coupling of Peptides and Proteins to Polysaccharides by Means of Cyanogen Halides , 1967, Nature.

[11]  J. Feder,et al.  The determination of the concentration of hydrolytic enzyme solutions: alpha-chymotrypsin, trypsin, papain, elastase, subtilisin, and acetylcholinesterase. , 1966, Journal of the American Chemical Society.

[12]  G. W. Anderson,et al.  Racemization Control in the Synthesis of Peptides by the Mixed Carbonic-Carboxylic Anhydride Method , 1966 .

[13]  J. Whitaker,et al.  Kinetics of Papain-Catalyzed Hydrolysis of α-N-Benzoyl-L-arginine Ethyl Ester and α-N-Benzoyl-L-argininamide1 , 1965 .

[14]  P. Hemmerich,et al.  SPECIFICITY OF LIVER FLAVOKINASE FOR 9-(1'-D-RIBITYL)ISOALLOXAZINES VARIOUSLY SUBSTITUTED IN POSITIONS 2, 6, AND 7. , 1963, The Journal of biological chemistry.

[15]  A. Glazer,et al.  Phenolic hydroxyl ionization in papain. , 1961, The Journal of biological chemistry.

[16]  G. Ellman,et al.  Tissue sulfhydryl groups. , 1959, Archives of biochemistry and biophysics.

[17]  E. L. Smith,et al.  Crystalline papain; number and reactivity of thiol groups; chromatographic behavior. , 1958, The Journal of biological chemistry.

[18]  E. L. Smith,et al.  Crystalline papain. I. Preparation, specificity, and activation. , 1954, The Journal of biological chemistry.

[19]  L. Lerman,et al.  A Biochemically Specific Method for Enzyme Isolation. , 1953, Proceedings of the National Academy of Sciences of the United States of America.

[20]  I. Silman,et al.  Water-insoluble derivatives of enzymes, antigens, and antibodies. , 1966, Annual review of biochemistry.

[21]  A. Glazer,et al.  THE SULFUR DISTRIBUTION OF PAPAIN. , 1965, The Journal of biological chemistry.

[22]  A. Pihl,et al.  Studies on the active--SH group of papain and on the mechanism of papain activation by thiols. , 1963, The Journal of biological chemistry.