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The purification and some physical-chemical studies of crystalline chymopapain B
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|Title:||The purification and some physical-chemical studies of crystalline chymopapain B|
|Authors:||Kunimitsu, Donald Kunio|
|Abstract:||Chymopapain was first isolated and crystallized from fresh papaya latex by Jansen and Balls (J. Biol. Chem., 137, 459, 1951). More recently, a proteolytic component with some of the properties described by these workers was isolated and crystallized from dried papaya latex by Ebata (J. Biol. Chem., 237, 1086, 1962). During the course of the purification of chymopapain from dried papaya latex, the appearance of several proteolytically-active components was consistently observed during chromatography on Amberlite IRC-50 (XE-64). We have been able to crystallize the protease present in one particular fraction which evidently differs from the chymopapain isolated by Ebata. This crystalline component (designated chymopapain B) is shown to be homogeneous by the criteria of ultra-centrifugation, electrophoresis, amino-terminal analysis and re-chromatography. Physical studies on chymopapain B indicate that it differs from the chymopapain described by Ebata (designated chymopapain A). Molecular weight determinations by the Archibald procedure yield a molecular weight of 35,000 for component A and 30,000 for component B. Electrophoretic studies indicate that both components are very basic, with their isoelectric points in the neighborhood of pH 10. Furthermore, both show relative stability to heat and acidic pH's. Like papain and chymopapain A, chymopapain B is also activated by thiol compounds, indicating that it is a sulfhydryl enzyme. Titration of the active enzyme with p-chloro-mercuribenzoate, mercuric chloride and N-ethylmaleimide reveals the presence of two free -SH groups but extended studies indicate that only one -SH is essential for activity. Preliminary investigations into the substrate specificity of this enzyme show that it resembles papain in its ability to hydrolyze a large variety of peptide and amide linkages. Thus far, N-benzoyl-L-arginine amide is found to be the most sensitive substrate for chymopapain B. Moreover, the ability of the chymopapain B to carry out anilide synthesis has been demonstrated, suggesting that this enzyme, like papain, can function as a transferase. Detailed kinetic studies with N-benzoyl-L-arginine amide as substrate show that chymopapain B hydrolyzes this compound at approximately one-third the rate of papain. Determinations of the Michaelis constant and maximal velocity at various pH's have also been undertaken.|
Thesis (Ph. D.)--University of Hawaii, 1964.
Bibliography: leaves 153-156.
xv, 156 leaves mounted ill., tables
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|Appears in Collections:||Ph.D. - Biomedical Sciences (Biochemistry)|
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