Papain crystalline

Porter, R.R. Hydrolysis of rabbit gamma-globul in and antibodies with crystalline papain. Biochem J. 73 119-126, 1959. 

The interest in papain increased enormously after the publication by Kimmel and Smith D ] of a modification of the purification procedure of Balls and Lineweaver [5j. This modification permitted the isolation of pure papain from papaya dried latex and was used for many years as the standard method for the production of papain. The crystalline papain of Kimmel and Smith consists of three components active papain, reversibly oxidized papain, and irreversibly oxidized papain. Reversibly oxidized papain can be converted into active papain by reduction of the active-site thiol by low molecular weight thiols [10], sodium borohydride [11], or CN [12]. In active papain, the Cys-25, which is essential far catalytic activity, is present in a reduced form, while in reversibly oxidized papain the Cvs-25 forms a mixed disulfide with cysteine. Drenth et al. reported that in irreversibly oxidized papain the Cys-25 has been oxidized to the sulfuric acid... 

A, K. Balls and R Linewcaver, Isolation and properties of crystalline papain. J. BioL E. F.lamen and A. K. Balk. Chymopapain a new crystalline proteinase from papaya... 

I. Drenth, EC. H. Kalk, and H. M. Swcn. Binding of chlooniclhyl ketone aibstnte analogues to crystalline papain. Biochemistry 75 3731 (1976). 

One milligram of crystalline papain or mercuripapain (Worthington Biochemical Corp., Freehold, New Jersey) is added to a solution of 100 mg of IgG in 10 ml of phosphate-buffered (10 mM phosphate, pH 7.3) 0.15 M NaCl, with 1 mM EDTA and 25 mM mercaptoeth-anol. The mixture is incubated for 1 hr at 37°. Further proteolysis is ended, and sulfhydryl groups are alkylated by adding iodoacetamide to a final concentration of 30 mM and incubating for an additional 15 min at 37°. 

Recently, Kang and Warner (102) fractionated crude papain into crystalline papain, chymopapain, and papaya peptidase A. Chymopapain constituted the major component of the crude papain, and it proved to be the most thermostable at pH values between 5 and 9. Table VIII shows the activity of the various purified enzymes on native and heat-denatured meat fractions after subtracting blank values. All three enzymes degraded meat fractions, but with somewhat different hydrolytic potencies. Chymopapain showed activity comparable to papain and papaya peptidase A on meat fractions. 

Treatment of rabbit IgG at 37°C and neutral pH with 0.1-1% by weight of crystalline papain, in the presence of reducing agent, cleaves... 

In addition to availability, crystalline papain has other properties which contribute to its importance in the field of enzymology. The molecular weight of papain is about 20,500, which is small enough to permit studies of the sequence of amino acids and the over-all structure by presently available chemical and enzymatic methods. The presence of one or more sulfhydryl groups which are concerned with the proteolytic activity gives some indication as to where to look for the active site of the enzyme. 

Crystalline papain can be prepared from fresh latex by the procedure of Balls and Lineweaver without modification. From limited experience (9,41,148) it appears that different batches of fresh latex are fairly uniform with respect to papain content. Such is not the case with commercial dried latex (100). Not all batches give good... 

Crystalline papain and mercuripapain have a specific proteolytic activity 5 to 6 times greater than the crude latex (100). However, electrophoretic studies by Smith, Kimmel, and Brown (153) have demonstrated that papain comprises about 7% of the soluble components of papaya latex. Thus the preparation of the crystals results in a 16-fold purification this represents a better estimate of... 

Fig. 2. Electrophoretic patterns of crystalline papain (153). A, tracings of the electrophoretic patterns of twice recrystallized papain, pH 3.9 protein concentration—1.0% in acetate buffer containing 0.001 M Versene and 0.02 M cysteine. Ascending and descending patterns are shown after 275 minutes of migration. The minor peak evident on the descending pattern represents less than 2% of the total protein and was absent in most of the preparations. B shows the descending pattern at 0.2% protein concentration in Veronal buffer at pH 7.5 after 150 minutes. C is the descending pattern after 150 minutes in acetate buffer at pH 6.0 at a protein concentration of 0.4%. Cysteine and Versene were omitted in B and C. The patterns in B and C are at approximately twice the linear enlargement of those in A.

Fig. 3. Electrophoretic mobility as a function of pH for crystalline papain (153). The runs were made at 1.5° in univalent buffers at 0.1 ionic strength. Ac. is acetate, V is Veronal, and G is glycine.

The effect of pH on the mobility of crystalline papain is shown in Figure 3. The apparent isoelectric point in buffers of 0.1 ionic strength is at pH 8.75 in good agreement with earlier findings which indicated an alkaline isoelectric point near pH 9 (9,41). 

Fig. 5. Sedimentation constants (iSjo, ) of crystalline papain as a function of protein concentration (153). A shows studies at pH 3.9 to 4.0 in acetate buffer (0.1 to 0.2 ionic strength) at a cysteine concentration of 0.02 M. Results with ( ) added Versene at 0.002 M and without (O) Versene. B shows results in acetate at pH 4.0 (0.1 ionic strength) in the absence of cysteine ( ) and for papain oxidized with iodine (O). C gives measurements with 0.02 M cysteine at 0.1 ionic strength in acetate at pH 5.4 (O) and in Veronal at pH 7.0 ( ). For B and C the straight lines have arbitrarily been drawn to an extrapolated value of 2.42 S for Sia,u at zero concentration this is the average value found at pH 4 in the presence of cysteine.

The diffusion constant of crystalline papain has been determined by Balls and Lineweaver (9), by Close (41), and by Smith, Kimmel, and Brown (153). The results of these studies are given in Table I. There is excellent agreement between the values cited by Close and by Smith, Kimmel, and Brown in both cases the measurements were made by the height-area method in the electrophoresis cell by the procedure of Longsworth (108). Balls and Lineweaver used the porous disk method of Northrop and Anson (128). Within the precision of this method the agreement is excellent. 

Crystalline papain is remarkably stable in urea solutions (107). Virtually no inactivation occurred in 9 iW urea solution after 24 hours at 30° and pH 6.5. Data presented later in this review suggest that exposure to high concentrations of urea such as this produce little configurational change in papain. 

The elementary composition of crystalline papain is given in Table II. The results from three different laboratories are shown for comparison. The value for the sulfur content corresponds to 8 atoms of sulfur per mole of papain. 

Winnick, Cone, and Greenberg (180) disputed the coenzyme function of activators after demonstrating that no inactivation of ficin occurred after removal of the activators by careful anaerobic dialysis. It is difficult to reconcile these findings with those of Irving et al. Indeed, results with crystalline papain to be presented later give no support to the coenzyme theory and can be interpreted in terms of the reversible oxidation-reduction of thiol groups alone. 

The reaction of papain with PCMB or PCMBS in 70% ethanol is different from that in water. Crystalline papain and the column-reduced material behave identically. The derivatives were obtained in the presence of an 8.5- to 20-fold excess of the mercurial and were partly crystalline. Analyses for mercury showed that as many as 6 moles of either mercurial can combine with papain (53,150). 

It is theoretically possible for the reducing column to reduce disulfide bridges in papain, thus enabling 6 moles of mercurial to combine with each mole of protein. This possibility seems remote since dialyzed crystalline papain in ethanolic solution combines in the same molar ratio. Moreover, such a reduction of S—S bonds does not occur with serum albumin (48). Previous experience with PCMB (74) has provided no evidence that this compound can split disulfide bonds. It should be noted that papain after combination with 6 moles of PCMB is inactive treatment with cysteine and Versene regenerates only a portion of the original proteolytic activity. It appears likely that some modification of the native structure of papain occurs in ethanolic solution in the presence of the phenyl mercury compounds which permits combination in the 6 to 1 ratio. 

Studies of the structure of papain were initiated by Thompson (165), who determined the amino or N-terminal amino acid sequence and the free amino groups. Crystalline papain and mercuripapain were allowed to react with l-fluoro-2,4-dinitrobenzene (FDNB) by the procedures of Sanger (136). The dinitrophenyl (DNP) proteins were subsequently hydrolyzed in 6 iV HCl and the DNP-amino acids isolated by partition chromatography of the ether extracts of the hydrolyzates. Quantitative estimates of the DNP-amino acids were made spectrophotometrically. The results are given in Table V. 

The action of crystalline papain on synthetic substrates was first studied by Balls and Lineweaver (9), who observed that hippuryl-amide and carbobenzoxy-L-isoglutamine were hydrolyzed. 

Fig. 15. Effect of pH on the hydrolysiB of four substrates by crystalline papain in the presence of 0.005 M cysteine and 0.001 M Versene (100). The substrates were carbobenzoxy-L-leucinamide (CLA), hippurylamide (HA), carbobenzoxy-i/-glutamic acid diamide (CGDA), and carbobenzoxy-L-isoglutamine (CIG).

The three crystalline proteolytic enzymes of pancreas—trypsin, chymotrypsin, and carbox3rpeptidase— possess a well-defined esterase activity on appropriate synthetic compounds which are closely related in structure to substrates possessing amide or peptide bonds (126). Crystalline papain also has an esterase action (99,100). 

Recently, Johnson and Herriott (92), using crystalline papain, have confirmed and extended the work of Behrens and Bergmann, and have shown that a variety of peptides may function as cosubstrates, e.g., glutathione. They could not demonstrate any of the expected intermediates resulting from synthesis prior to hydrolysis. 

From the foregoing discussion it is apparent that crystalline papain appears to satisfy the usual criteria that have been applied to determine the homogeneity of proteins. The sedimentation and diffusion patterns, the stoichiometry of the amino acid composition, the end group analysis, the chromatographic behavior, and the immunochemical studies offer strong evidence in favor of homogeneity. The minor electrophoretic asymmetry has been discussed. The variability in specific activity and in the sulfhydryl to protein ratio in different preparations of crystalline papain raised some question in this regard. It should be reemphasized that the specific activity... 

From all evidence, it appears that crystalline papain is homogeneous in the chemical sense. From this standpoint it represents a satisfactory substance for the determination of structure and the correlation of structure and activity. The observation that much of the molecule at the amino-terminal end is not essential for the activity strongly supports the view that enzymes, in general, possess an active site and that intact protein structure is unnecessary for catalytic activity. The presence of an essential thiol group is in accord with the idea that this group is part of the active site and may participate in the catalytic activities of the enzyme through the formation of a thiol ester. 

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