The Sulfur distributin of papain and related studies.
Creators: name: Glazer, Alexander Namiot
Publisher: name: University of Utah
Year: 2012
Subjects: Biochemistry
Disulfide Interchange

description: In the General Introduction to this Thesis, it was pointed out that the work of previous investigators posed two major problems.  The first of these is that of the two "missing" sulfur atoms, and the second, that of the chemical nature of the "active site" of papain.  The results of the present investigation provide the answer to the first of these two problems.  The investigation of the sulfhydryl content of native, unactivated papain by a number of methods reveals only a fraction of a mole of sulfhydryl per mole of protein.  The disulfide interchange reaction clearly indicates the presence of eight groups of papain which, after exposure to 9.6N HC1 at 39°, behave in a manner analogous to the cysteine and cysteine residues in other proteins.  In fact, it appears to be reasonable to assume that under the conditions of the interchange reaction, we are dealing with eight half-cystine residues.  The determination of the disulfide content of papain by argentimetric amperometric titration with sulfite as the cleaving agent clearly shows the presence of three disulfide bonds in papain at near-neutral pH, whether in the native, urea-denatured, or detergent denatured protein.  When papain is exposed to pH 1.7 for a short period of time and then titrated at near-neutral pH in 8M urea, four disulfide bonds may be titrated with silver ion in presence of sulfite.  Finally, determination of cysteic acid in acid hydrolysates of papain oxidized with preformic acid clearly shows the presence of eight moles of cysteic acid per mole of papain.  The above evidence indicates that after acid treatment or oxidation (which is also performed under acidic conditions), the sulfur content of papain can be accounted for completely on the basis of the half-cystine content of the protein.  The answer to the first of the two problems posed above unfortunately has not automatically provided an answer to the second.  The studies on the thiol content of active papain in clearly indicate that the activation of papain is associated with the appearance of on thiol group per mole of protein.  The hypothesis that activation of papain involves the cleavage of a disulfide bond is subject to a number of serious criticisms.  1. A reductive cleavage of an intramolecular disulfide by column reduction should lead to the appearance of two thiol groups.  Actually, Finkle and Smith have shown that only one such group appears as a result of this treatment.  This point has been confirmed in the present study.  2. The assumption of an inter-molecular disulfide bond requires the concomitant assumption that inactive papain is a dimer.  No evidence for the existence of such a dimer has been found.  3. The fact that acid treatment is required before the fourth disulfide bond can be titrated with silver ion in the presence of sulfite is plausibly explained on the basis of the simple assumption that the denaturation resulting from this treatment renders the bond accessible to sulfite, or changes its environment.  The finding that 8M urea at 37° does not bring this about argues against this explanation.  In 1958, Smith has suggested that inactive papain exists in an oxidized from, corresponding to the "sulfenic" state.  Activation could then be considered as representing reduction to the thiol.  This suggestion has received support from the results obtained with the streptococcal proteinase.  This enzyme contains only one half-cystine residue per mole.  In analogy with papain, the streptococcal proteinase requires activation by thiol compounds.  No sulfhydryl can be detected in the inactive enzyme.  This situation is clearly best explained by assuming that in the inactive enzyme the sulfhydryl has been oxidized to the -SOH form and that activation consists in the reduction to the sulfhydryl.  If we assume that inactive papain does have a sulfenic acid group, the remaining problem is that of its partner - the eight sulfur atom.  It is possible that the latter is present in acid-labile covalent linkage, R-S-X, between the half-cystine epsilon-amino group of lysine, an anhydride linkage with a gamma-COOH or a beta-COOH of a glutamic or aspartic acid residue, or an ester linkage between a sulfenic acid and a phenolic hydroxyl group of tyrosine or the -OH group of a serine or threonine residue.  The appearance of the fourth disulfide bond after acid treatment could then be interpreted as follows:  R-S-X + H+ ?RSH + X+ (1)  | RSH + RSOH ? RSSR (2) where R-SOH is the oxidized form of the sulfhydryl involved in the active site of papain.  The study of the region of sulfur absorption of proteins in the ultraviolet region, which may have led to the detection of unusual sulfur linkages, is unfortunately greatly complicated by a variety of effects due to other groupings under the conditions necessary for cleavage of acid-labile linkages.  The studies on partial acid hydrolysates of oxidized papain have indicated that ac cyteic-cysteic sequence in probably present.  Further, the studies on tryptic and chymotryptic hydrolysates of papain, S-carboxymethyl papain and oxidized papain indicate that there is a high concentration of half-cystine residues in some portion of the molecule.  A large number of reactions, e.g., dismutations, or reactions such as reaction 2 shown above, which depend on steric factors, could be visualized as being favored by the proximity of the half-cystine residues resulting both from sequential arrangement and the folding of the peptide chain.  The ultimate answer to the problem of chemical nature of the "active site" in papain will have to await the determination of the complete amino acid sequence of this protein,  The development of specific methods for the demonstration of highly labile linkages in proteins may also be necessary.