The production of papain—an agricultural industry for tropical America

In tropical America there is very little interest in the cultivation ofCarica papaya for the production of papain. This article, dealing primarily with field production and processing of crude papain, demonstrates that where coffee is grown commercially, papain may be produced profitably.     

Emerging roles of low-molecular-weight thiols at the host–microbe interface

Low-molecular-weight (LMW) thiols are an abundant class of cysteine-derived small molecules found in all forms of life that maintain reducing conditions within cells. While their contributions to cellular redox homeostasis are well established, LMW thiols can also mediate other aspects of cellular physiology, including intercellular interactions between microbial and host cells. Here we discuss emerging roles for these redox-active metabolites at the host–microbe interface. We begin by providing an overview of chemical and computational approaches to LMW-thiol discovery. Next, we highlight mechanisms of virulence regulation by LMW thiols in infected cells. Finally, we describe how microbial metabolism of these compounds may influence host physiology.     

Crystal structure of tarocystatin–papain complex: implications for the inhibition property of group-2 phytocystatins

Tarocystatin (CeCPI) from taro (Colocasia esculenta cv. Kaohsiung no. 1), a group-2 phytocystatin, shares a conserved N-terminal cystatin domain (NtD) with other phytocystatins but contains a C-terminal cystatin-like extension (CtE). The structure of the tarocystatin–papain complex and the domain interaction between NtD and CtE in tarocystatin have not been determined. We resolved the crystal structure of the phytocystatin–papain complex at resolution 2.03 Å. Surprisingly, the structure of the NtD–papain complex in a stoichiometry of 1:1 could be built, with no CtE observed. Only two remnant residues of CtE could be built in the structure of the CtE–papain complex. Therefore, CtE is easily digested by papain. To further characterize the interaction between NtD and CtE, three segments of tarocystatin, including the full-length (FL), NtD and CtE, were used to analyze the domain–domain interaction and the inhibition ability. The results from glutaraldehyde cross-linking and yeast two-hybrid assay indicated the existence of an intrinsic flexibility in the region linking NtD and CtE for most tarocystatin molecules. In the inhibition activity assay, the glutathione-S-transferase (GST)-fused FL showed the highest inhibition ability without residual peptidase activity, and GST-NtD and FL showed almost the same inhibition ability, which was higher than with NtD alone. On the basis of the structures, the linker flexibility and inhibition activity of tarocystatins, we propose that the overhangs from the cystatin domain may enhance the inhibition ability of the cystatin domain against papain.     

Protected immobilization of Taq DNA polymerase by active site masking on self-assembled monolayers of ω-functionalized thiols

A new efficient immobilization method that enables oriented immobilization of biologically active proteins was developed based on concepts of active site masking and kinetic control. Taq DNA polymerase was immobilized covalently on mixed self-assembled monolayers (SAMs) of ω-carboxylated thiol and ω-hydroxylated thiol through amide bonds between the protein and the carboxyl group in SAMs. Activity of the immobilized enzyme as large as 70% of solution-phase enzyme was achieved by masking the active site of the Taq DNA polymerase prior to the immobilization. In addition, the number of immobilization bonds was controlled by optimizing the carboxyl group concentration in the mixed monolayer. The maximum activity of immobilized Taq DNA polymerase was achieved at 5% of 12-mercaptododecanoic acid. The activity observed with protected immobilized enzyme was approximately 20 times higher than that observed with randomly immobilized enzyme. The maximum activity was acquired at a 1:1 DNA/enzyme masking ratio, immobilization pH 8.3, and within 10 min of reaction time. This concept of the active site masking and kinetic control of the number of covalent bonds between proteins and the surface can be generally applicable to a broad range of proteins to be immobilized on the solid surface with higher activity.     

The reaction of papain with Ellman''s reagent [5,5′-dithiobis-(2-nitrobenzoate) dianion]

1. The report by Robyt et al. (1971) that the 2-nitro-5-mercaptobenzoate dianion (Nbs(2-)) produced by reaction of papain with the 5,5'-dithiobis-(2-nitrobenzoate) dianion (Nbs(2) (2-); Ellman's reagent) cleaves the three disulphide bonds in papain is shown to be incorrect. 2. When partially active papain containing approx. 0.4 mol of thiol/mol of protein is incubated with excess of Nbs(2) (2-) at pH8, Nbs(2) (2-) reacts with the protein in an amount stoicheiometric with the cysteinyl thiol group of papain to produce Nbs(2-) in an amount stoicheiometric with the original papain cysteinyl thiol group, and the catalytically inactive mixed disulphide, papain-Nbs(-). 3. Papain catalyses the hydrolysis of Nbs(2) (2-) at pH10.5 probably by nucleophilic catalysis involving the enzyme's thiol group. 4. These results cast very serious doubts on the claim by Robyt et al. (1971) to have established a new general method for the determination of cystinyl disulphide residues in proteins.     

The products from papain and pepsin hydrolyses of guinea-pig immunoglobulins γ1G and γ2G

1. The products from papain and pepsin hydrolyses of the guinea-pig immunoglobulins gamma(1)G and gamma(2)G were isolated and characterized with regard to molecular weight, amino acid composition, hexose content and antigenic specificity. 2. Fragments Fab and (Fab')(2) from immunoglobulins gamma(1)G and gamma(2)G have similar electrophoretic and antigenic properties, but show some class-specific differences in amino acid composition. 3. Three Fc fragments were obtained after papain digestion of immunoglobulin gamma(2)G, namely, fragment Fc dimer (mol.wt. 58000), fragment Fc monomer (mol.wt. 29000) and fragment Fc' (mol.wt. 8000). A single crystalline fragment, namely fragment Fc' (mol.wt. 11000), was isolated after papain digestion of immunoglobulin gamma(1)G. 4. Peptic digestion of immunoglobulins gamma(1)G and gamma(2)G releases C-terminal fragments, namely, fragments pFc', of similar molecular weight (13000) but different amino acid compositions and distinct antigenic specificities. 5. Digestion-time studies show that immunoglobulin gamma(1)G is far more susceptible to proteolysis than is immunoglobulin gamma(2)G and suggest that at least a proportion of molecules are split primarily at a site that liberates fragment gamma(1)Fc'.     

Evidence that binding to the S2-subsite of papain may be coupled with catalytically relevant structural change involving the cysteine-25–histidine-159 diad. Kinetics of the reaction of papain with a two-protonic-state reactivity probe containing a hydrophobic side chain

A method is proposed by which site-specific reactivity probes that exhibit different reactivities in two ionization states can be used to detect association-activation phenomena that involve repositioning of acid/base groups in enzyme active centres. The pH-dependences of the apparent second-order rate constants (k) for the reactions of the thiol group of papain (EC 3.4.22.2) with a series of two-protonic-state reactivity probes are compared. The short-chain probes, 2,2'-dipyridyl disulphide and n-propyl 2-pyridyl disulphide, react at pH6 in adsorptive complexes and/or transition states with geometries that do not permit hydrogen-bonding of the pyridyl nitrogen atom with the active-centre imidazolium ion, as evidenced by the rate minima at pH6 and the rate maxima at pH4 provided by reagent protonation. Only when the probe molecule, e.g. 4-(N-aminoethyl 2'-pyridyl disulphide)-7-nitrobenzo-2-oxa-1,3-diazole [compound(III)], contains a long hydrophobic side chain is the reaction characterized by maximal rates at about pH6, as in the acylation step of the catalytic act (at pH6, k(compound III)/k(2,2'-dipyridyl disulphide) approximately 100). It is proposed that this striking difference in profile shape may result from binding of the hydrophobic side chain of compound (III) possibly in the S(2)-subsite of papain, which promotes a change in catalytic-site geometry involving repositioning of the imidazolium ion of histidine-159 and hydrogen-bonding with the N atom of the leaving group, as has been postulated to occur in the acylation step of substate hydrolysis.     

Separation of two functional domains of the family F/10 β-xylanase from Streptomyces olivaceoviridis E-86 limited proteolysis with papain and some of their properties

Limited proteolysis of papain on the intact family F/10 -xylanase (45 kDa) led to the isolation of catalytic domain (32 kDa) and xylan-binding domain (15 kDa). The two truncated protein fragments were purified by gel filtration to homogeneity. The purified catalytic domain was fully active against 4-O-methyl-d-glucurono-d-xylan, and the action mode on xylan was the same as the intact xylanase. However, the removal of xylan-binding domain reduces dramatically the capability of adsorption for xylan.     

Vascular non-protein thiols: prooxidants or antioxidants in atherogenesis?

Abstract-cell-mediated lipoprotein oxidation may be due to generation of non-protein thiols (NP-SH) from cystine with formation of oxidizing species. However, NP-SH, especially GSH, may also exert antioxidant effects in vitro and in vivo. To further investigate whether vascular NP-SH could be prooxidants or antioxidants in atherosclerosis, we have correlated the aortic content of NP-SH with that of lipoperoxides in 10 rabbits fed on a fat-enriched atherogenic diet for 9 weeks. As compared to 7 control rabbits, aortic NP-SH and lipoperoxides were significantly increased in the fat-fed animals. The levels of NP-SH were strongly and inversely correlated with those of lipid peroxidation in the atherosclerotic aorta (r(s) -0.92, P < 0.0001 for thiobarbituric acid reactive substances, and r(s) -0.80, P < 0.01 for fluorescent damage products of lipid peroxidation). A similar trend was evident also in the control rabbits (r(s) -0.60 for both indices of lipid peroxidation). Thus, the present data suggest that vascular NP-SH exert significant antioxidant-antilipoperoxidative effects in vivo especially in fat diet-related atherogenic conditions.     

A reinvestigation of residues 64–68 and 175 in papain. Evidence that residues 64 and 175 are asparagine

The tryptophan-containing peptides were isolated from the chymotryptic digest of S-carboxymethylated papain. Residue 175, which is strongly hydrogen-bonded to the active-site histidine residue in the tertiary structure of papain, is asparagine, confirming the work of Kimmel, Rogers & Smith (1965). Its function is probably to maintain the orientation and tautomeric state of the imidazole ring of histidine-159. The amino acid sequence predicted from the electron-density map of papain for residues 64-68 was confirmed, but residue 64 is asparagine, not aspartic acid. This residue, which is about 10 A from the thiol group of the active-site cysteine-25, cannot therefore be a site of electrostatic attraction for substrates of basic amino acids.     

Quantitative microspectrophotometrical determination of protein thiols and disulfides with 2,2′-dihydroxy-6,6′-dinaphthyldisulfide (DDD)

Summary 2,2-dihydroxy-6,6-dinaphthyldisulfide (DDD) reacts with both protein thiol groups and with protein disulfides (Nöhammer 1977). By varying the pH of the DDD-reaction, as well as the reaction times, the complex reaction became specific with respect to the histochemical demonstration of protein-SH groups. Furthermore, the application of the histochemical DDD-reaction following quantitative blockade of the protein-SH groups enabled the demonstration of distinctive DDD-reactive disulfides. The specifity and the extent of the different histochemical DDD-staining methods were investigated by comparing macroscopically determined values of the protein-SH-contents, and the contents of the different kinds of disulfides in Ehrlich-ascites-tumor cells (EATC) (Modig 1968; Hofer 1975), with microspectrometrical values determined with the MCN-method of Nöhammer et al. (1981), and with microspectrometrical values measured on EATC after staining with the modified DDD-methods. Also, the method for the histochemical demonstration of protein-SH with DDD after the reduction of the disulfides with thioglycolate was investigated and conditions were found by which the protein-SH content could be determined quantitatively with DDD and Fast blue B after the reduction of the disulfides. With the aid of the MCN-method (Nöhammer et al. 1981), the intracellular disulfide interchange reaction was investigated, leading to pH-dependent changes of the SH-SS-ratio of fixed cells during their incubation in aqueous media. In addition the possibility of protein loss during the long incubation times of the fixed cells in the DDD-solutions was investigated. For the quantitative microscpecrometrical determination of the protein content of EATC the so-called tetrazonium-coupling method, optimized by Nöhmmer (1978) and calibrated by Nöhammer et al. (1981), was used.Dedicated to Prof. Dr. E. Ziegler on the occasion of his 70^th birthday     

Reversible addition of thiols to steroid α,β-unsaturated carbonyl groups: Possible role in steroid-protein interactions

The equilibrium constant (M^?1) for addition of 2-mercaptoethanol at 25°C was found to be 7 × 10³ with 2-cyclohexenone, to be near 2 with 3-methyl-2-cyclohexenone, 19-norprogesterone, and 19-nortestosterone, and to be in the range of 0.01–0.03 with progesterone and testosterone. The results demonstrate that thiol addition to steroid α,β-unsaturated carbonyl groups is sufficiently favorable to be of potential importance in steroid-protein interactions.     

1,8-Naphthalimide–Cu(ІІ) ensemble based turn-on fluorescent probe for the detection of thiols in organic aqueous media

A 1,8-naphthalimide–Cu(II) ensemble was rationally designed and synthesized as a new turn-on fluorescent probe utilizing the ‘chemosensing ensemble’ method for detections of thiols (Cys, Hcy and GSH) with high selectivity over other α-amino acids at pH 7.4 in organic aqueous media (EtOH/HEPES, v/v = 9:1). The recognition mechanism was attributed to the remove Cu(II) from the 1,8-naphthalimide–Cu(II) ensemble by thiols and the release of flurescence of ligand 1. Remarkable fluorescence enhancements were therefore observed in the sensing process of thiols by the 1,8-naphthalimide–Cu(II) ensemble. Furthermore, the 1,8-naphthalimide–Cu(II) ensemble was successfully applied to the fluorescence imaging of thiols in CHO cells with high sensitivity and selectivity.     

Synthesis of Nα,Nδ-dicarbobenzoxy-L-ornithyl-β-alanine benzyl ester,a derivative of salty peptide,in 1,1,1-trichloroethane with polyethylene glycol-modified papain

Summary N,N-Dicarbobenzoxy-L-ornithyl--alanine benzyl ester, a derivative of salty peptide, was synthesized from N,N-dicarbobenzoxy-L-ornithine ethyl ester and -alanine benzyl ester in 1,1,1-trichloroethane using papain modified with polyethylene glycol. The peptide bond formation proceeded in a transparent organic solvent at room temperature and the product was obtained as precipitates from the reaction system.     

p-Chloromercuribenzoate specifically modifies thiols associated with the active sites of β-Ketoadipate enol-lactone hydrolase and succinyl CoA: β-Ketoadipate CoA transferase

-Ketoadipate enol-lactone hydrolase (EC 3.1.1.24) and succinyl CoA:-ketoadipate transferase (EC 2.8.3.6) catalyze consecutive metabolic reactions in bacteria. The enzymes appear to be members of different families of related proteins. Enzymes within the enol-lactone hydrolase family appear to have diverged so extensively that common ancestry sometimes is not directly evident from comparison of NH₂-terminal amino acid sequences of the proteins. Amino acid sequences at or near the active sites of the enzymes are likely to have been conserved, and hence a chemical proble that reacted specifically near the active sites of the enzymes might identify regions of amino acid sequence in which evolutionary affinities among widely divergent proteins could be identified. p-Chloromercuribenzoate appears to be such a probe because enol-lactone hydrolases and CoA transferases from Acinetobacter calcoaceticus and Pseudomonas putida were completely inhibited by stoichiometric quantities of the compound which appears to modify selectively cysteinyl side chains at or near the active sites of the enzymes. Stoichiometric inhibition of P. putida enol-lactone hydrolase was observed in the presence of excess dithiothreitol; therefore the reactive cysteinyl residue in this enzyme appears to be nucleophilic. The hydrolase is inhibited by -ketoadipate, but the compound must be supplied at 10 mM concentrations in order to achieve 50% inhibition, so the product inhibition is unlikely to be significant under physiological conditions.Dedicated to Roger Stanier with whom biochemistry was without tears (Stanier 1980)     

Isolation and characterization of the four major cysteine-proteinase components of the latex of carica papaya L. reactivity characteristics towards 2,2′-dipyridyl disulfide of the thiol groups of papain,chymopapains A and B,and papaya peptidase A

High-quality spray-dried latex of Carica papaya L was fractionated by using SP-Sephadex-C50. The four major cysteine-proteinase components—papain(E.C.3.4.22.2), chymopapains A and B(jointly designated currently as E.C.3.4.22.6), and papaya peptidase A—were isolated and characterized by protein chemical methods and by study of their thiol groups using2,2′-dipyridyl disulfide as a two-protonic-state titrant and reactivity probe. Papain and papaya peptidase A each contain one thiol group/molecule, which in each case is part of the catalytic site, as evidenced by high reactivity toward2,2′-dipyridyl disulfide in acidic media. Chymopapains A and B each contain two thiol groups/molecule, only one of which is essential for catalytic activity. The reactivities of the thiol groups of these enzymes toward2,2′-dipyridyl disulfide at pH4 and10 and activity loss analysis by Tsou Chen-Lu plots each provides a ready means of distinguishing among the four cysteine proteinases. The nonessential thiol groups of chymopapains A and B readily undergo irreversible oxidation. The reactivity characteristics of the essential thiol groups of the four enzymes suggest the presence of somewhat similar interactive cysteine-histidine catalytic center systems in papain, papaya peptidase A, and chymopapain B but a different type of catalytic center environment in chymopapain A.     

Reactions of thiols and selenols with the FeMoS cluster [Fe(MoS4)2]3−

The known complex [Et₄N]₃[Fe(MoS₄)₂] has been shown by EPR and visible spectral studies to react with both thiophenol and selenophenol. The reaction results in a change in the characteristic S=3/2 EPR spectrum of this species from a complex rhombic pattern to one of a very simple axial appearance. Although this effect is similar to that observed for reaction of these species with the iron- molybdenum cofactor of nitrogenase, a moiety known to consist of a FeMoS cluster species, the large excesses of reagents and the long reaction times required for complete formation of product indicate that these reactions are of questionable direct relevance to the biological system. The reaction corresponding to the EPR spectral change from rhombic to axial in the [Fe(MoS₄)₂]³⁻/PhSeH system has also been partially characterized by product isolation which indicates that attack by selenol of the two terminal MoS₂ moieties in the starting material has occurred.     

Defense response in non-genomic model species: methyl jasmonate exposure reveals the passion fruit leaves’ ability to assemble a cocktail of functionally diversified Kunitz-type trypsin inhibitors and recruit two of them against papain

Main conclusion

Multiplicity of protease inhibitors induced by predators may increase the understanding of a plant’s intelligent behavior toward environmental challenges. Information about defense mechanisms of non-genomic model plant passion fruit (Passiflora edulis Sims) in response to predator attack is still limited. Here, via biochemical approaches, we showed its flexibility to build-up a broad repertoire of potent Kunitz-type trypsin inhibitors (KTIs) in response to methyl jasmonate. Seven inhibitors (20–25 kDa) were purified from exposed leaves by chromatographic techniques. Interestingly, the KTIs possessed truncated Kunitz motif in their N-terminus and some of them also presented non-consensus residues. Gelatin-Native-PAGE established multiple isoforms for each inhibitor. Significant differences regarding inhibitors’ activity toward trypsin and chymotrypsin were observed, indicating functional polymorphism. Despite its rarity, two of them also inhibited papain, and such bifunctionality suggests a recruiting process onto another mechanistic class of target protease (cysteine-type). All inhibitors acted strongly on midgut proteases from sugarcane borer, Diatraea saccharalis (a lepidopteran insect) while in vivo assays supported their insecticide properties. Moreover, the bifunctional inhibitors displayed activity toward midgut proteases from cowpea weevil, Callosobruchus maculatus (a coleopteran insect). Unexpectedly, all inhibitors were highly effective against midgut proteases from Aedes aegypti a dipteran insect (vector of neglected tropical diseases) opening new avenues for plant-derived PIs for vector control-oriented research. Our results reflect the KTIs’ complexities in passion fruit which could be wisely exploited by influencing plant defense conditions. Therefore, the potential of passion fruit as source of bioactive compounds with diversified biotechnological application was strengthened.     

Glutathione and other low-molecular-weight thiols relax guinea pig trachea ex vivo: interactions with nitric oxide?

The aim of this study was to determine the effects of glutathione (GSH) on trachea smooth muscle tension in view of previously reported interactions between GSH and nitric oxide (NO) (Gaston B. Biochim Biophys Acta 1411: 323-333, 1999; Kelm M. Biochim Biophys Acta 1411: 273-289, 1999; and Kharitonov VG, Sundquist AR, and Sharma VS. J Biol Chem 270: 28158-28164, 1995) and the high (millimolar) concentrations of GSH in trachea epithelium (Rahman I, Li XY, Donaldson K, Harrison DJ, and MacNee W. Am J Physiol Lung Cell Mol Physiol 269: L285-L292, 1995). GSH and other thiols (1.0-10 mM) dose dependently decreased the tension in isolated guinea pig tracheas. Relaxations by GSH were paralleled with sevenfold increased nitrite levels (P < 0.05) in the tracheal effluent, suggesting an interaction between GSH and NO. However, preincubation with a NO scavenger did not reduce the relaxations by GSH or its NO adduct, S-nitrosoglutathione (GSNO). Inhibition of guanylyl cyclase inhibited the relaxations induced by GSNO, but not by GSH. Blocking potassium channels, however, completely abolished the relaxing effects of GSH (P < 0.05). Preincubation of tracheas with GSH significantly (P < 0.05) suppressed hyperreactivity to histamine as caused by removal of tracheal epithelium. These data indicate that GSH plays a role in maintaining tracheal tone. The mechanism is probably an antioxidative action of GSH itself rather than an action of NO or GSNO.