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PubMed1984

Peptides of human erythrocyte band 3 protein produced by extracellular papain cleavage.

The structure of the human red blood cell membrane band 3 protein has been investigated by proteolysis of intact cells with papain. Papain cleaves the 35,000-dalton chymotryptic peptide (CH35) of band 3 into two integral fragments of Mr 7,500 (P7) and 28,000 (P28). The papain cleavage of CH35 causes inhibition of band 3-catalyzed Cl transport. The peptide P28 carries the band 3 carbohydrate and also contains the 2 cysteine residues in CH35. The anion transport inhibitor H2DIDS (4,4'-diisothiocyanodihydrostilbene-2,2'-disulfonate) reacts covalently with a lysine residue on P28, rather than P7, in native band 3. However, if the cells are first digested with papain and then reacted with H2DIDS, there is significant covalent reaction with P7, producing a covalent cross-link between P7 and the 60,000-dalton chymotryptic peptide (CH60). Graded papain digestion experiments and end group determinations indicate that the alignment of the band 3 peptides is N-CH60-P7-P28-COOH. The NH2-terminal sequence of P7 is identical with a segment of a peptic fragment of band 3 recently sequenced (Brock, C.J., Tanner, M.J.A., and Kempf, C. (1983) Biochem. J. 213, 577-586). This published sequence, plus our sequence data on CH35 and P7, show that papain cleaves the outer surface of CH35 at two sites, which are 6 residues and 71 residues from the chymotrypsin cleavage site. The 65-residue peptide (P7) between these sites is the best characterized segment of band 3 thus far described: its sequence and location in the band 3 primary structure are now known, and both ends of the peptide are unambiguously exofacial.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1975

The repair, protection and sensitization of papain with respect to inactivation by H2O2 and OH: effects of dithiothreitol, penicillamine, cystine and penicillamine disulphide.

While dithiothreitol repairs the peroxide-produced sulphenic acid derivative of papain in a fast reaction involving only one dithiothreitol molecule, penicillamine reacts with it to form papainCys25SSPen. Disulphide is also formed in the absence of peroxide (see article) by reactions of papainCys25S- AND PenS- radicals derived from -OH reactions in penicillamine-papain mixtures. A similar formation of papainCys25SSCys occurs in mixtures of cysteine and papain. However, unlike papainCys25SSCys, papainCys25SSPen cannot easily be restored to the active form of papain by the exchange reaction with CysSH, and this may have significance for an understanding of the sensitizing action of penicillamine observed in some in vivo systems. Under the action of OH radicals dithiothreitol has less of a tendency to form mixed disulphides and is more effective in repairing papain-OH intermediates than either cysteine or penicillamine. Due to secondary reactions of RSOH and other oxidized species the disulphides of cysteine and penicillamine are less effective than the sulphydryls in protecting papain against inactivation by -OH.

Cystine↗
PubMed1965

DIRECT EVIDENCE FOR AN ACYLATED THIOL AS AN INTERMEDIATE IN PAPAIN- AND FICIN-CATALYSED HYDROLYSES.

1. Methyl thionohippurate was prepared and shown to be a specific substrate for both papain and ficin. 2. The ultraviolet-absorption properties of the acyl-enzyme intermediate for both papain and ficin with methyl thionohippurate was that expected of an acyl-thiol. The possibility that other functional groups present in papain or ficin might be the site of acylation has been excluded. 3. The change in extinction at the absorption maximum with time was as expected for the acyl-enzyme on the basis of the known Michaelis-Menten parameters for methyl thionohippurate. 4. The variation of extinction with initial substrate concentration for both papain and ficin was that expected from the Michaelis-Menten parameters. 5. The extinction of the absorption maximum of the thionohippuryl-enzyme intermediate was suppressed by the addition of methyl hippurate to the extent predicted from the Michaelis-Menten parameters. 6. The decay of the extinction for the acyl-enzyme was arrested by adjusting the pH of the solution to 2.5. 7. These experiments provide compelling evidence that the acylation by substrate of both papain and ficin takes place through a thiol residue.

Acylation↗
PubMed2002

Effect of metal ions on structure and activity of papain from Carica papaya.

Papain, a powerful proteolytic enzyme, is an endoprotease belonging to cysteine endopeptidase family. It is used extensively in food processing especially in tenderization of meat. In this study, we have made an attempt to show the structure activity relationship of this enzyme and the role of calcium and magnesium ions in the activity and stability of the enzyme. Results of activation and stabilization of the enzyme by these cations showed concentration dependent effect. The enzymatic activity of papain increases to a maximum of 18% and 24% in presence of calcium and magnesium ions at 1 x 10(-3) M concentration, respectively. Thermal denaturation studies showed that the binding of calcium and magnesium ions bring about change in the thermal stability of papain at various concentrations of these metal ions. Far ultraviolet circular dichroic studies showed no significant change in the alpha-helix and beta-sheet structure of the papain upon binding of these metal ions. The mechanism underlying the structure activity relationship of papain in presence of these metal ions have been discussed here with reference to the ionic radii, ligand binding preference, coordination numbers and the electrostatic forces between the protein molecule and cations present in the microenvironment of the enzyme.

Binding Sites↗
PubMed2006

Stability of protease in organic solvent: structural identification by solid-state NMR of lyophilized papain before and after 1-propanol treatment and the corresponding enzymatic activities.

Lyophilized enzyme powder is often used in organic solvents. However, the enzymatic activity decreases during the reaction process. In the present study, the relation between structural stability and enzymatic activity in an organic solvent was investigated. 13C cross-polarization magic angle spinning NMR spectroscopy was used to determine the secondary structure of lyophilized papain in the solid-state. Deconvolution of the peaks of the backbone carbonyl carbons suggested that the proportion of beta-sheet conformation increased after lyophilization from a phosphate buffer solution. The esterification of N-benzyloxycarbonyl phenylalanylalanine amide was attempted using the lyophilized papain as a catalyst in anhydrous 1-propanol. The yield of ester was 46.1% after 48 h at 50 degrees C, but this reaction slowed remarkably after 48 h. When the lyophilized papain was suspended in anhydrous 1-propanol for 7 days without the substrate, the proportion of beta-sheet conformation was further increased and the suspended papain had no activity. These results suggest that the increase in beta-sheet conformation caused inactivation of papain. The increase in beta-sheet conformation caused by both lyophilization and suspension in propanol was found, which was related to a decrease in enzymatic activity.

1-Propanol↗
PubMed2000

Differential effect toward inhibition of papain and cathepsin C by recombinant human salivary cystatin SN and its variants produced by a baculovirus system.

Human salivary cystatin SN (CsnSN) is a member of the cystatin superfamily of cysteine proteinase inhibitors. In this study we used a baculovirus expression system to produce a full-length unaltered CsnSN and its variants. The variants were constructed with the changes in the three predicted proteinase-binding regions: the N-terminus (variant N(12-13), G12A-G13A), beta-hairpin loop I (variant L(56-58), Q56G-T57G-V58G) and beta-hairpin loop II (variant L(106-107), P106G-W107G). The secreted CsnSNs were purified using sequential spiral cartridge ultrafiltration and DE-52 radial flow chromatography. The purified proteins were examined for papain- and cathepsin C-inhibition. The wild-type CsnSN, and variants N(12-13) and L(106-107) bound tightly to papain (K(i) < 10 pM), whereas mutation in the loop I reduced binding affinity 5700-fold (K(i) = 57 nM). On the other hand, the wild-type CsnSN bound to cathepsin C less tightly (K(i) = 100 nM). The mutation in the N-terminus or loop I reduced binding affinity by 16 (K(i) = 1.6 microM)- and 19-fold (K(i) = 1.9 microM), respectively, while mutation in loop II resulted in an ineffective cathepsin C inhibitor (K(i) = 14 microM). Collectively, these results suggest that the N-terminal G12-G13 residues of CsnSN are not essential for papain inhibition but play a role in cathepsin C inhibition; residues Q56-T57-V58 in the loop I are essential for both papain and cathepsin C inhibitions, and residues P106-W107 in the loop II are not important for papain inhibition but essential for cathepsin C inhibition. These results demonstrated that CsnSN variants have different effects toward different cysteine proteinases.

Animals↗
PubMed2001

mRNA expression of basic fibroblast growth factor from a single intratracheal instillation of papain-induced emphysema in rats.

The relations between mRNA expression of basic fibroblast growth factor (bFGF) and the changes in collagen I and collagen III in pulmonary tissues from a single intratracheal instillation of papain-induced emphysema in rats were investigated. Wistar rats (n = 42) were randomly divided into normal group and emphysema model 1, 3, 5, 7, 15, 30-day groups (n = 6 in each group). The rat model of emphysema was induced by a single intratracheal instillation of papain. The results of immunohistochemistry SABC and in situ hybridization with bFGF probe were quantitatively analyzed to examine the changes of collagen I and collagen III and bFGF mRNA expression in lung tissues and the percent of positive expression of bFGFmRNA in alveolar macrophages. The results were as follows: (1) In the emphysema model groups the optical densities of collagen I and collagen III began to increase after 3 days, reached the highest at the 7th day, and began to reduce at the 15th day; (2) No expression of bFGFmRNA in pulmonary tissues was detectable in the normal group. The positive expression of bFGFmRNA was detectable in lung tissues one day after the intratracheal instillation of papain. The average optical densities reached the peak (41.895 +/- 7.017) at the 7th day, significantly higher than in the normal group (0.581 +/- 0.139, P < 0.01). The positive expression of bFGFmRNA in lung tissues began to reduce at the 15th day; (3) Positive expression of bFGFmRNA in alveolar macrophages of instillation papain rats was detectable 3 days after the intratracheal instillation of papain, and reached the highest at the 7th day with the percent of positive expression of bFGF mRNA in alveolar macrophages being 70.13 +/- 11.21, higher than in the normal group (5.12 +/- 0.18, P < 0.01); (4) The expression of bFGF mRNA in the lung tissues and macrophages was positively related with the changes in collagen I and collagen III (P < 0.01 or P < 0.05) respectively. It was suggested that the up-regulation of bFGF mRNA expression during the development of emphysema can lead pulmonary interstitial fibrosis, which may take part in the injury and repair and the lung tissue reconstruction.

Animals↗
PubMed1986

L-cystine transport by papain-treated rat renal brush-border membrane vesicles.

In papain-treated rat renal brush-border membrane vesicles, cystine uptake was enhanced under sodium gradient conditions. This effect was not observed when sodium was equilibrated across the vesicle membrane or when sodium was completely absent from the incubation medium. The increased rate of cystine uptake occurred within the first two minutes of incubation and coincided with the period of increased flux of sodium known to occur after papain treatment. Under sodium gradient conditions, the Vmax of cystine uptake by treated vesicles was 65% greater while the Km was 25% lower than the value observed in untreated membranes. The increased cystine uptake after papain treatment occurred when medium cystine was in the electroneutral form. In the absence of a sodium gradient, cystine uptake by control membranes was insensitive to changes in membrane potential and this was unaltered after papain treatment. Exposure of the membranes to papain also resulted in a profound decrease in cystine binding which occurs in native membranes incubated with cystine. The fact that cystine uptake is unchanged under sodium equilibration and even enhanced under sodium gradient conditions suggests that the component of cystine binding is not essential for cystine transport and may represent non-specific binding to membrane proteins.

Animals↗
PubMed1977

Active water-insoluble derivatives of papain and other enzymes based on preformed diazonium-type supports.

Papain (EC 3.4.22.2) has been coupled to supports of titanium (IV) oxide and cellulose, which are particulate and pre-coated with diazotised 1,3-diaminobenzene, giving water-insoluble and stable derivatives which possess low proteolytic activity but high esterolytic activity. In addition the reversible binding of zinc (II) at the active site of papain has been exploited to inhibit protectively the enzyme during its linkage to the aforementioned supports, thereby yielding water-insoluble derivatives of papain having superior activity upon reactivation with EDTA. Application of the improved procedure of enzyme coupling to macroporous cellulose particles gave a water-insoluble derivative of papain having further enhanced proteolytic activity. Other properties of the water-insoluble derivatives of papain and of similarly prepared water-insoluble conjugates of urease (EC 3.5.1.5) and cholinesterase (EC 3.1.1.8) with cellulose are also reported.

Cellulose↗
PubMed1991

Putative papain-related thiol proteases of positive-strand RNA viruses. Identification of rubi- and aphthovirus proteases and delineation of a novel conserved domain associated with proteases of rubi-, alpha- and coronaviruses.

A computer-assisted comparative analysis of the amino acid sequences of (putative) thiol proteases encoded by the genomes of several diverse groups of positive-stranded RNA viruses and distantly related to the family of cellular papain-like proteases is presented. A high level of similarity was detected between the leader protease of foot-and-mouth-disease virus and the protease of murine hepatitis coronavirus which cleaves the N-terminal p28 protein from the polyprotein. Statistically significant alignment of a portion of the rubella virus polyprotein with cellular papain-like proteases was obtained, leading to tentative identification of the papain-like protease as the enzyme mediating processing of the non-structural proteins of this virus. Specific grouping between the sequences of the proteases of alpha-viruses, and poty- and bymoviruses was revealed. It was noted that papain-like proteases of positive-stranded RNA viruses are much more variable both in their sequences and in genomic locations than chymotrypsin-related proteases found in the same virus class. A novel conserved domain of unknown function has also been identified which flanks the papain-like proteases of alpha-, rubi- and coronaviruses.

Alphavirus↗
PubMed1995

Hairpin loop mutations of chicken cystatin have different effects on the inhibition of cathepsin B, cathepsin L and papain.

Five recombinant hairpin loop variants of chicken cystatin (delta V55, delta V55-S56, delta P103-L105, delta I102-Q107, loop2-KD2) were constructed by cassette mutagenesis, expressed in E. coli, purified to homogeneity, characterized by protein-chemical means and by their inhibitory properties. The variant forms, modified in two of the three postulated cysteine proteinase binding regions, were inhibitorily active. However, the equilibrium dissociation constants of the complexes between papain as well as human cathepsin B or L and the cystatin variants show a weaker affinity for all three enzymes compared with recombinant chicken cystatin. These results prove the contribution of both hairpin loops to complex formation with the three enzymes. Furthermore, the kinetic constants indicate discrete differences in the molecular mechanism of interaction between chicken cystatin and papain, cathepsin B, and cathepsin L. Inhibition of cathepsin L was much less affected than inhibition of papain or cathepsin B by the modifications achieved in the five variants. Remarkably, at high enzyme concentration (above 0.5 nM) inhibition of papain by these variants was 'temporary', that means, active papain was released from the enzyme-inhibitor complex within minutes to hours (compare [1]).

Amino Acid Sequence↗
PubMed1986

Amino acid sequence of the variable region of heavy chain in immunoglobulin (Mot) having unusual papain cleavage sites.

An IgGl(lambda) Mot myeloma protein showed a unique susceptibility toward papain digestion. The Fab fragment of Mot was more digestible with papain than the Fc fragment. This phenomenon was found to occur by unusual cleavage of the Fd fragment with papain. Determination of the complete primary structure of the V region of the H chain of Mot identified two papain cleavage sites in the second complementarity-determining region (CDR). Amino acid sequence of the cleavage sites was Ser(55)-Asp-Asp-Argdecrease-Thr-Thr-Tyr-Gly-Pro-Argdecrease- Ser-Gln- (decrease = cleavage site). In the vicinity of these cleavage sites, many hydrophilic and polar residues are present and the predicted secondary structure near these cleavage sites suggested that this region was exposed on the surface of the molecule, and that the unusual papain cleavage of the IgG Mot might be caused by a unique conformation of the molecule, making it highly susceptible to enzyme digestion.

Amino Acid Sequence↗
PubMed1987

Papain fragmentation of the gastric (H+ + K+)-ATPase.

Membrane-bound (H+ + K+)-ATPase purified from hog gastric mucosa was exposed to limited papain digestion. Such treatment resulted in a rapid inhibition of the K+-stimulated adenosine triphosphatase and p-nitrophenyl phosphatase activities, with about 90% of these activities lost after 3 min incubation at 37 degrees C with 0.1 units of papain per mg of enzyme protein. Parallel to the inhibition of the enzyme activities, there was a production of a 77 kDa membrane-bound fragment containing the aspartyl phosphate residue of the phospho-intermediate. This fragment accounted for about 45% of the total enzyme protein after the 3 min papain treatment. The digestion barely affected the steady-state level of phosphorylation, allowed the aspartyl phosphate of the 77 kDa fragment to undergo the transition to the E2P form, and did not significantly alter the fraction of ADP-sensitive phosphoenzyme. The presence of KCl, however, depressed the steady-state level of phosphoenzyme formed from [gamma-32P]ATP considerably less than that of the control enzyme. With further exposure to papain the 77 kDa peptide became fragmented into a 28 kDa soluble peptide that retained the phosphorylating site. Binding of fluorescein 5'-isothiocyanate (FITC) to the native enzyme did not affect the sites of papain hydrolysis because the same peptide fragments were obtained. The FITC reaction site was also in the 28 kDa soluble peptide fragment.

Adenosine Diphosphate↗
PubMed1994

Clarification of substrate specificity of papain by crystal analyses of complexes with covalent-type inhibitors.

In order to investigate the stereo specificity of papain Sn subsites (n = 1-4) at the atomic level, two kinds of covalent-type inhibitors were designed based on the previous results on papain-E-64 and papain-E-64-c interactions, and their complex crystals with papain were analyzed by X-ray diffraction. The results show that the hydrophobic regions consisting of Val-133, Val-157 and Asp-158 and of Tyr-61, Gly-66 and Tyr-67 residues interact with the hydrophobic P2 and P3 side chains of inhibitors, thus indicating the function of the former and latter binding pockets as S2 and S3 subsites, respectively. Furthermore, the X-ray analysis suggests that the papain has no definite Sn subsite of n > or = 4, and the S3-P3 hydrophobic interaction is significantly affected by the Pn side chain (n > or = 4) of both the substrate and the inhibitor.

Cysteine Proteinase Inhibitors↗
PubMed1994

New substrates of papain, based on the conserved sequence of natural inhibitors of the cystatin family.

A series of peptide substrates with different fluorogenic leaving groups has been synthesized. The peptide moiety in these substrates mimics a highly conserved sequence (QVVAG) in the natural reversible inhibitors of cysteine proteinases, the cystatins, that participates to the tight binding of target proteinases. This sequence is invariably cleaved at the A-G bond when synthetic peptides containing it were incubated with papain. AEC and AMC fluorophores were therefore attached to the Ala residue to construct new substrates for cysteine proteinases. The solubility of the resulting substrates was improved by attaching a N-terminal gluconoyl group, or by introducing an arginyl residue at P5 (nomenclature of Schechter I, Berger A (1967) Biochem Biophys Res Commun 27, 157-162). Neither induced significant changes in the kcat/Km values with papain. Those values were all in the 10(5) M-1 s-1 range. The kcat/Km was increased 10-50-fold by using substrates with intramolecularly quenched fluorescence. With these, the enzyme specificity on both sides of the scissile bond can be investigated. The substrate Abz-QVVAGA-EDDnp is among the most sensitive papain substrates ever reported, with a kcat/Km value of 29 10(6) M-1 s-1. The positioning and conformation of the bound QVVA moiety within the active site of papain were predicted by molecular modelling using the X-ray coordinates of a peptide inhibitor-papain complex.

Amino Acid Sequence↗
PubMed2004

Characterization of a partially folded intermediate of papain induced by fluorinated alcohols at low pH.

A systematic investigation of the effects of aqueous 1,1,1,3,3,3-hexafluoroisopropanol (HFIP) and 2,2,2-trifluoroethanol (TFE) on the structure of acid-unfolded papain (EC. 3.4.22.2) was made using circular dichroism (CD), intrinsic tryptophan fluorescence, and 1-anilino 8-sulfonic acid (ANS) binding. At pH 2, papain exhibits substantial secondary structure as beta-sheet and is relatively less denatured as compared to 6 M guanidine hydrochloride (GdnHCl) but loses the persistent tertiary structure of the native state. Addition of HFIP and TFE caused an induction of alpha-helical structure as evident from the increase in the mean residue ellipticity value at 208 and 222 nm. Induction was 20% more in HFIP than TFE. Interestingly, at 13% (v/v) HFIP and 30% (v/v) TFE a near-UV CD spectrum approaches the native-like spectral features. Tryptophan fluorescence studies indicate the change in the environment of the tryptophan residues on the addition of HFIP and TFE to acid-unfolded papain. Maximum ANS binding occurs at 13% (v/v) HFIP and 30% (v/v) TFE, suggesting a compact "molten globule"-like conformation with enhanced exposure of hydrophobic surface area. Acid-unfolded papain in presence of 13% (v/v) HFIP and 30% (v/v) TFE showed the recovery of enzymatic activity by 54 and 61%, respectively. Thermal stability of these states was assessed by changes in fluorescence emission maximum and absorbance at 292 nm. Temperature-induced unfolding of papain at pH 2 was non-cooperative and the transition curves were biphasic in nature. Temperature-induced unfolding of HFIP and TFE-induced state was weakly cooperative in comparison to cooperative transition of native.

Alcohols↗
PubMed2005

Nitric oxide-related species-induced protein oxidation: reversible, irreversible, and protective effects on enzyme function of papain.

Protein oxidation, irreversible modification, and inactivation may play key roles in various neurodegenerative disorders. Therefore, we studied the effects of the potentially in vivo occurring nitric oxide-related species on two different markers of protein oxidation: protein carbonyl generation on bovine serum albumine (BSA) and loss of activity of a cysteine-dependent protease, papain, in vitro by using Angeli's salt, papanonoate, SIN-1, and S-nitrosoglutathione (GSNO) as donors of nitroxyl, nitric oxide, peroxynitrite, and nitrosonium ions, respectively. Angeli's salt, SIN-1, and papanonoate (0-1000 microM) all generated a concentration-dependent increase in carbonyl formation on BSA (107, 60, and 45%, respectively). GSNO did not affect carbonyl formation. Papain was inhibited by Angeli's salt, SIN-1, papanonoate, and GSNO with IC50 values of 0.62, 2.3, 54, and 80 microM, respectively. Angeli's salt (3.16 microM)-induced papain inactivation was only partially reversible, while the effects of GSNO (316 microM) and papanonoate (316 microM) were reversible upon addition of excess DTT. The Angeli's salt-mediated DTT-irreversible inhibition of papain was prevented by GSNO or papanonoate pretreatment, hypothetically through mixed disulfide formation or S-nitrosylation of the catalytically critical thiol group of papain. These results, for the first time, compare the generation of carbonyls in proteins by Angeli's salt, papanonoate, and SIN-1. Furthermore, these results suggest that S-nitrosothiols may have a novel function in protecting critical thiols from irreversible oxidative damage.

Antioxidants↗
PubMed2003

Differential binding characteristics of protein G and protein A for Fc fragments of papain-digested mouse IgG.

It has been previously reported that staphylococcal protein A (SPA) bound only to the Fc region of mouse immunoglobulin G (IgG) and streptococcal protein G (SPG) bound to both Fab and Fc regions of mouse IgG and the binding sites for SPG and SPA on Fc were overlapped. In this study the binding characteristics of SPG and SPA for papain-digested mouse IgG were analysed. Papain digestion of mouse IgG purified from CAy-IFNg99C hybridoma (secreting IgG1 monoclonal antibody specific for human interferon gamma)-induced ascites resulted in Fab and two major Fc fragments referred to as the high molecular weight (HMW) and the low molecular weight (LMW) Fc fragments. SPG bound to Fab and the LMW Fc fragments of the papain-digested IgG. However SPG did not bind to the HMW Fc fragment. SPA showed practically no reactivity with the Fab and the LMW Fc fragments of the papain-digested mouse IgG but only to the HMW Fc fragment. SPG and SPA binding assays showed that papain digestion discriminated the SPG and SPA binding sites in the Fc fragment of mouse IgG. These results demonstrated a clear evidence for the presence of two independent SPG and SPA binding sites in the Fc fragment of mouse IgG.

Animals↗