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PubMed1976

The chemical modification of papain with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide.

The reaction of the water-soluble carbodimide, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), with active papain in the presence of the nucleophile ethyl glycinate results in an irreversible inactivation of the enzyme. This inactivation is accompanied by the derivatization of the catalytically essential thiol group of the enzyme (Cys-25) and by the modification of 6 out of 14 of papain's carboxyl groups and up to 9 out of 19 of the enyzme's tyrosyl residues. No apparent irreversible modification of histidine residues is observed. Mercuripapain is also irreversibly inactivated by EDC/ethyl glycinate, again with the concomitant modification of 6 carboxyl groups, up to 10 tyrosyl residues, and no histidine residues; but in this case there is no thiol derivatization. Treatment of either modified native papain or modified mercuripapain with hydroxylamine results in the complete regeneration of free tyrosyl residues but does not restore any activity. The competitive inhibitor benzamidoacetonitrile substantially protects native papain against inactivation and against the derivatization of the essential thiol group as well as 2 of the 6 otherwise accessible carboxyl groups. The inhibitor has no effect upon tyrosyl modification. These findings are discussed in the context of a possible catalytic role for a carboxyl group in the active site of papain.

Amino Acids↗

PubMed1978

Modification of papain with tetranitromethane.

Papain [EC 3.4.22.2] polymerizes readily upon treatment with tetranitromethane (TNM) by forming intermolecular covalent linkages through its tyrosine residues (Tsukamoto, S. & Ohno, M. (1974) J. Biochem. 75, 1377-1380). Polymerization occurred optimally at pH 9.0 with S-sulfenylsulfonate papain. Circular dichroic spectra of polymerized papains showed a small change in ellipticity when compared with that of unmodified papain. Esterolytic activity of the modified enzyme toward benzoyl-L-arginine ethyl ester (BAEE) was almost fully retained, at least up to the formation of hexamer, with an unchanged Km value. Spectrophotometric and amino acid analyses indicated that two or three tyrosine residues are involved in intermolecular crosslinks depending on the amount of TNM used. The tyrosine residues nitrated were identified as those at positions 61, 116, 103, and 4, the extent of nitration decreasing in this order. When activated papain was treated with increasing molar ratios of TNM, an essential sulfhydryl function was first oxidized and, at a 2-fold molar excess of the reagent, restoration of activity was no longer observed even after addition of dithiothreitol (DTT). The evidence obtained in the present study eliminates the possibility of inactivation due to nitration of a tryptophan residue, which had been suggested previously.

Amino Acid Sequence↗

PubMed1976

Effect of the immediate environment on the reactivity of the essential -SH group of papain.

The effect of the microenvironment on the reactivity of the essential -- SH group of papain was studied by alkylation with methyl iodide and with the more polar iodoacetamide. Rate and activation parameters for these reactions were determined with two forms of the -- SH group: the free mercaptide ion at pH 10.0, and the mercaptide-imidazolium ion-pair at pH 5.5. The ion-pair of papain reacts with methyl iodide at a rate 1470 times less than that of thiolsubtilisin. This surprising difference between the reactivities of the two enzymes suggests that in contrast to thiolsubtilisin, where a non-polar environment enhances the rate, in the case of papain a more polar environment somewhat inhibits the reaction with the non-polar methyl iodide. The positive activation entropy for the papain reaction may indicate an 'ordered' structure of bound water around the sulfur atom. The high rate and the low activation entropy (organized transition state) of the reaction of papain with iodoacetamide can be explained in terms of hydrogen-bond formation between the enzyme and the amide group of the alkylating agent.

Alkylation↗

PubMed1977

Inhibition of papain by N-acyl-aminoacetaldehydes and N-acyl-aminopropanones. Evidence for hemithioacetal formation by a cross-saturation technique in nuclear-magnetic resonance spectroscopy.

N-Acyl-aminoacetaldehydes are potent inhibitors of the proteolytic enzyme, papain. Although they exist predominantly in their hydrated form in aqueous solution only the aldehyde is an effective inhibitor. The binding constants for related amides and methyl ketones confirm that it is principally the lower steric requirement of the aldehyde rather than its increased electrophilicity which is responsible for its powerful inhibitor properties. Using nuclear magnetic resonance spectroscopy, evidence is provided for an N-acetyl-aminoacetaldehyde-papain complex. Using a cross-saturation technique evidence is also provided for a hemithioacetal, formed from the aldehyde and the active-site thiol group. Hemithioacetal formation has also been detected between N-benzoyl-aminoacetaldehyde and papain. This provides the first direct evidence for a tetrahedral adduct with papain and supports the proposed involvement of such intermediates in papain-catalysed hydrolyses.

Acetaldehyde↗

PubMed1978

Identification of the functional ionic groups of papain by pH/rate profile analysis.

The pH dependence of papain catalysis was analyzed by a scheme which evaluates the kinetic contribution of both protonated and unprotonated species of functional groups involved in catalysis. Kinetic measurements were made at constant pH, without buffers, by automatic titration. The rate-determining step for papain-catalyzed hydrolysis of alpha-N-benzoyl-L-arginine ethyl ester, determined by nucleophile competition, changed from acylation below pH 6.5 to mixed acylation-deacylation above pH 6.5. Kinetic analysis indicated that three prototropic groups governed the pH-specificity of alpha-N-benzoyl-L-arginine ethyl ester hydrolysis. These prototropic groups had pKa values of 4.8, 6.5 to 6.7, and 8.7. Theoretical treatment of the kinetics provided an excellent fit with the experimentally found profile when the contribution of all three prototropic groups was considered. Analysis showed that, in acid, the pathways of papain catalysis were functional with either two or three active-site protons. In base, a single functional ionic pathway is associated with an active site with only one proton. Pathways involving an unprotonated active site are catalytically inoperative in both acid and base. These results indicate that papain exhibits several catalytically functional ionic pathways. The results are discussed in terms of pKa assignments, and the mechanism of papain catalysis.

Arginine↗

PubMed1975

Effect of papain-induced emphysema on permeability of rat lung to drugs.

To investigate the effect of a papain-induced emphysema-like condition on pulmonary absorption of drugs, rats were exposed to either papain aerosol or distilled water aerosol (control) intermittently for 2 wk, and rates of drug absorption from damaged and control lungs were compared. To measure absorption rates, 0.1 ml of drug solution (0.1-10 mM) was administered through a tracheal cannula to anesthetized animals, and after various times lungs were assayed for unabsorbed compound. In absorption experiments with the lipoid-insoluble compounds, mannitol, p-aminohippuric acid, and procaine amide ethobromide, all three drugs were absorbed from the lungs about twice as rapidly in papain-treated rats as in control. In contrast, procaine amide, a relatively lipoid-soluble drug, was absorbed at the same rate in both control and papain-treated animals. The results suggest that papain-induced lung damage increases the porosity of the pulmonary epithelium.

Aerosols↗

PubMed1975

[Pulmonary function in papain induced emphysema in dogs (author's tranls)].

By intratracheal injection of the protease Papain to experimental animals parenchymal changes in the lung can be induced, that resemble human emphysema. Papain (dosage 26 to 112 mg, 1 to 4 injections) was given intratracheally to 8 bastard dogs, (weighing from 12.5 to 20 kg) during light general anesthesia. Pulmonary function was assessed in weekly intervals and related to morphologic changes in the lung. Static compliance of the lung and FRC measured during respiratory arrest were increased after papain, bronchial resistance, measured while artificially ventilated at constant pressure was also increased. Changes of static lung compliance and FRC were seen after the first administration of papain, but further increased with time of observation and after multiple doses of papain. Increase of resistance was not found before 5 weeks. At quiet breathing resistance was not increased at all. No significant changes were found of arterial pO2 and pCO2, pH, standard and actual bicarbonate, diffusion capacity for O2, tidal volume, minute ventilation and ventilatory rate. Morphological findings confirmed the changes described by others. Pulmonary function appears to be pathological at a time when morphology still seems to be normal. The question is discussed to what extent the model of experimental emphysema induced by proteolytic enzymes can contribute to the understanding of human pulmonary emphysema. Lung function in the course of experimental emphysema is compared with function in different clinical types of emphysema.

Airway Resistance↗

PubMed1978

Pathogenesis of papain-induced emphysema in the hamster.

In an effort to determine the mechanism of papain action in causing an emphysema-like lesion in hamsters, the number and types of cells and the activities of two lysosomal enzymes in the lung were determined after papain exposure. Three and four weeks after a 3-h exposure to an aerosol of 3% papain the following alterations in lung structure and function were observed: (1) the mean linear intercept, or average distance between adjacent alveoli, was increased; (2) the internal surface area declined; (3) the dynamic compliance was elevated at low breathing frequencies. The numbers of cells present free in the lung increased from a control value of 2.0 +/- 0.2 x 10 (6) to 6.6 +/- 0.5 x 10 (6) 5 days after exposure. The free beta-glucuronidase, alysosomal enzyme, likewise increased over threefold during the first 3 days after exposure. These results are consistent with the hypothesis that papain induces an inflammatory-type responses, and this may be in part responsible for inducing the lesion.

Animals↗

PubMed1979

Papain-induced asthma--physiological and immunological features.

Increasing reports of respiratory disease associated with exposure to papain prompted clinical, physiological, and immunological studies of the supervisor of a meat tenderizer factory who developed asthma after long-term contact with papain dust. His symptoms were worse at work and better on weekends and vacations. Bronchial inhalation challenges produced both immediate and late asthma to papain but not to the other ingredients in the food product. Immunological studies revealed the presence of specific IgE antibodies by direct and passive transfer skin tests and the radioallergosorbent test, and specific precipitating antibodies by immunodiffusion tests. These findings are indicative of a dual type I and III hypersensitivity. Papain acting as an allergen in an occupational setting is a risk factor for eliciting asthma even in a nontropic individual.

Administration, Intranasal↗

PubMed1976

Kinetics of the action of papain on fluorescent peptide substrates.

Kinetic measurements have been performed on the action of papain on mansyl-Gly-Val-Glu-Leu-Gly and on mansyl-Gly-Gly-Val-Glu-Leu-Gly, both of which are cleaved solely at the Glu-Leu bond under the conditions of our experiments. Stopped-flow experiments have shown that, under conditions of enzyme excess, the enhancement of the fluorescence of the mansyl group upon association of each of the oligopeptide substrates with papain is a biphasic process. A very rapid initial increase in fluorescence is followed by a slower first-order fluorescence enhancement. The observed rate constant for the latter process is greater with the mansyl pentapeptide than with the mansyl hexapeptide. A similar biphasic fluorescence change is seen upon the interaction of the mansyl peptides with mercuripapain, but the second step is much slower than in the case of the active enzyme. The rate of the second step in the association of active papain with the mansyl paptides shows saturation with increasing enzyme concentration, supporting the view that an initial enzyme-substrate complex (ES) is converted in a first-order process to the complex (ES) that undergoes cleavage to form products. The hydrolysis of the Glu-Leu bond is associated with a first-order decrease in fluorescence, as a consequence of the formation of the mansyl peptide product, which is bound less strongly than the substrate. The rate constant for this process is about 140 times greater with the mansyl hexapeptide than with the mansyl pentapeptide, thus giving further indication of the importance of secondary enzyme-substrate interactions in the efficiency of papain catalysis. For each of the two mansyl peptides, the values of the rate constants and the apparent Michaelis constants associated with the cleavage of the Glu-Leu bond, as determined by stopped-flow measurements under conditions of enzyme excess, were the same, within the precision of the data, as those estimated from experiments under conditions of substrate excess, where the formation of Leu-Gly was determined by means of the fluorescamine reaction. This indicates that, with these substrates, the rate-limiting step in the overall catalytic process is associated with the breakdown of ES. Estimates are given of the dissociation constant of ES and of the rate constants in the interconversion of ES and ES.

Fluorescence↗

PubMed1976

Papain-catalyzed reactions at subzero temperatures.

As a first step in the investigation of papain catalysis using subzero temperatures to detect, accumulate, and characterize enzyme-substrate intermediates, we have studied some potential cryosolvents and carried out preliminary intermediate trapping experiments. The effects of subzero temperatures and aqueous dimethyl sulfoxide solutions on the papain-catalyzed hydrolysis of Nalpha-carbobenzoxy-L-lysine p-nitrophenyl ester have been investigated in detail. At 0 degrees C, the value of kcat decreases with increasing dimethyl sulfoxide concentration, decreasing in proportion to the decreased water concentration; however, the value of Km increases exponentially. The effect on Km can be accounted for by a combination of both dielectric and competitive inhibition effects. The Arrhenius plot for the deacylation reaction in 7.65 M (60% v/v) dimethyl sulfoxide is linear over the temperature range 0 to -45 degrees C and extrapolates to a calculated value of kcat at 25 degrees C in excellent agreement with that obtained in the absence of organic solvent. The pH-rate profile is not substantially perturbed by the presence of 7.65 M dimethyl sulfoxide. At -45 degrees C and below, turnover occurs extremely slowly, and is essentially negligible, although acylation is still quite rapid. Consequently, the acyl enzyme, Na-carbobenzoxy-L-lysyl-papain, can be readily accumulated and trapped at temperatures below -50 degrees C. At these low temperatures, under conditions of excess substrate, the amount of p-nitrophenol liberated in the acylation reaction is equivalent to the active-site normality of the enzyme, indicating a 1:1 stoichiometry in formation of the acyl enzyme. The effect of dimethyl sulfoxide up to 7.65 M, on the intrinsic ultraviolet, fluorescence, and circular dichroic properties of the enzyme shows no evidence of any solvent-induced structural changes. All experimental observations are consistent with the conclusion that 7.65 M dimethyl sulfoxide and subzero temperatures have no deleterious effects on papain-catalyzed reactions. A related series of experiments indicate that aqueous ethanol cryosolvents up to 13.7 M (80% v/v) are also suitable. Preliminary experiments at subzero temperatures using Na-carbobenzoxy-L-lysine methyl ester suggest the existence of three enzyme-substrate intermediates which can be detected and accumulated.

Circular Dichroism↗

PubMed1976

4-Chloro-7-nitrobenzo-2-oxa-1,3-diazole as a reactivity probe for the investigation of the thiol proteinases. evidence that ficin and bromelain may lack carboxyl groups conformationally equivalent to that of aspartic acid-158 of papain.

1. 4-Chloro-7-nitrobenzo-2-oxa-1,3-diazole (Nbd chloride) was used as a reactivity probe to characterize the active centres of papin (EC 3.4.22.2), ficin (EC 3.4.22.3) and bromelain (EC 3.4.22.4). 2. In the pH range 0-8 Nbd chloride probably exists mainly as a monocation, possibly with the proton located on N-1 of the oxadiazole ring. 3. Spectroscopic evidence is presented for the intermediacy of Meisenheimer-type adducts in the reaction of Nbd chloride with nucleophiles. 4. The pH-dependence of the second-order rate constants (k) of the reactions of the three enzymes with Nbd chloride was determined at 25 degrees C, I = 0.1 mol/litre in 6.7% (v/v) ethanol in the pH range 2.5-5, where, at least for papain and ficin, the reactions occur specifically with their active-centre thiol groups. The pH-k profile for the papain reaction is bell-shaped (pKaI = 3.24, pKaII = 3.44 and k = 86M(-1)-s(-1), whereas that for ficin is sigmoidal (pKa = 3.6, k = 0.36M(-1)-s(-1), the rate increasing with increasing pH. The profile for the bromelain reaction appears to resemble that for the ficin reaction, but is complicated by amino-group labelling. 5. The bell-shaped profile of the papain reaction is considered to arise from the reaction of the thiolate ion of cysteine-25, maintained in acidic media by interaction with the side chain of histidine-159, with the Nbd chloride monocation hydrogen-bonded at its nitro group to the un-ionized form of the carboxyl group of aspartic acid-158. The lack of acid catalysis in the corresponding reactions of ficin and probably of bromelain suggests that these enzymes may lack carboxyl groups conformationally equivalent to that of aspartic acid-158 of papain. The possible consequences of this for the catalytic sites of these enzymes is discussed.

4-Chloro-7-nitrobenzofurazan↗

PubMed1979

Interaction of papain with derivatives of phenylalanylglycinal: fluorescence studies.

Fluorescence studies have been performed on the interaction of papain with active-site-directed inhibitors of the type mansyl-(Gly)n-Phe-glycinal, where n = 0, 1, 2. It has been found that whereas the mansyl [6-(N-methylantilino)-2-naphthalene sulfonyl] fluorescence of mansyl-Phe-glycinal is greatly enhanced, that of the two longer mansyl compounds is not, although all three are equally effective as inhibitors of papain action. Measurements of fluorescence polarization and rotational relaxation time support the conclusion that the fluorescent probe group of the two longer mansyl compounds protrudes into the solvent to a greater degree than that of mansyl-Phe-glycinal. Considerable energy transfer from papain tryptophan to the mansyl group is evident for all three inhibitors, however, although it is most marked with mansyl-Phe-glycinal. Stopped-flow fluorescence measurements have shown that, after initial rapid interaction, the first-order conformational changes in the active-site region of papain in the complex with mansyl-Phe-glycinal are approximately 1/10(4) those observed with comparable mansyl oligopeptide substrates, and approximately 1/10(2) those with acetyl-Phe-glycinal.

Dipeptides↗

PubMed1977

B-cell tolerance. III. Effect of papain-mediated cleavage of cell surface IgD on tolerance susceptibility of murine B cells.

Under defined conditions, papain removes IgD from cells while leaving IgM, H-2, Ia, Lyb-2, and complement receptor intact. The effect of such treatment with papain on the induction of tolerance in murine splenic B cells was determined in an in vitro system. Treatment of the cells with papain has no effect on subsequent antibody responsiveness presumably because surface receptors regenerate before and during incubation with immunogen. Removal of increasing amounts of IgD results in increasing susceptibility of thymus-dependent responsive cells to tolerance induction. The tolerance susceptibility of thymus-independent responsive cells, which we have previously suggested are immature cells that bear only IgM, is unaffected by cleavage of IgD. If cells are incubated for 24 h after treatment with papain, cell surface IgD and tolerance resistance return. These results indicate that a surface molecule affects susceptibility of B cells to induction of tolerance and suggest that this molecule may be IgD.

Animals↗

PubMed1979

Papain-induced asthma: diagnosis by skin test, RAST and bronchial provocation test.

Seven out of eleven workers occupationally exposed to airborne papain developed immediate hypersensitive reactions, predominantly asthma and rhinitis. Skin tests and RAST with papain were positive in all symptomatic workers, but not in the four asymptomatic workers. Furthermore, out of forty non-exposed asthmatics, thirty-eight had negative RAST results and all had negative skin test results. Bronchial provocation tests with 0.15-0.5 mg papain performed in five patients with a positive case history showed in each case an immediate asthmatic reaction; in addition to that, one patient developed signs of a dual asthmatic reaction. Our results suggest that airborne papain is a highly immunogenic agent in humans, which induces type I allergic reactions in a large percentage of the exposed subjects.

Asthma↗

PubMed1979

Studies on the specificity of human IgE-antibodies to the plant proteases papain and bromelain.

The sera of seven patients clinically hypersensitive to papain--in one case also to baromelain--and the sera of sixty asthmatic patients with allergies to other inhalant and food allergens were investigated for IgE antibody activity to the plant proteases papain and bromelain and to common allergens by RAST, confirmed in some sera by RAST inhibition. There seems to be a relation between the antibody reactions to papain and bromelain, in several cases also between the reactions to these proteases and to grass pollen and flour. Studies by RAST inhibition showed that papain, bromelain, wheat flour, rye flour, grass pollen and birch pollen mutually inhibit IgE antibody to each antigen; but the degree of inhibition varies among the different sera and allergens. Our results suggest that these allergens from various plants, besides having specific antigenic determinants, also possess similar or even identical antigenically active regions, leading to immunological cross-reactivity.

Antibody Specificity↗

PubMed1975

Effects of treating immune lymphocytes with a proteolytic enzyme (papain): reduction in immune potential and recovery after incubation.

Immune spleen cells were treated with papain, washed, and boosted with homologous antigen (4-hydroxy-3-iodo-5-nitrophenylacetic acid coupled to chicken globulin) for 1 hr at 4 degrees C. When transferred to irradiated mice, they produced up to 20-fold less anti-hapten antibody than non-papain-treated cells. The lymphocytes could recover their ability to respond if incubated at 37 degrees C after papain treatment but before antigen boosting. Incubation for 30 min at 37 degrees C gave complete or almost complete recovery. Although papain usually reduced the response, in 31% of experiments the reduction was not significant. Possible reasons for this variability are discussed.

Animals↗

PubMed1979

Affinity chromatographic purification of papain.

The reinvestigation of the affinity chromatographic method of purifying papain has been carried out. It has been reported that papain could be purified by taking advantage of the affinity of the enzyme for the insolubilized peptide inhibitor, agarose-Gly-Gly-Tyr(Bz)-Arg. Using pure tetrapeptide obtained commercially and standard coupling procedures, a significant purification of papain could not be achieved. Both active and nonactivatible enzyme bound to a column prepared in this manner were eluted together by the use of deionized water. An affinity medium with properties similar to those reported by Blumberg et al. was obtained by removal of the benzyl group on tyrosine prior to coupling with agarose. The deprotected tetrapeptide was also synthesized by an independent route and inhibition constants for the binding of the protected and deprotected tetrapeptide to papain were determined in kinetic experiments.

Amino Acid Sequence↗