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At least 19 recordsLinked to original sources
PubMed1994

Structure of chymopapain M the late-eluted chymopapain deduced by comparative modelling techniques and active-centre characteristics determined by pH-dependent kinetics of catalysis and reactions with time-dependent inhibitors: the Cys-25/His-159 ion-pair is insufficient for catalytic competence in both chymopapain M and papain.

Chymopapain M, the monothiol cysteine proteinase component of the chymopapain band eluted after chymopapains A and B in cation-exchange chromatography, was isolated from the dried latex of Carica papaya and characterized by kinetic and chromatographic analysis. This late-eluted chymopapain is probably a component of the cysteine proteinase fraction of papaya latex discovered by Schack [(1967) Compt. Rend. Trav. Lab. Carlsberg 36, 67-83], named papaya peptidase B by Lynn [(1979) Biochim. Biophys. Acta 569, 193-201] and partially characterized by Polgár [(1981) Biochim. Biophys. Acta 658, 262-269] and is the enzyme with unusual specificity characteristics (papaya proteinase IV) that Buttle, Kembhavi, Sharp, Shute, Rich and Barrett [Biochem. J. (1989) 261, 469-476] claimed to be a previously undetected cysteine proteinase eluted from a cation-exchange column near to the early-eluted chymopapains. A study of the time-dependent chromatographic consequences of thiol-dependent proteolysis of the components of papaya latex is reported. Chymopapain M was isolated by (i) affinity chromatography followed by separation from papain using cation-exchange f.p.l.c. on a Mono S HR5/5 column and (ii) cation-exchange chromatography followed by an unusual variant of covalent chromatography by thiol-disulphide interchange. The existence in chymopapain M of a nucleophilic interactive Cys/His catalytic-site system analogous to those in papain (EC 3.4.22.2) and other cysteine proteinases was deduced from the characteristics shape of the pH-second-order rate constant (k) profiles for its reactions with 2,2'-dipyridyl disulphide and ethyl 2-pyridyl disulphide. Analysis of the pH-k data for the reactions of chymopapain M with the 2-pyridyl disulphides and with 4,4'-dipyridyl disulphide permits the assignment of molecular pKa values of 3.4 and 8.7 to the formation and subsequent dehydronation of the Cys-S-/His-Im+H state of the catalytic site and reveals three other kinetically influential ionizations with pKa values 3.4, 4.3 and 5.6. The pH-dependences of kcat./Km for the hydrolysis of N-acetyl-L-Phe-Gly-4-nitroanilide at 25.0 degrees C and I0.1 M catalysed by chymopapain M and papain were determined. For both enzymes, little catalytic activity (5-7% of the maximal) develops consequent on formation of the catalytic site Cys-S-/His-Im+H ion-pair state (across pKa 3.4 for both enzymes). For papain, full expression of Kcat./Km for the uncharged substrate requires only the additional hydronic dissociation with pKa 4.2. By contrast, full expression of kcat./Km for chymopapain M requires additional hydronic dissociation with pKa values of 4.3 and 5.6.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1995

The structural origins of the unusual specificities observed in the isolation of chymopapain M and actinidin by covalent chromatography and the lack of inhibition of chymopapain M by cystatin.

1. The selectivity observed when the potentially general technique for the isolation of fully active forms of cysteine proteinases, covalent chromatography by thiol-disulphide interchange, is applied to chymopapain M and to actinidin was investigated by a combination of experimentation and computer modelling. Neither of these enzymes is able to react with the original Sepharose-GSH-2-dipyridyl disulphide gel, but fully active forms of both enzymes are obtained by using Sepharose-2-hydroxypropyl-2'-dipyridyl disulphide gel, which is both electrically neutral and sterically less demanding than the GSH gel. Electrostatic potential calculations, minimization and molecular-dynamics simulations provide explanations for the unusual, but different, specificities exhibited by actinidin and chymopapain M in the interactions of their active centres with ligands. 2. The unique behaviour of chymopapain M in exerting an almost absolute specificity for substrates with glycine at the P1 position and in resisting inhibition by cystatin was examined by the computer-modelling techniques. A new, modelled, structure of the complete chicken egg-white cystatin molecule based on the crystal structure of a short form of cystatin was deduced as a necessary prerequisite. The results suggest that electrostatic repulsion prevents reaction of actinidin with the GSH gel, whereas a steric 'cap' resulting from a unique arginine-65-glutamic acid-23 interaction in chymopapain M prevents reaction of the gel with this enzyme and accounts for the lack of its inhibition by cystatin and its specificity in catalysis. 3. Use of chymopapain M as a structural variant of papain demonstrates the validity of the predictions of Lowe and Yuthavong [Biochem. J. (1971) 124, 107-115] relating to the structural requirements and binding characteristics of the S1 subsite of papain.

Binding Sites↗
PubMed1988

Prevention of chymopapain anaphylaxis by screening chemonucleolysis candidates with cutaneous chymopapain testing.

With chymopapain at a concentration of 10 mg/ml, the authors skin tested 540 chemonucleolysis candidates; six were positive, and 534 were negative. None of the positive patients received therapeutic injections of chymopapain. There were no instances of unequivocal anaphylaxis to chymopapain in the patients with negative skin tests treated with chymopapain. When this 0% incidence of systemic reactions in skin test negative patients is compared with the historical rate of 1%, this difference is statistically significant (p less than 0.05). Restriction of chymopapain treatment to patients with negative prick tests can reduce the incidence of systemic reactions.

Anaphylaxis↗
PubMed1987

Prevention of chymopapain anaphylaxis by screening chemonucleolysis candidates with cutaneous chymopapain testing. A preliminary report.

The risk of anaphylaxis in candidates for chemonucleolysis for herniated lumbar discs is approximately 1%. An investigation was designed to eliminate or reduce the incidence of chymopapain anaphylaxis. The procedure was to restrict chemonucleolysis to patients who are prick test-negative to chymopapain at a concentration of 10 mg/ml. The authors skin-tested 292 chemonucleolysis candidates; five were positive and 287 were negative. None of the positive patients were injected with chymopapain. There were no instances of unequivocal chymopapain anaphylaxis in the skin test-negative group. This 0% incidence of anaphylaxis in skin test-negative patients was compared with a population estimate of 1%, based on historical data. The resulting value of p = 0.08 value fell short of the conventional level of significance of 0.05. The p value will reach 0.05 when the series of skin test-negative patients without anaphylaxis reaches 360. It is important that this information is readily available to physicians concerned about anaphylactic reactions to chymopapain.

Adult↗
PubMed1984

Chymopapain allergy: case reports and identification of patients at risk for chymopapain anaphylaxis.

Chymopapain has been approved for intradiscal injection in the United States and is expected to be used in approximately 100,000 patients per year. The need to identify the population at risk for anaphylaxis is obvious. Both in vivo and in vitro methods are available for measurement of IgE against chymopapain. This is a report of two cases of chymopapain allergy. One case discusses a 25-year-old woman who had rhinitis, asthma, and urticaria associated with occupational health hazards who was rejected for chemonucleolysis. The other case describes a 59-year-old man who had a predictably severe anaphylactic reaction to chymopapain and responded to treatment with epinephrine. Both patients had IgE antibody against chymopapain.

Adult↗
PubMed1978

Correlation between hypersensitivity to parenteral chymopapain and the presence of IgE anti-chymopapain antibody.

A solid phase radioimmunoassay, similar to the RAST, was developed in an attempt to predict anaphylactic reactions in patients injected with the proteolytic enzyme chymopapain, used in therapy for prolapsed intervertebral disc. The test measured the serum content of anti-chymopapain antibodies of the IgE class. Of 1263 patients tested, twelve gave anaphylactic reactions. The test was predictive for seven of them (58%), while sixty were false positives. Measurements were also made of anti-chymopapain IgE or other classes of antibodies which developed in the sera of patients after chymopapain injection. The presence of antibodies to chymopapain in individuals who had not been injected was also demonstrated.

Anaphylaxis↗
PubMed1978

A spectrophotometric method for the detection of contaminant chymopapains in preparations of papain. Selective modification of one type of thiol group in the chymopapains by a two-protonic-state reagent.

A simple spectrophotometric method for the detection of chymopapains A and B, contaminants in some preparations of papain (EC 3.422.2), was devised. If the amount of rapidly reacting thiol in the preparation, as determined by increase in A343 consequent on reaction with the two-protonic-state thiol titrant, 2,2' -dipyridl disulphide, is greater at PH8-9 than at pH4, contamination by the chymopapains is in dicated.

Chymopapain↗
PubMed1993

Immunohistochemical study of chymopapain injected into the rabbit intervertebral disc using anti-chymopapain antibody.

This study was undertaken to investigate the morphological action of chymopapain (CP) in intervertebral discs. Of 20 mature Japanese white rabbits, 19 lumbar intervertebral discs were used for electron microscopic examination, and 36 discs, for immunohistochemical examination. Discs from 3 hours to 8 weeks after injection were observed by light and electron microscopy using anti-CP rabbit antibody labeled with horseradish peroxidase. Using Kagami's DMF-dehydration, two types of fine fibers were observed in the extracellular matrix of normal nucleus pulposus, in addition to collagen fiber. After injection of CP, the thinner fibers disappeared, while the thicker fibers remained. The injected CP spread from the nucleus pulposus to the annulus fibrosus within several hours and remained as long as 4 weeks. By immunoelectron microscopy, positive granules were divided into Type 1 (lacking relationship to collagen fibers) and Type 2 (adhering to or surrounding collagen fibers). The thinner fibers appear to be proteoglycan monomer and are the target for CP.

Animals↗
PubMed1996

Cervical chymopapain nucleolysis: MR imaging assessment of chymopapain efficacy.

Soft cervical disc herniations can be treated by minimally invasive procedures with excellent results. Chemonucleolysis with chymopapain is one alternative to surgery that is shown to be innocuous within a strict technical framework. MR imaging allows an accurate diagnosis of the cause of cervical root compression, thus facilitating the choice of conventional surgery or minimally invasive procedures.

Adult↗
PubMed1984

Chymopapain, chemonucleolysis, and nucleus pulposus regeneration. A biochemical and biomechanical study.

In the adult mongrel dog, in vivo injection of chymopapain into the intervertebral disc resulted, at two weeks, in disc space narrowing. However, [35S]sulfate labeling and proteoglycan characterization demonstrate that the nucleus retains the ability to synthesize proteoglycans, although they were degraded rapidly by residual proteolytic activity. Three months following chymopapain treatment, the intervertebral dog disc shows that an increase in disc height, return of nuclear material, and proteoglycan aggregate is present. At six months following chymopapain treatment, proteoglycans of similar characteristics to normal canine intervertebral disc are identified with a glucosamine/galactosamine ratio approaching normal values. Biomechanically, the short-term (30-120 minutes) in vitro effects of chymopapain appear to be the same as the carrier causing increased disc height, stiffness values, and creep rates. In the vivo study, after three weeks, chymopapain-injected discs had significant reductions in disc height and compressive stiffness, but the creep rate was increased substantially. However, at three months postinjection, these biomechanical properties began to reverse and approached those of the uninjected controls. The observations reported in this study suggest that chymopapain has a profound but reversible effect on normal canine intervertebral disc. The radiographic narrowing of the intervertebral disc following chymopapain injection correlates with loss of proteoglycan content, structure, and biomechanical properties. The restoration of normal disc height following chymopapain injection is explained by reconstitution of normal intervertebral disc. EDTA and cysteine used alone have no discernable in vivo enzymatic effect on intervertebral disc proteoglycan biochemistry. Chemonucleolysis with chymopapain would appear less likely to alter permanently proteoglycan biochemistry and the biomechanical properties of the disc than surgical excision in experimental animals.

Animals↗
PubMed1996

Adolescent disc protrusions. A long-term follow-up of surgery compared to chymopapain.

STUDY DESIGN: This study compared chymopapain with primary surgery in the treatment of 60 radiologically proven adolescent lumbar disc protrusions and symptoms of low back pain and sciatica; the failures of intradiscal therapy were treated by surgical discectomy. OBJECTIVES: To establish whether chymopapain is as good as primary surgery in treating adolescents with proven lumbar disc protrusions. SUMMARY OF BACKGROUND DATA: Symptomatic lumbar disc protrusions are rare in white adolescents; the reported incidence varies from 0.8% to 3.2% of all lumbar disc protrusions. This is the largest study with long-term follow-up in the world literature. METHODS: Forty-two patients between the ages of 13 and 19 years with proven lumbar disc protrusions were initially treated with chymopapain; the failures of intradiscal therapy were treated by surgical discectomy. Eighteen patients were treated with surgical discectomy. After initial review at 1, 3, 6, and 12 months, the patients were assessed using a postal questionnaire and telephone interview at a minimum of 5 years' follow-up (means: 8.5 years for chymopapain group, 7.2 years for surgery group). RESULTS: Full replies were received from 16 of 18 (89%) in the surgery group and 42 of 42 (100%) in the chymopapain group. The long-term outcome is classed as good or excellent in 81% of the surgical group and 64% in the chymopapain group. If chymopapain is used as a first-line treatment, with surgery reserved for the failures, the long-term outcome is good or excellent in 82%. The chymopapain group had a shorter hospital stay. The surgical group were more likely to be unemployed and were less able to perform manual work and less able to engage in sporting activity. CONCLUSIONS: Back pain, radicular pain, and tension signs are common, but neurologic signs are less frequent in this age group. Long-term results of surgery are no better than the results of first-line chymopapain treatment with surgery being reserved for the failures. In 60% to 70% of patients, the morbidity, cost, and hospital stay were lessened. The patient is more likely to be in satisfactory employment after chemonucleolysis than after primary surgery.

Activities of Daily Living↗
PubMed1991

[Sensitization to chymopapain in patients treated with chemonucleolysis].

BACKGROUND: Chemonucleolysis (intradisk administration of chymopapain) is a procedure to treat intervertebral disk hernia. Recently, its use has been questioned due to the development of anaphylactic reactions in patients sensitized to chymopapain. The prevalence of sensitization to chymopapain has been evaluated before and after chemonucleolysis, and the possibility to establish risk groups through the allergy history has been assessed. METHODS: 104 consecutive patients who were candidates to chemonucleolysis were evaluated with an allergy questionnaire, cutaneous tests to aeroallergens and to chymopapain, and chymopapain-specific IgE. The two latter tests were repeated one month after chemonucleolysis. RESULTS: Only 2 patients (1.9%) showed evidence of chymopapain sensitization before the procedure. Sixteen patients (16%) were sensitized after chemonucleolysis. None of the possible risk factors evaluated in the allergy questionnaire (atopy, drug allergy, papaya occupational exposure or use of additives, cosmetics or drugs containing papaine) were significantly related with the risk of sensitization to chymopapain. CONCLUSIONS: The prevalence of chymopapain sensitization in the study group was low. The allergy questionnaire (atopy, drug allergy, use of papaya, occupational history did not identify sensitized patients. Cutaneous tests and specific IgE are the best method to detect chymopapain sensitization. The remarkable rate of sensitization after chemonucleolysis may partially limit the usefulness of the procedure.

Adult↗
PubMed1986

Chymopapain A. Purification and investigation by covalent chromatography and characterization by two-protonic-state reactivity-probe kinetics, steady-state kinetics and resonance Raman spectroscopy of some dithioacyl derivatives.

Chymopapain A was isolated from the dried latex of papaya (Carica papaya) by ion-exchange chromatography followed by covalent chromatography by thiol-disulphide interchange. The latter procedure was used to produce fully active enzyme containing one essential thiol group per molecule of protein, to establish that the chymopapain A molecule contains, in addition, one non-essential thiol group per molecule and to recalculate the literature value of epsilon 280 for the enzyme as 36 000 M-1 X cm -1. The Michaelis parameters for the hydrolysis of L-benzoylarginine p-nitroanilide and of benzyloxy-carbonyl-lysine nitrophenyl ester at 25 degrees C, and I 0.1 at several pH values catalysed by chymopapain A, papaya proteinase omega, papain (EC 3.4.22.2) and actinidin (EC 3.4.22.14) were determined. Towards these substrates chymopapain A has kcat./km values similar to those of actinidin and of papaya proteinase omega and significantly lower than those of papain or ficin. The environment of the catalytic site of chymopapain A is markedly different from those of other cysteine proteinases studied to date, as evidenced by the pH-dependence of the second-order rate constant (k) for the reaction of the catalytic-site thiol group with 2,2'-dipyridyl disulphide. The striking bell-shaped component that is a characteristic feature of the reactions of S-/ImH+ (thiolate/imidazolium) ion-pair components of many cysteine-proteinase catalytic sites with the 2,2'-dipyridyl disulphide univalent cation is not present in the pH-k profile for the chymopapain A reaction. The result is consistent with the presence of an additional positive charge in, or near, the catalytic site that repels the cationic form of the probe reagent. Resonance Raman spectra were collected at pH values 2.5, 6.0 and 8.0 for each of the following dithioacyl derivatives of chymopapain A: N-benzoylglycine-, N-(Beta-phenylpropionl)glycine- and N-methoxycarbonylphenylalanylglycine-. The main conclusion of the spectral study is that in each case the acyl group binds as a single population known as conformer B in which the glycinic N atom is in close contact with the thiol S atom of the catalytic-site cysteine residue, as is the case also for papain and other cysteine proteinases studied. Thus the abnormal catalytic-site environment of chymopapain A detected by the reactivity-probe studies, which may have consequences for the acylation step of the catalytic act, does not perturb the conformation of the bound acyl group at the acyl-enzyme-intermediate stage of catalysis.

2,2'-Dipyridyl↗
PubMed1983

Chymopapain, chemonucleolysis, and nucleus pulposus regeneration.

UNLABELLED: In the adult mongrel dog, in vivo injection of chymopapain into the intervertebral disc resulted in disc-space narrowing at two weeks, with a complete loss of proteoglycan (as indicated by safranin-O staining) from the nucleus pulposus, the cartilaginous end-plates, and the annulus fibrosus. As demonstrated by [35S]sulphate-labeling and proteoglycan isolation, the nucleus pulposus retained the ability to synthesize proteoglycans, but these were degraded by endogenous proteolytic activity. Three months after chymopapain treatment the intervertebral disc showed an increase in height. There was a return of intense safranin-O staining in the annulus and the cartilaginous end-plates, and very prominently in the nucleus. The proteoglycans that were present were recovered as aggregates, with the proteoglycan monomer being slightly larger than in the controls. Six months after chymopapain treatment the intervertebral disc had increased further in height, and normal histology had been restored. The chemical composition and physical properties of the proteoglycans that were isolated from the nucleus pulposus were essentially the same as those from the controls. These observations suggest that the nucleus can regenerate following the injection of chymopapain. CLINICAL RELEVANCE: Our observations demonstrate that chymopapain has a profound but reversible effect on the intervertebral disc. The radiographic narrowing of the intervertebral disc following chymopapain injection correlates with the loss of proteoglycan content and structure. The restoration of normal disc height following chymopapain injection is explained by reconstitution of the intervertebral disc with normal proteoglycans. In experimental animals, chemonucleolysis with chymopapain appears to be less likely than surgical excision to permanently alter the biochemistry of the nucleus pulposus.

Animals↗
PubMed1984

Proteins: chymopapain and insulin.

We studied clinical and immunologic aspects of the reactions to two newly introduced drugs, chymopapain and human recombinant deoxyribonucleic acid insulin (HI), in patients demonstrating allergies to one of these two drugs. We then used this information to improve our ability to diagnose and prevent chymopapain allergy and to further our understanding of systemic insulin allergy and its management. Of the patients who were sensitive to chymopapain, one had severe anaphylaxis to intradisc injection while the other had rhinitis, asthma, and urticaria with occupational exposure. The latter demonstrated cutaneous reactivity to papain; the former refused skin testing. Both demonstrated immunoglobulin (Ig) E and IgG to chymopapain as measured by enzyme-linked immunosorbent assay. We have prospectively skin tested 61 patients with chymopapain. Sixty-one patients have had negative skin tests and have tolerated the intradisc injection of chymopapain without incident. We are continuing our prospective skin test study in order to identify a population at risk for allergy to chymopapain. Two patients with systemic allergic reactions to animal insulin have at least as much cutaneous reactivity and IgE and IgG antibodies to HI as to porcine insulin. A large local reaction occurred during an attempt to desensitize one of them to HI; the patient was subsequently desensitized without difficulty to porcine insulin, to which she was less skin reactive. We conclude that HI will not eliminate insulin allergy in patients with systemic allergy to animal insulin and that such patients will continue to require the usual therapeutic measures for insulin allergy.

Adult↗
PubMed1993

Comparison of tissue reaction with chondroitinase ABC and chymopapain in rabbits as the basis of clinical application in chemonucleolysis.

Although chemonucleolysis with chymopapain is an approved treatment for lumbar intervertebral disk herniation, recent serious complications have raised doubt concerning its safety. It is therefore necessary to search for a safer and more selective agent than chymopapain for chemonucleolysis. Experimental chemonucleolysis with chondroitinase ABC was thus tested and compared with chymopapain. Acute tissue reactions to chondroitinase ABC were investigated and compared with chymopapain. The epidural space, yellow ligament, sciatic nerve, knee joint, and Achilles tendon were examined. Chymopapain damaged nervous and ligamentous tissues as well as cartilaginous tissue. Chondroitinase ABC did not damage nervous and ligamentous tissue. Chondroitinase ABC affected only cartilaginous tissue, and its action was chiefly limited to digestion of the matrix. Chondroitinase ABC has high enzymatic specificity for matrix in vivo. In addition, chondroitinase ABC is less toxic to noncartilaginous tissues than chymopapain. Chondroitinase ABC might be a more suitable and safer enzyme than chymopapain for chemonucleolysis.

Achilles Tendon↗