Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “PAPAIN”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 649 records · Page 36Linked to original sources
PubMed2002

Preparation and characterization of papain-modified sesame (Sesamum indicum L.) protein isolates.

Defatted sesame meal ( approximately 40-50% protein content) is very important as a protein source for human consumption due to the presence of sulfur-containing amino acids, mainly methionine. Sesame protein isolate (SPI) is produced from dehulled, defatted sesame meal and used as a starting material to produce protein hydrolysate by papain. Protein solubility at different pH values, emulsifying properties in terms of emulsion activity index (EAI) and emulsion stability index (ESI), foaming properties in terms of foam capacity (FC) and foam stability (FS), and molecular weight distribution of the SPI hydrolysates were investigated. Within 10 min of hydrolysis, the maximum cleavage of peptide bonds occurred as observed from the degree of hydrolysis. Protein hydrolysates have better functional properties than the original SPI. Significant increase in protein solubility, EAI, and ESI were observed. The greatest increase in solubility was observed between pH 5.0 and 7.0. The molecular weight of the hydrolysates was also reduced significantly during hydrolysis. These improved functional properties of different protein hydrolysates would make them useful products, especially in the food, pharmaceutical, and related industries.

Chemical Phenomena↗
PubMed1984

Structure-activity relationship of the ficin hydrolysis of phenyl hippurates. Comparison with papain, actinidin, and bromelain.

A study of the hydrolysis of 30 substituted-phenyl hippurates by the enzyme ficin has been made. From the results the following quantitative structure--activity relationship (QSAR) has been derived: log 1/Km = 0.79 pi'3 + 0.58 sigma + 0.28 MR4,5 + 3.70. In this expression Km is the Michaelis constant, pi'3 refers to the more hydrophobic of the two meta substituents, and MR4,5 is the molar refractivity of substituents in the 4- and 5-positions of the phenyl ring. This QSAR is compared with those from papain, actinidin, bromelain B, and bromelain D.

Bromelains↗
PubMed2005

Interaction of papain-like cysteine proteases with dipeptide-derived nitriles.

A series of 44 dipeptide nitriles with various amino acids at the P2 position and glycine nitrile at position P1 were prepared and evaluated as inhibitors of cysteine proteinases. With respect to the important contribution of the P2-S2 interaction to the formation of enzyme-inhibitor complexes, it was focused to introduce structural diversity into the P2 side chain. Nonproteinogenic amino acids were introduced, and systematic fluorine, bromine, and phenyl scans for phenylalanine in the P2 position were performed. Moreover, the N-terminal protection was varied. Kinetic investigations were carried out with cathepsin L, S, and K as well as papain. Changes in the backbone structure of the parent N-(tert-butoxycarbonyl)-phenylalanyl-glycine-nitrile (16), such as the introduction of an R-configured amino acid or an azaamino acid into P2 as well as methylation of the P1 nitrogen, resulted in a drastic loss of affinity. Exemplarily, the cyano group of 16 was replaced by an aldehyde or methyl ketone function. Structure-activity relationships were discussed with respect to the substrate specificity of the target enzymes.

Cathepsin K↗
PubMed2000

(R)-tert-Butoxycarbonylamino-fluorenylmethoxycarbonyl-glycine from (S)-benzyloxycarbonyl-serine or from papain resolution of the corresponding amide or methyl ester.

The enantiospecific synthesis of (R)-Boc-(Fmoc)-aminoglycine 7 was achieved. (S)-Cbz-serine 1 was reacted with diphenylphosphoryl azide in the presence of triethylamine to yield cyclic (S) carbamate 2. The ring nitrogen of 2 was protected with a Boc group (3). The cyclic carbamate of 3 was hydrolyzed with benzyltrimethylammonium hydroxide to yield the (R)-enantiomer of alcohol 4. The oxidation of 4 with pyridinium dichromate yielded the enantiomerically pure (97% ee) (R)-Boc-(Cbz)-aminoglycine 5, which was converted to 7 with retention of optical purity. Similarly, starting from (S)-Boc-serine 9, cyclic (S) carbamate 10 was obtained. The ring nitrogen of 10 was protected with a Cbz group (11) with retention of configuration. The cyclic carbamate of 11 was base hydrolyzed to yield 12, the (S)-enantiomer alcohol. Independently, Boc-(Fmoc)-aminoglycine amide 13 and Boc-(Fmoc)-aminoglycine methyl ester 14 were resolved using papain. The stereochemistry of the isolated acid was determined to be (R) by coelution on HPLC of its derivative with Marfey's reagent and that of an authentic sample (7) obtained by enantiospecific synthesis.

Amides↗
PubMed2003

Inhibition of papain-like cysteine proteases and legumain by caspase-specific inhibitors: when reaction mechanism is more important than specificity.

We report here that a number of commonly used small peptide caspase inhibitors consisting of a caspase recognition sequence linked to chloromethylketone, fluoromethylketone or aldehyde reactive group efficiently inhibit other cysteine proteases than caspases. The in vitro studies included cathepsins B, H, L, S, K, F, V, X and C, papain and legumain. Z-DEVD-cmk was shown to be the preferred irreversible inhibitor of most of the cathepsins in vitro, followed by Z-DEVD-fmk, Ac-YVAD-cmk, Z-YVAD-fmk and Z-VAD-fmk. Inactivation of legumain by all the inhibitors investigated was moderate, whereas cathepsins H and C were poorly inhibited or not inhibited at all. Inhibition by aldehydes was not very potent. All the three fluoromethylketones efficiently inhibited cathepsins in Jurkat and human embryonic kidney 293 cells at concentrations of 100 microM. Furthermore, they completely inhibited cathepsins B and X activity in tissue extracts at concentrations as low as 1 microM. These results suggest that data based on the use of these inhibitors should be taken with caution and that other proteases may be implicated in the processes previously ascribed solely to caspases.

Aldehydes↗
PubMed1968

The location of the active-site histidine residue in the primary sequence of papain.

Papain that had been irreversibly inhibited with 1,3-dibromo[2-(14)C]acetone was reduced with sodium borohydride and carboxymethylated with iodoacetic acid. After digestion with trypsin and alpha-chymotrypsin the radioactive peptides were purified chromatographically. Their amino acid composition indicated that cysteine-25 and histidine-106 were cross-linked. Since cysteine-25 is known to be the active-site cysteine residue, histidine-106 must be the active-site histidine residue.

Acetone↗
PubMed1969

Stepwise cleavage of rabbit immunoglobulin G by papain and isolation of four types of biologically active Fc fragments.

Four types of Fc fragments of different sizes were isolated by papain treatment of rabbit immunoglobulin G under various conditions and by subsequent chromatographic procedures. 1. Brief digestion at neutral pH without reduction produced a molecule in which the Fab and Fc fragments were still linked by a pair of labile disulphide bridges, and the Fc fragment released by cleaving these bonds, called 1Fc fragment, contained a portion of the ;hinge' region including an interchain disulphide bridge. Both complement-binding and guinea-pig skin-binding activities were retained by this fragment, which had mol. wt. 48000. 2. Prolonged digestion at neutral pH of immunoglobulin G whose labile inter-heavy-chain disulphide bridges had been reduced removed the ;hinge' region, giving mFc fragments (mol. wt. 46000), which lacked the capacity to bind guinea-pig skin but retained the antigenic as well as the complement-binding activities of 1Fc fragment completely. 3. Digestion at pH5.0 yielded a smaller fragment, sFc (mol. wt. 40000), which was no longer able to bind complement. Though the antigenic structure was intact, sFc fragment was curiously unable to precipitate with antibodies to the N-terminal determinants. 4. Fragment stFc (mol. wt. 25000), representing the C-terminal portion of Fc fragment, was formed from all the larger fragments by digestion at pH4.5. Only the C-terminal antigenic determinants were retained by stFc fragment.

Amino Acids↗
PubMed1971

Kinetic specificity in papain-catalysed hydrolyses.

The specificity of the proteolytic enzyme, papain, for the peptide bond of the substrate adjacent to that about to be cleaved and for the acyl residue of some N-acylglycine derivatives is manifest almost exclusively in the formation of the acyl-enzyme from the enzyme-substrate complex. Models for the enzyme-substrate complex and acyl-enzyme intermediate are suggested that account for these observations. In particular it is suggested that the peptide bond of the substrate adjacent to that about to be cleaved, is bound in the cleft of the enzyme between the NH group of glycine-66 and the backbone C=O group of aspartic acid-158, and provides a sensitive amplification mechanism through which the specificity of the enzyme for hydrophobic amino acids such as l-phenylalanine is relayed. It is also suggested that the distortion in the enzyme-substrate complex and the binding of the peptide bond adjacent to that about to be cleaved are also linked and behave co-operatively, the distortion of the protein facilitating binding and the stronger binding facilitating distortion. The results imply that between the enzyme-substrate complex and the acyl-enzyme a relaxation of the protein conformation must occur.

Anilides↗
PubMed1977

Evaluation of benzofuroxan as a chromophoric oxidizing agent for thiol groups by using its reactions with papain, ficin, bromelain and low-molecular-weight thiols.

1. Benzofuroxan (benzofurazan 1-oxide, benzo-2-oxa-1,3-diazole N-oxide) was evaluated as a specific chromophoric oxidizing agent for thiol groups. 2. Aliphatic thiol groups both in low-molecular-weight molecules and in the enzymes papain (EC 3.4.22.2), ficin (EC 3.4.22.3) and bromelain (EC 3.4.22.4) readily reduce benzofuroxan to o-benzoquinone dixime; potential competing reactions of amino groups are negligibly slow. 3. The fate of the thiol depends on its structure: a mechanism is proposed in which the thiol and benzofuroxan form an adduct which, if steric factors permit, reacts with another molecule of thiol to form a disulphide; when the thiol is located in the active site of a thiol proteinase and steric factors preclude enzyme dinner formation, the adduct reacts instead with water or HO- to form a sulphenic acid; attack on the sulphur atom of the adduct by either a sulphur or oxygen nucleophile releases o-benzoquinone dioxine. 4. Benzofuroxan contains n o proton-binding sites with pKa values in the range 3-10 and probably none in the range 0-14; o-benzoquinone dioxine undergoes a one-proton ionization with pKa=6.75.5. o-benzoquinone dioxime absorbs strongly at wavelengths greater than 410nm, where absorption by benzofuroxan, proteins and simple thiol compounds is negligible; 416 nm is an isosbestic point (epsilon 416 = 5110 litre. mol-1-cm-1); epsilon430=3740+[1460/(1+[H+]/Ka)] where pKa=6.75. 6. The possibility of acid-base catalysis of the oxidation by active-centre histidine residues of the thiol proteinases is discussed.

Benzoxazoles↗
PubMed1977

Benzofuroxan as a thiol-specific reactivity probe. Kinetics of its reactions with papain, ficin, bromelain and low-molecular-weight thiols.

1. The characteristics of benzofuroxan (benzofurazan 1-oxide, benzo-2-oxa-1,3-diazole N-oxide) that relate to its application as a reactivity probe for the study of environments of thiol groups are discussed. 2. To establish a kinetic and mechanistic basis for its use as a probe, a kinetic study of its reaction with 2-mercaptoethanol was carried out. 3. This reaction appears to proceed by a rate-determining attack of the thiolate ion on one of the electrophilic centres of benzofuroxan (possibly C-6) to provide a low steady-state concentration of an intermediate adduct; rapid reaction of this adduct with a second molecule of thiol gives the disulphide and o-benzoquinone dioxime. 4. The effects of the different types of environment that proteins can provide on the kinetic characteristics of reactions of thiol groups with benzofuroxan are delineated. 5. Benzofuroxan was used as a thiolspecific reactivity probe to investigate the active centres of papain (EC 3.4.22.2), ficin (EC 3.4.22.3) and bromelain (EC 3.4.22.4). The results support the concept that the active centres of all three enzymes either contain a nucleophilic thiolate ion whose formation is characterized by a pKa of 3-4 and whose reaction with an electrophile can be assisted by interaction of a site of high electron density in the electrophile with active-centre imidazolium ion of pKa 8-9, or can provide such ions by protonic redistribution in enzyme-reagent or enzyme-substrate complexes.

Benzoxazoles↗
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↗
PubMed1979

Reactivities of neutral and cationic forms of 2,2'-dipyridyl disulphide towards thiolate anions. Detection of differences between the active centres of actinidin, papain and ficin by a three-protonic-state reactivity probe.

The second-order rate constants (k) for the reactions of 2,2'-dipyridyl disulphide (pKa2,45) with 2-mercaptoethanol (pKa9.6) and with benzimidazol-2-ylmethanethiol (pKa values 5.6 and 8.3) were determined at 25 degrees C at I 0.1 by stopped-flow spectral analysis over a wide range of pH. These were used to calculate the pH-independent second-order rate constants (k) for the reactions of neutral 2,2'-dipyridyl disulphide and of its monocation with the 2-mercaptoethanol thiolate anion (associated pKa9.6) and with the benzimidazol-2-ylmethanethiol zwitterion (associated pKa5.6). For both thiolate ions, the rate-enhancement factor (kmonocation/kneutral disulphide) is about 1.5x10(3). The dependence on pH in acidic media of k for the reaction of 2,2'-dipyridyl disulphide with actinidin, the thiol proteinase from Actinidia chinensis, was shown to differ from the forms of pH-dependence observed for the analogous reactions with papain (EC 3.4.22.2) and ficin (3.4.22.3). The reactivity of the 2,2'-dipyridyl disulphide dication and its apparent sensitivity to the presence and location of a positive charge in the attacking thiol are discussed.

2,2'-Dipyridyl↗
PubMed1982

Half-time analysis of the integrated Michaelis equation. Simulation and use of the half-time plot and its direct linear variant in the analysis of some alpha-chymotrypsin, papain- and fumarase-catalysed reactions.

Substitution of half-time parameters in the integrated form of the Michaelis-Menten equation for any enzyme-catalysed reaction yields an equation that gives a linear relationship between the half-time of the reaction and the substrate concentration at that point of the reaction. The logarithmic term of the integrated equation becomes a constant as a result of the substitution, which means that the use of the half-time plot of the equation requires calculation only of half-time and substrate-concentration values at various stages of the reaction. The half-time method is both simple and exact, being analogous to an [S(0)]/v(i) against [S(0)] plot. A direct linear form of the half-time plot has been devised that allows very simple estimation of Michaelis parameters and/or initial velocities from progress-curve data. This method involves no approximation and is statistically valid. Simulation studies have shown that linear-regression analysis of half-time plots provides unbiased estimates of the Michaelis parameters. Simulation of the effect of error in estimation of the product concentration at infinite time [P(infinity)] reveals that this is always a cause for concern, such errors being magnified approximately an order of magnitude in the estimate of the Michaelis constant. Both the half-time plot and the direct linear form have been applied to the analysis of a variety of experimental data. The method has been shown to produce excellent results provided certain simple rules are followed regarding criteria of experimental design. A set of rules has been formulated that, if followed, allows progress-curve data to be acquired and analysed in a reliable fashion. It is apparent that the use of modern spectrophotometers in carefully designed experiments allows the collection of data characterized by low noise and accurate [P(infinity)] estimates. [P(infinity)] values have been found, in the present work, to be precise to within +/-0.2% and noise levels have always been below 0.1% (signal-to-noise ratio approximately 1000). As a result of the considerations above, it is concluded that there is little to be feared with regard to the analysis of enzyme kinetics using complete progress curves, despite the generally lukewarm recommendations to be found in the literature. The saving in time, materials and experimental effort amply justify analysis of enzyme kinetics by progress-curve methods. Half-time plots linear to >/=90% of reaction have been obtained for some alpha-chymotrypsin-, papain- and fumarase-catalysed reactions.

Chymotrypsin↗
PubMed1982

Characterization of papaya peptidase A as a cysteine proteinase of Carica papaya L. with active-centre properties that differ from those of papain by using 2,2'-dipyridyl disulphide and 4-chloro-7-nitrobenzofurazan as reactivity probes. Use of two-protonic-state electrophiles in the identification of catalytic-site thiol groups.

1. The proteinase papaya peptidase A, one of the major components of the latex of Carica papaya L., was shown to contain 1 thiol group per molecule; this thiol group is essential for catalytic activity and is part of the catalytic site. 2. The usefulness of two-protonic-state reactivity probes coupled with modification/activity-loss data in assigning a thiol group as an integral part of the catalytic site as against merely 'essential' for activity is discussed. 3. The active centre of papaya peptidase A was investigated by using 2,2'-dipyridyl disulphide and 4-chloro-7-nitrobenzofurazan as reactivity probes. The presence in the enzyme in weakly acidic media of an interactive system containing a nucleophile S atom (pKI3.9,pKII7.9) was demonstrated. 5. Papaya peptidase A resembles ficin (EC 3.4.22.3) and actinidin (the cysteine proteinase from Actinidin chinenis) in that it does not appear to possess a carboxy group able to influence the reactivity of the thiol group by change of ionization state at pH values of about 4, a situation that contrasts markedly with that which obtains in papain. 6. Implications of the results for possible variations in cysteine proteinase mechanism are discussed.

2,2'-Dipyridyl↗
PubMed1983

Effects of conformational selectivity and of overlapping kinetically influential ionizations on the characteristics of pH-dependent enzyme kinetics. Implications of free-enzyme pKa variability in reactions of papain for its catalytic mechanism.

The effects of selection by a small molecule, when binding to a protein, of a particular conformation from an equilibrium stereopopulation on the characteristics of the pH-dependence of reaction with a reactivity probe or substrate were determined by analysis of an appropriate kinetic model. For reaction in one protonic state containing an equilibrium mixture of two conformational isomers, the pH-second-order rate constant (k) profile is of conventional sigmoidal form. The apparent pKa value is a composite of the pKa values of the two conformational states. The value of pKapp. for a given enzyme under given experimental conditions will always be the same (provided that the site-specificity assumed in the model is maintained) irrespective of whether only one conformation reacts or both react, with the same or with different rate constants. The experimentally determined pH-independent rate constant (kapp.) is an average of the reactivities of the two conformational states weighted in favour of the predominant form. The presence of an additional but unreactive conformational state also affects the value of kapp. The possibility that overlapping acid dissociations that affect the reactivity of the enzyme might provide pH-k profiles often indistinguishable in practice from simple sigmoidal dissociation curves and subject to variability in apparent pKa values was evaluated by a simulation study. If two reactive protonic states of the enzyme respond differently to changes in the structure of the substrate or site-specific reactivity probe, differences in apparent pKa values of up to approx. 1 unit can be exhibited without deviation from sigmoidal behaviour being reliably observed. Differences in apparent pKa values observed in some site-specific reactions of papain and their possible consequences for its catalytic mechanism are discussed.

Hydrogen-Ion Concentration↗
PubMed1989

The interplay of electrostatic fields and binding interactions determining catalytic-site reactivity in actinidin. A possible origin of differences in the behaviour of actinidin and papain.

1. The pH-dependence of the second-order rate constant (k) for the reaction of actinidin (EC 3.4.22.14) with 2-(N'-acetyl-L-phenylalanylamino)ethyl 2'-pyridyl disulphide was determined and the contributions to k of various hydronic states were evaluated. 2. The data were used to assess the consequences for transition-state geometry of providing P2/S2 hydrophobic contacts in addition to hydrogen-bonding opportunities in the S1-S2 intersubsite region. 3. The P2/S2 contacts (a) substantially improve enzyme-ligand binding, (b) greatly enhance the contribution to reactivity of the hydronic state bounded by pKa 3 (the pKa characteristic of the formation of catalytic-site-S-/-ImH+ state) and pKa 5 (a relatively minor contributor in reactions that lack the P2/S2 contacts), such that the major rate optimum occurs at pH 4 instead of at pH 2.8-2.9, and (c) reveal the kinetic influence of a pKa approx. 6.3 not hitherto observed in reactions of actinidin. 4. Possibilities for the interplay of electrostatic effects and binding interactions in both actinidin and papain (EC 3.4.22.2) are discussed.

Binding Sites↗
PubMed1989

Affinity purification of the novel cysteine proteinase papaya proteinase IV, and papain from papaya latex.

A procedure is described for the purification of a previously undetected cysteine proteinase, which we have called papaya proteinase IV, from spray-dried latex of the papaya (Carica papaya) plant. The purification involves affinity chromatography on Gly-Phe-aminoacetonitrile linked to CH-Sepharose 4B, with elution by 2-hydroxyethyl disulphide at pH 4.5. The product thus obtained is a mixture of almost fully active papain and papay proteinase IV, which are then separated by cation-exchange chromatography. A preliminary characterization of papaya proteinase IV showed it to be very similar to chymopapain in both molecular size and charge. However, the new enzyme is immunologically distinct from the previously characterized cysteine proteinases of papaya latex. It also differs in its lack of activity against the synthetic substrates of the other papaya proteinases, in its narrow specificity against protein substrates and its lack of inhibition by chicken cystatin. Papaya proteinase IV is abundant, contributing almost 30% of the protein in spray-dried papaya latex, and contamination of chymopapain preparations with this enzyme may account for some of the previously reported heterogeneity of chymopapain.

Catalysis↗