Papain Preparation Research Articles

Preparation and characterization of a S-monomethoxypoly-(ethylene glycol) thioderivative of papain

A derivative of papain in which the free thiol group of the proteinase is attached to a poly(ethylene glycol) chain via a disulphide bond has been prepared. The poly(ethylene glycol) chain confers to the conjugate new physico-chemical properties, including a looser binding to ion exchange chromatographic supports such as CM-Sephadex C-50. The conjugate is thus readily separated from the irreversibly oxidized (unconjugated) forms of the enzyme. A papain preparation titrating 0.97 mol of thiol per mol of enzyme is obtained from the conjugate.

Investigation on Assay for the Proteolytic Activity of Protease and Papain Preparations for Food Manufacturing

Investigation on Assay for the Proteolytic Activity of Protease and Papain Preparations for Food Manufacturing

Review of the Problems Involved in Using Enzymes in Blood Group Serology - Provision of Freeze- Dried ICSH/ISBT Protease Enzyme and Anti-D Reference Standards

Proteolytic enzyme preparations and techniques used routinely in blood group serology for the detection of atypical patient antibodies prior to transfusion vary widely and are often poorly standardised. Recent advances have been made in the use of biochemical methods to standardise and stabilise the potency of the enzyme preparations used. A joint working party of the International Council for Standardization in Haematology (ICSH) and the International Society of Blood Transfusion (ISBT) has investigated possibilities for the provision of standards for the protease preparations and techniques. The specification for these standards was that the performance of enzyme reference preparation in the reference technique should be of equivalent sensitivity to the ICSH/ISBT LISS spin indirect antiglobulin test using a titration series of a reference weak anti-D, and be free from false-positive reactions. The working party circulated materials for evaluation in inter-laboratory trials, followed by a laboratory workshop meeting to achieve agreement on the specification for reference materials and methods. Reference freeze-dried papain at 0.6 azoalbumin units and weak anti-D preparations (91/562) have been prepared and validated to meet these specifications. The performance of a test enzyme preparation in the technique for which it is recommended for use should be at least equal to that of the reference papain preparation, by the reference two-stage technique in terms of sensitivity, using a titration series of the reference anti-D, and freedom from false-positive reactions, using six fresh inert sera. The reference papain and weak anti-D can also be used to calibrate the level of proteolytic activity required in other procedures in blood group serology, such as new technology methods for antibody detection, and automated and microplate cell grouping procedures. These preparations and an agreed method for their use are now available from listed centres as ICSH/ISBT and Food and Drug Administration reference materials.

Enzymatic preparation of optically active silylmethanol derivatives having a stereogenic silicon atom by hydrolase-catalyzed enantioselective esterification

Kinetic resolution of ethylmethylphenylsilylmethanol, a primary alcohol having a stereogenic silicon atom, was tried by hydrolase-catalyzed enantioselective reactions. Among twenty kinds of hydrolases examined, a commercial crude papain preparation was found to exhibit the highest enantioselectivity with moderate activity toward the silicon-containing alcohol on esterification with 5-phenylpentanoic acid in an organic solvent system, and the (+)-enantiomer of 92 %ee was obtained as the remaining substrate. Several silylmethanol derivatives could be also resolved by this enantioselective esterification, even though it was difficult to synthesize such chiral quaternary silanes with high optical purity by chemical methods due to the absence of leaving groups on the silicon atom. These results demonstrate that enzymes can recognize the configuration not only of carbon atoms but also of silicon atoms, and indicate the usefulness of biocatalysts for preparing optically active silanes.

Selection of Commercial Enzymes for Soymilkcurd Production by Limited Proteolysis with Immobilized Enzyme Reactor.

Commercial proteases suitable for soymilk-curd production by limited proteolysis with immobilized enzyme reactor were selected from three plant and a microbial proteases. Limited proteolysis of soy-protein with the enzymes at 30, 5 and 0°C was examined by an electrophoresis. Two commercial preparations of papain and a preparation of bromelain, which were plant proteases, limitedly digested native soyprotein even under the low temperature conditions. The soy-protein solution and soymilk treated with the plant proteases at 5°C gave curd by heat treatment. The plant enzymes were immobilized on SP-Sephadex C-25. The immobilized enzymes digested the soy-protein similarly to the free enzymes. Soymilk-curd was also obtained from the soymilk hydrolyzed with the immobilized enzymes.

Proteolytic elimination of decay-accelerating factor (DAF): Lytic abnormality coincides with removal of DAF in papain-treated human erythrocytes

Erythrocytes (E) from patients with paroxysmal nocturnal hemoglobinuria (PNH) lack decay-accelerating factor (DAF) and this partly causes increasing susceptibility of the E to complement. Several reagents have been used to convert normal E to the complement-sensitive (PNH-like) cells. The relationship between DAF amounts and complement susceptibility of these PNH-like cells has been examined. Of the reported reagents for preparation of PNH-like cells, 2-amino-ethylisothiouronium bromide (AET), papain, and periodate efficiently converted normal E to the complement-sensitive cells, but only papain reduced the quantity of DAF on the cells. Further, of the proteases we tested only papain cleaved DAF to liberate its major fragment from the cells. The papain-treated cells lysed in a similar fashion to PNH cells as the serum concentration increased. The major papain-digested product of DAF had M r 55,000, lacked hydrophobicity, and retained the ability to inhibit the C3 convertases. These findings suggest that papain allows liberation from cells of functional domains as well as most of the antigenic epitopes of DAF to generate a PNH-like cell.

Highly efficient proteinase assay with chromogenic substrates and its application in a study of enzyme inhibitors

A simple and rapid method for the determination of enzyme activities with chromogenic substrates is described. Trypsin and papain were used as model proteinases and N-benzoyl- dl-arginine p-nitroanilide (BAPNA) was applied as substrate. The enzyme assay was performed on a multi-scale using 96-well microtitration plates and product release was detected with the aid of an automatic plate reader, widely used in ELISA tests. The procedure was used for electrophoretic studies of trypsin and a crude papain preparation. It was also applied for the investigation of N-peptidyl- O-acylhydroxylamine proteinase inhibitors. In comparison with commonly used procedures with chromogenic substrates, the proposed approach consumes markedly reduced amounts of all reagents. It allows an almost unlimited number of samples to be assayed in a short time and should be applicable to the detection and determination of any enzyme activitiy where chromogenic substrates are applicable.

Standardization of papain reagents by measurement of active sites using a synthetic inhibitor, E-64.

L-trans-epoxysuccinyl-leucylamido (4-guanidino) butane (E-64) reacts rapidly and irreversibly in a one-to-one ratio with the active site of papain, causing complete inhibition of the enzyme. After the addition of various concentrations of E-64 to a papain preparation, the residual enzyme activity can be measured using an azoprotein technique. The molarity of E-64 required to cause complete inhibition of papain activity is equal to the molarity of papain-active sites. Preparations of papain from various sources were assayed for protease activity by hydrolysis of azoalbumin using several variants of the basic technique and also by hydrolysis of azocasein. For each variant of azoprotein assay procedure, the active sites of the papain were measured using E-64. All variations of the azoprotein technique yielded similar estimates of the active site molarity of the papain preparations, whereas the azoprotein assay results alone showed wide variation. Quantitation of the active-site molarity of various papain preparations using E-64 correlated with serologic efficacy.

Induction of differentiation in mouse erythroleukaemia cells by the action of papain at the cell surface.

The addition of one of several proteases to cultures of mouse erythroleukaemia (MEL) or human K-562 leukaemia cells can induce a substantial portion of the cells to undergo erythroid differentiation. This effect is due, at least in part, to the proteolytic action of these enzymes. The critical substrate(s) for this proteolytic action is not a component of the medium or a long-lived substance(s) released from the cells. In order to determine if the substrate(s) is located on the cell surface or intracellularly, a comparison of the ability of non-immobilized papain and immobilized papain (i.e. covalently linked to Sepharose beads which were larger than the cells) to induce MEL cell differentiation was undertaken. Both papain preparations induced the same level of differentiation. The proteolytic activity of the bead-linked papain remained associated with the beads. Therefore, proteases induce erythroid differentiation in these cells by acting proteolytically on a substrate(s) that is exterior to the cell.

Identification of the probable inhibitory reactive sites of the cysteine proteinase inhibitors human cystatin C and chicken cystatin.

When an excess of human cystatin C or chicken cystatin was mixed with papain, an enzyme-inhibitor complex was formed immediately. The residual free cystatin was then progressively converted to a form with different electrophoretic mobility and chromatographic properties. The modified cystatins were isolated and sequenced, showing that there had been cleavage of a single peptide bond in each molecule: Gly11-Gly12 in cystatin C, and Gly9-Ala10 in chicken cystatin. The residues Gly11 (cystatin C) and Gly9 (chicken cystatin) are among only three residues conserved in all known sequences of inhibitory cystatins. The modified cystatins were at least 1000-fold weaker inhibitors of papain than the native cystatins. An 18-residue synthetic peptide corresponding to residues 4-21 of cystatin C did not inhibit papain but was cleaved at the same Gly-Gly bond as cystatin C. When iodoacetate or L-3-carboxy-trans-2,3-epoxypropionyl-leucylamido-(4-guanidin o)butane was added to the mixtures of either cystatin with papain, modification of the excess cystatin was blocked. Papain-cystatin complexes were stable to prolonged incubation, even in the presence of excess papain. We conclude that the peptidyl bond of the conserved glycine residue in human cystatin C and chicken cystatin probably is part of a substrate-like inhibitory reactive site of these cysteine proteinase inhibitors of the cystatin superfamily and that this may be true also for other inhibitors of this superfamily. We also propose that human cystatin C and chicken cystatin, and probably other cystatins as well, inhibit cysteine proteinases by the simultaneous interactions with such proteinases of the inhibitory reactive sites and other, so far not identified, areas of the cystatins. The cleavage of the inhibitory reactive site glycyl bond in mixtures of papain with excess quantities of cystatins is apparently due to the activity of a small percentage of atypical cysteine proteinase molecules in the papain preparation that form only very loose complexes with cystatins under the conditions employed and degrade the free cystatin molecules.

Clinical biochemistry and pathology of mature beef cattle following ante-mortem intravenous administration of a commercial papain preparation

Ten healthy beef cattle in a commercial abbatoir were treated intravenously before slaughter with a commercial papain-based tenderising injection (Pro Ten). Animals were observed for behavioural and clinical abnormalities following treatment. Serum enzyme activities were measured pre-treatment and post-treatment immediately pre-slaughter < 6 min later to detect liver and muscle damage. Carcases were examined grossly post mortem. Histological examination of liver, kidney and muscle followed. Nine contemporary, age-matched controls were similarly examined. It was concluded that ProTen treatment did not cause any detectable hepatocellular or renal damage and there was no significant difference in the parameters examined between treated and untreated cattle. A decision to ban the use of ProTen in cattle could not therefore be based on the premise that it interfered with the animal's welfare in the period following injection under the conditions pertaining in this experiment.

Applicability of glutaminyl-tRNA cyclotransferase in the peptide synthesis

Peptides with amino-terminal glutamine and their derivatives on treatment with crude papain preparation, containing glutaminyl-tRNA cyclotransferase (E.C. 2.3.2.5.), are converted into the corresponding compounds with pyroglutamic acid. This enzymic cyclization does not take place with glutamic acid or its γ-derivatives. The reaction has been optimized for the preparation of pyroglutamic acid phenylhydrazide. Glutaminyl-leucine phenylhydrazide and glutaminyl-histidyl-proline amide reacted to give pyroglutamyl-leucine phenylhydrazide and TRH, respectively. Treatment of crude papain with iodoacetamide completely inhibited its activity without affecting the glutaminyl-cyclotransferase activity.

Chromatographic separation of papain evaluated by immunochemical methods

The chromatographic separation of crude papain preparations on Sephadex G-50 (fine) enables pure papain to be obtained in a single step. Immunochemical techniques have been found to be very convenient for testing the purity of the individual chromatographic fractions. A general approach is presented that makes it possible to follow the course of the chromatographic purification of any immunogenic compound by simple qualitative immunochemical techniques that can be applied in any laboratory.

A necessary modification to the preparation of papain from any high-quality latex of Carica papaya and evidence for the structural integrity of the enzyme produced by traditional methods

A method of preparation of papain (EC 3.4.22.2) from relatively soluble types of latex of Carica papaya, including spray-dried latex produced by a controlled and relatively mild process, was devised. Spray-dried latex dissolves easily in water up to 350mg/ml at 22 degrees C, which corresponds to approx. 230mg of protein/ml. When the usual method of preparation of crystalline papain contaminated only by its oxidation products, developed by Kimmel & Smith [J. Biol. Chem. (1954) 207, 515-531], is applied to spray-dried latex, the result is a preparation of papain heavily contaminated by chymopapains A and B (EC 3.4.22.6), and to a lesser extent by papaya peptidase A. This applies also to other types of papaya-latex currently commercially available, which, though less soluble than spray-dried latex, are more soluble than the types of latex available when the method of Kimmel & Smith (1954) was developed. This contamination is avoided by adjusting the concentration of the initial latex extract to 65mg of protein/ml (or less) before salt fractionation. For spray-dried latex this corresponds to 100mg of latex/ml. Papain isolated from spray-dried latex was characterized by using 2,2'-dipyridyl disulphide and 4-chloro-7-nitrobenzofurazan as thiol-specific reactivity probes and alpha-N-benzoyl-l-arginine ethyl ester as substrate. Papain isolated from this source appears to have the same catalytic-centre characteristics as papain isolated previously from latex produced by harsher methods. The catalysis of the hydrolysis of alpha-N-benzoyl-l-arginine ethyl ester by the mixture of thiol proteinases extracted from spray-dried latex by application of the method of Kimmel & Smith (1954) appears to obey Michaelis-Menten kinetics. The presence of the other enzymes results in an increase in the value of K(m) and a decrease in the catalytic-centre activity (k(cat.)) relative to the values for the catalysis by papain.

On a rabbit hyperlipemia induced by a fungic galactomannane peptide.

I.v. injection into rabbits of a fungic galactomannane peptide isolated from the culture medium of Aspergillus oryzae induced the apparition, 20 h later, of an hypertriglyceridemia, with a concomittant decrease of about 70% of the post-heparin lipoprotein lipase activity. The same effect had been obtained earlier with a carbohydrate-rich fraction purified from a crude papain preparation. The 2 fractions are compared.

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.

Papain denaturation is not a two-state transition

. Even proteins such as lysozyme [2] which has a cleft in the middle and parvalbumin [3] which consists of two distinct domains are denatured without stable intermediate states. This led to a tempting conclusion that all small globular proteins built of one poly- peptide chain represent a single cooperative system. In this connection it was very interesting to examine papain in which two structural domains are separated by a deep cleft [4] . These parts of the molecule are linked by a number of secondary bonds and seem to be tightly fastened. However, consideration of the course of the chains in each part suggests that they fold independently [S] . Then the question arises whether these parts are indeed independent. Thermodynamic analysis of the calorimetric data on papain thermal denaturation carried out in this paper has shown that papain denaturation is not a two-state transition and the two parts of this macromolecule represent quasi-independent cooper- ative systems. Thus it follows that both parts of papain are folded independently. 2. Materials and methods A commercial preparation of papain (Fluka AG, Buchs SG, Switzerland) was twice recrystallized according to [6]. Papain was activated by reducing with 0.01 M dithiothreitol in 0.1 M Tris-HCl (PH 8.0) for 2 h at room temperature. Then the

Ultraviolet inactivation of papain.

Abstract— Flash photolysis transient spectra (Λ &gt; 250 nm) of aqueous papain show that the initial products are the neutral tryptophan radical Tip (Λmax 510 nm), the tryptophan triplet state 3Trp (Λm., 460nm), the disulfide bridge electron adduct –ṠS‐— (Λmax 420nm) and the hydrated electron eaq‐. The –ṠS‐– yield was not altered by nitrous oxide or air, indicating that the formation of this product does not involve electrons in the external medium. The original papain preparation was activated by irradiating under nitrogen. The action spectrum supports previous work attributing the low initial activity to blocking of cysteinyl site 25 with a mixed disulfide. Flash lamp irradiation in nitrogen led to activation at low starting activities and inactivation at higher starting activities, while only inactivation at the same quantum yield was observed with air saturation. The results are consistent with photoionization of an essential tryptophyl residue as the key inactivating step.

Preparation of insoluble papain through cyanoethylated starch anthranilates.

Preparation of insoluble papain through cyanoethylated starch anthranilates.

The induction of nonspecific resistance in mice by papain.

various types of heterologous challenge have also been reported (Rowley, 1955; Landy, 1956a; Dubos and Schaedler, 1957; and others). The purpose of this report is to discuss the nonspecific resistance induced in mice by a substance present in crude commercial preparations of papain. Papain is a proteolytic enzyme prepared from the fruit of the papaya (Carica papaya). This study was initiated when, in the course of various experiments concerning the effect of enzymatic digestion on the immunologic properties of bacterial cells, it was noted that a papain suspension (experimental enzyme control) induced a significant degree of protection in mice challenged with Staphylococcus aureus. In that study it was noted that when 0.5 ^0 papain was injected into mice approximately 60*^0 of the animals survived a