Immobilization of papain on cotton fabric by sol–gel method

A method has been developed to immobilize papain on cotton fabric by means of sol–gel technique. The activity of free papain and papain in silica sol under sonication was studied. Scanning electron microscopy, energy dispersive spectrometer and the Bradford method were used to characterize papain immobilization. The efficiency of the immobilization was investigated by examining the relative enzymatic activity of free and immobilized papain, respectively. The results show that the optimum pH value in the medium for immobilized papain is shifted to alkaline side. In addition, the adaptability of papain to environmental acidity is significantly increased. The thermostability of immobilized papain shows no significant change compared to the free enzyme. The papain immobilized on fabric by sol–gel technique retains more than 30% of the original activity after six reuses continuously.     

Cysteine proteinase activity regulation. A possible role of heparin and heparin-like glycosaminoglycans.

Papain is considered to be the archetype of cysteine proteinases. The interaction of heparin and other glycosaminoglycans with papain may be representative of many mammalian cysteine proteinase-glycosaminoglycan interactions that can regulate the function of this class of proteinases in vivo. The conformational changes in papain structure due to glycosaminoglycan interaction were studied by circular dichroism spectroscopy, and the changes in enzyme behavior were studied by kinetic analysis, monitored with fluorogenic substrate. The presence of heparin significantly increases the alpha-helix content of papain. Heparin binding to papain was demonstrated by affinity chromatography and shown to be mediated by electrostatic interactions. The incubation of papain with heparin promoted a powerful increase in the affinity of the enzyme for the substrate. In order to probe the glycosaminoglycan structure requirements for the papain interaction, the effects of two other glycosaminoglycans were tested. Like heparin, heparan sulfate, to a lesser degree, was able to decrease the papain substrate affinity, and it simultaneously induced alpha-helix structure in papain. On the other hand, dermatan sulfate was not able to decrease the substrate affinity and did not induce alpha-helix structure in papain. Heparin stabilizes the papain structure and thereby its activity at alkaline pH.     

Studies on quantum dots synthesized in aqueous solution for biological labeling via electrostatic interaction

3-Mercaptopropyl acid-stabilized CdTe nanoparticles synthesized in aqueous solution are effectively bound to a biomacromolecule, papain, via electrostatic interaction. The conjugation between the nanoparticles and the papain is demonstrated by UV-Vis absorption, photoluminescence spectroscopy, transmission electron microscopy, and fluorescence micrographs. The biological activity of papain is maintained after the conjugation. The effects of the quantity of papain and the size of nanoparticles on the fluorescence characteristics of the CdTe-papain bioconjugates were studied.     

Properties of papain, immobilized on organosilica

Papain, a proteolytic enzyme, is used in the reactions of organic synthesis for preparing peptides. The use of immobilized papain with this aim is very promising. Preparations of papain immobilized by organosilica have been studied for their physicochemical properties as well kinetics of the papain immobilization by amino-organosilica activated by cyanuric chloride. Retention of the enzyme activity of immobilized papain reached 40% and depended on the amount of enzyme bound with the carrier. Kmobs of the immobilized enzyme did not differ significantly from that of the soluble enzyme. After immobilization the pH-optimum an pH-profile of the catalytical activity of papain remained unchangeable. For the period of 20 days immobilized papain has lost 20-50% activity.     

Papain reduces gastric acid secretion induced by histamine and other secretagogues in anesthetized rats

We studied the effect of papain on rats' gastric acid secretion and found that: 1. Feeding of latex of unripe papaya fruit significantly reduced gastric acid secretion induced by methacholine; 2. Feeding of crystalline papain in doses of 3.2 mg/kg reduced gastric acid secretion induced by histamine, methacholine and tetragastrin; 3. The reduction of gastric acid secretion was observed as early as 2 hours after papain feeding, lasted up to 48 hours, and waned within 96 hours; 4. Intraperitoneal injection of papain had no effect on acid secretion. These results led us to believe tha the effect of papain on gastric acid secretion is a local one acting directly on the gastric mucosa, and this local effect of a single dose of papain is reversible, causing no permanent damage to the mucosa.     

高分子膜载体固定化木瓜蛋白酶

在浸润条件下,以0.5%(v/v)戊二醛交联的高分子膜尼龙载体固定化木瓜蛋白酶。对固定化条件进行了优化,比较了固定化酶与游离酶的酶学参数。结果表明,4℃、pH6.0条件下,将膜载体浸润于2mg/mL酶液中5h,固定化酶活为303.4U/g。固定化酶最适反应pH为6.0～7.0,最适反应温度为65℃。其pH稳定性、热稳定性均比游离酶高。     

Study of a papain activation factor present in the dialysable fraction of soyabean extract

Soyabean extract contains an activator which passes through parchment paper. The increase in the activity of a mixture of urease and papain with increasing amount of papain in the mixture is due to the increase in the non-dialysable fraction of papain.     

Protease inhibitors from ripened and unripened bananas.

The proteolysis of casein by trypsin, chymotrypsin and papain was inhibited by ripened and unripened bontha, poovan, nendran, cavendish and rasthali bananas. The inhibition of trypsin, chymotrypsin and papain by different ripened banana cultivars was much more than that of unripened banana cultivars. The trypsin and chymotrypsin inhibitory activity of ripened poovan was heat stable, resistant to pronase and partly stable to trypsin but the trypsin and chymotrypsin inhibitory activity of unripened poovan was stable to heat and resistant to pronase only. The partial stability of trypsin inhibitory activity and instability of papain inhibitory activity of ripened poovan to alkaline pH suggests that the inhibitory factors of trypsin and papain were dissimilar. The probable role of unripened banana papain inhibitors in curing stomach ulcers and antinutritional role of ripened banana trypsin inhibitors is discussed.     

Single disulfide bond reduced papain exists in a compact intermediate state

Partially reduced proteins and other chemically modified derivatives are very useful model systems to understand the protein folding in vivo. Upon reduction, proteins attain different conformations with varying degrees of compactness. The reduction of papain in the presence of 8 M urea leads to the partial reduction of one disulfide bond. This derivative (single disulfide reduced carboxymethylated 1RCM papain (3RCM papain)) was characterized by spectroscopic methods and the effect of this reduction on the unfolding of the protein was investigated. Under this partial reduction, papain exhibits more than half of the tertiary and most of the secondary structures relative to the non-reduced molecule (free cysteine reduced and carboxymethylated papain (1RCM papain)). Hydrophobic regions are exposed to the solvent as observed through 8-anilino-1-naphthalene sulfonic acid binding which was absent in the fully intact and unfolded protein, at neutral pH. Hydrodynamic studies indicated that 3RCM papain, under neutral conditions, possess expanded conformation as compared to the native protein. Tryptophan fluorescence quenching studies suggested the exposure of aromatic residues to solvent. Guanidine hydrochloride induced unfolding of this derivative, at neutral pH, showed a non-cooperative transition contrary to the cooperativity seen with intact protein. Thermal unfolding indicates that 3RCM papain is less stable compared to the intact protein. These findings suggest that partial reduction of papain has a significant effect on the unfolding behavior of papain.     

甲醇溶液对木瓜蛋白酶催化活性的影响

试验结果表明,木瓜蛋白酶在一定浓度甲醇溶液中水解酪蛋白的活性显著上升;动力学测定表明,该酶在甲醇溶液中Km下降;在甲醇溶液中木瓜蛋白酶催化酪蛋白水解反应的最适pH为8.5,最适温度为70℃;紫外差示光谱研究表明,在甲醇溶液作用下木瓜蛋白酶的二级结构发生了变化;荧光发射光谱研究表明,木瓜蛋白酶在甲醇溶液中发射峰位向低波长移动,荧光峰值明显增高。     

Cysteine enhances activity and stability of immobilized papain

Immobilization of papain on Sepharose 6B in the presence of different concentrations of cysteine affected the enzyme activity depending on cysteine concentration. The maximum specific activity was observed when papain was immobilized with 200 mM cysteine. The immobilization process brought significant enhancement of stability to temperature and extreme pH values with respect to free papain. After immobilization, the optimum temperature of papain activity increased by 20°C (from 60 to 80°C) and its optimum pH activity shifted from 6.5 to 8.0. Catalytic efficiency (k_cat/K_m) and specific activity of the immobilized enzyme do not significantly change after immobilization. The temperature profile of this form of immobilized papain showed a broad range of activity compared with both free and immobilized form of papain in the absence of cysteine. This significant behavior in terms of activation energy is also discussed.     

The Surface Plasmon Resonance Imaging sensor for papain based on immobilized cystatin

The Surface Plasmon Resonance Imaging (SPRI) sensor for papain determination based on the interaction between the immobilized cystatin and aqueous papain solution has been developed. The development of the sensor is a stage in the development of the sensor for the determination of cystein proteinases of the papain group. Cystatin was immobilized onto a gold chip by means of a thiol underlayer (cysteamine) and EDS/NHS reaction. Conditions of cystatin- papain interaction were optimized (pH, time of interaction and cystatin concentration). The developed sensor works within the range of 1 - 10 ng ml(-1). However, the precision of measurements (RSD equal to 45%) should be improved. The response of the sensor to papain is specific. This was checked using BSA as a reference.     

High-performance affinity chromatography for characterization of human immunoglobulin G digestion with papain

Reactive continuous rods of macroporous poly(glycidyl methacrylate-co-ethylene dimethacrylate) were prepared within the confines of a stainless steel column. Then papain was immobilized on these monoliths either directly or linked by a spacer arm. In a further step, a protein A affinity column was used for the characterization of the digestion products of human immunoglobulin G (IgG) by papain. The results showed that papain immobilized on the monolithic rod through a spacer arm exhibits higher activity for the digestion of human IgG than that without a spacer arm. The apparent Michaelis-Menten kinetic constants of free and immobilized papain, K(m) and V(max), were determined. The digestion conditions of human IgG with free and immobilized papain were optimized. Comparison of the thermal stability of free and immobilized papain showed that the immobilized papain exhibited higher thermal stability than the free enzyme. The half-time of immobilized papain reaches about a week under optimum pH and temperature conditions.     

Activation of glutaraldehyde-crosslinked chymotrypsinogen-A. Enzymatic activity and circular dichroism studies

Phenylhydrazine does not inactivate papain or glyceraldehyde-3-phosphate dehydrogenase under anaerobic conditions. The inactivation of papain and glyceralde-hyde-3-phosphate dehydrogenase under aerobic conditions is ascribed to the oxidation of phenylhydrazine by O₂ which generates phenyldiimide and H₂O₂, both of which react with the essential sulfhydryl groups and inactivate the enzymes. Phenyldiimide generated from methyl phenylazoformate inactivates both of the sulfhydryl enzymes under anaerobic conditions. The inactivation of papain and GPD with aerobic, aqueous solutions of [¹⁴C]phenylhydrazine introduces a small amount of radioactivity into the enzymes which is discharged by dithiothreitol. The amount of radioactivity bound to papain is increased when cyanide is present in the inactivation mixture.When the β-[¹⁴C]thiocyanoalanine derivative of papain is treated with phenylhydrazine the radioactivity is discharged from the enzyme. Cyanide evidently reacts with the sulfenic acid derivative of papain to form a thiocyanate derivative. Phenylhydrazine presumably displaces cyanide from the thiocyanate derivative to form a sulfenyl hydrazide derivative to account for the increased incorporation of [¹⁴C]phenylhydrazine when papain is inactivated with aerobic solutions of [¹⁴C]-phenylhydrazine in the presence of cyanide. When the sulfhydryl group of papain is oxidized to a sulfenic acid with H₂O₂ and then treated with [¹⁴C]phenylhydrazine, ¹⁴C is not incorporated into the enzyme. These experiments suggest that the H₂O₂ in the aerobic solutions of phenylhydrazine oxidizes the sulfhydryl group at the active site of papain to a sulfenic acid. The [¹⁴C]phenyldiimide in these solutions reacts to some extent with the active sulfhydryl group to form a sulfenyl hydrazide derivative.     

Nonionic tensides modify papain structure and proteolytic activity

The effect of 33 polyethoxylated nonionic tensides with various hydrophobic moieties was studied on the proteolytic activity and phase transition behaviour of papain. Tensides with longer ethyleneoxide chain markedly increased the phase transition temperatures of papain indicating possible hydrogen bond formation between the hydrophilic ethyleneoxide chain and the polar substructures of papain. The character of lipophilic moiety of tensides influences also each physicochemical parameter of papain suggesting the existence of hydrophobic interactions too. The significant positive linear correlations between the activity increasing and structure stabilizing effects of tensides make probably that the structure modification of papain accounts for its enchanced proteolytic activity.     

Conversion of the active-site cysteine residue of papain into a dehydro-serine, a serine and a glycine residue.

Photolysis of papain which had been inhibited with 2-bromo-2',4'-dimethoxyacetophenone regenerated papain, but also formed [deltaSer25]-papain (i.e. papain in which the active-site cysteine residue 25 was replaced by dehydroserine) via the intermediate dehydrocysteine analogue, [deltaCys25]-papain. Reduction with sodium borohydride gave [Ser25]papain. Both [Ser25]papain and [deltaSer25]-papain had binding properties similar to those of papain, but were devoid of enzymic activity. Their fluorescence properties were also investigated. Incubation of [deltaSer25]papain at pH 9.0 gave [Gly25]papain.     

Inhibitory mechanism of a cross-class serpin,the squamous cell carcinoma antigen 1

The squamous cell carcinoma antigen (SCCA) 1 and its homologous molecule, SCCA2, belong to the ovalbumin-serpin family. Although SCCA2 inhibits serine proteinases such as cathepsin G and mast cell chymase, SCCA1 targets cysteine proteinases such as cathepsin S, K, L, and papain. SCCA1 is therefore called a cross-class serpin. The inhibitory mechanism of the standard serpins is well characterized; those use a suicide substrate-like inhibitory mechanism during which an acyl-enzyme intermediate by a covalent bond is formed, and this complex is stable against hydrolysis. However, the inhibitory mechanism of cross-class serpins remains unresolved. In this article, we analyzed the inhibitory mechanism of SCCA1 on a cysteine proteinase, papain. SCCA1 interacted with papain at its reactive site loop, which was then cleaved, as the standard serpins. However, gel-filtration analyses showed that SCCA1 did not form a covalent complex with papain, in contrast to other serpins. Interaction with SCCA1 severely impaired the proteinase activity of papain, probably by inducing conformational change. The decreased, but still existing, proteinase activity of papain was completely inhibited by SCCA1 according to the suicide substrate-like inhibitory mechanism; however, papain recovered its proteinase activity with the compromised level, when all of intact SCCA1 was cleaved. These results suggest that the inhibitory mechanism of SCCA1 is unique among the serpin superfamily in that SCCA1 performs its inhibitory activity in two ways, contributing the suicide substrate-like mechanism without formation of a covalent complex and causing irreversible impairment of the catalytic activity of a proteinase.     

Conformational changes of papain induced on interaction with thiol proteinase inhibitors from newborn rat epidermis

The conformational changes of the papain molecular on interaction with two thiol proteinase inhibitors (TPI(1) and TPI(2] from newborn rat epidermis were studied by measuring circular dichroism (CD), the difference absorption spectrum, and the fluorescence spectrum due to tryptophan residues in papain. The far-ultraviolet CD band of papain between 210 and 230 nm was distinctly reduced on interaction with both inhibitors. Also, the near-ultraviolet CD spectrum of TPI(1)-bound papain changed between 285 and 320 nm as well as that of the TPI(2)-bound enzyme. The difference absorption spectrum for TPI(1)-bound papain exhibited two distinct peaks at 276.5 and 282 nm, indicating perturbation of aromatic amino acid residues. The fluorescence intensity of papain was significantly decreased on interaction with both inhibitors, which showed pH-dependency on an ionizable group, with pK values of 8.5 and 7.9 for TPI(1) and TPI(2), respectively. The complex formation of papain with both inhibitors caused a reduction of the susceptibility of a tryptophan residue, probably tryptophan-177, to chemical modification with N-bromosuccinimide. These results suggest that the active site involving histidine-159 in the papain molecule was much influenced by the alteration of the microenvironment of tryptophan-177 as a part of the interaction site for these two thiol proteinase inhibitors.     

Mechanism of solvent induced thermal stabilization of papain

In the present study an attempt is made to elucidate the effects of various cosolvents, such as sorbitol, sucrose, xylose and glycerol, on papain. The stabilizing effects of these cosolvents on the structure and function of papain is determined by the activity measurements, fluorescence spectroscopy and differential scanning calorimetry (DSC). The enzyme activity measurements indicate several fold increase in the thermal stability of the enzyme in all the cosolvents used. The thermal denaturation studies of papain in presence of various concentrations of cosolvents indicated a shift in the apparent thermal denaturation temperature (app Tm) suggesting increased thermal stability of papain in presence of cosolvents. The app Tm shifted from a control value of 83+/-1 degrees C to a value of >90+/-1 degrees C in presence of 40% sorbitol. The DSC thermogram for native papain can be clearly deconvoluted into two transitions corresponding to left and right domain and in presence of cosolvents both transitions A and B shift to higher temperature. Maximum stabilization was seen in case of 30% sorbitol where the thermal transition temperatures increased compared to control. The results from partial specific volume measurements of papain in presence of cosolvents suggest that the preferential interaction parameter (xi3) was negative in all cosolvents and maximum hydration was observed in the case of glycerol where the preferential interaction parameter was 0.165g/g. These above results suggest that there is a considerable increase in the thermal stability of papain in presence of these cosolvents as a result of preferential hydration.     

A two-step procedure for purification of papain from extract of papaya latex

A method is presented for purifying papain from extracts of papaya latex. The procedure involves precipitation of the extract of papaya latex with sodium chloride followed by affinity chromatography of the redissolved precipitate. Precipitation of the protein from the latex extract is necessary to separate the papain from material which interferes with the binding of papain to the affinity column. During affinity chromatography, the affinity column is overloaded to insure absence in the final product of impurities which are capable of binding to the affinity column.The papain prepared by this procedure yielded an amino acid analysis and an N-terminal amino acid analysis expected for a sample of pure papain. No Met was detected on amino acid analysis nor was the presence of N-terminal residues other than He detected. On polyacrylamide disc gel electrophoresis at pH 4.3, papain prepared by the method described in this work was indistinguishable from crystalline papain which was prepared by the method of Kimmel and Smith, and further purified by affinity chromatography. Both disc gel patterns consisted of a single band and a trailing shadow which was less than 5% of the main band. In routine spectrophotometric assays, the specific activity toward N,α-benzoyl-l-arginine ethyl ester of papain prepared by the procedure described in this work was indistinguishable from crystalline papain prepared by the method of Kimmel and Smith, and further purified by affinity chromatography. Values of 24 sec^?1' and 15 mm were obtained from the turnover number and K_m for the papain-catalyzed hydrolysis of N,α-benzoyl-l-arginine ethyl ester at 25 °C, pH 6.00, $\text{Γ}\text{2 0.30}$ using a pH stat.