Synthesis of redox-active ferrocene pyrazole conjugates and their cytotoxicity in human mammary adenocarcinoma MCF-7 cells

The redox-active ferrocenoyl modified pyrazole ligand (3-Fc-AMP) readily coordinates to a variety of transition metal ions. Here, we describe our findings regarding the synthesis and structural characterization of iron and cobalt complexes of Fc-AMP, and the cytotoxicity profiles of these chemicals in vitro. Using the human mammary adenocarcinoma MCF-7 cell line we show that the free ligand and three metal complexes induced cytotoxicity with calculated LC₅₀s ranging from 45.8 to 73.2 μM. The toxicity of the complexes decreased as the redox potential increased. The present study demonstrates the potential chemotherapeutic promise of metal complexes of a redox-active ferrocenoyl modified pyrazole ligand on human cancer cells.     

Synthesis and evaluation of ferrocenoyl pentapeptide (Fc-KLVFF) as an inhibitor of Alzheimer's Aβ₁-₄₂ fibril formation in vitro

Aggregation and fibril formation of β-amyloid peptides (Aβ) is the key event in the pathogenesis of Alzheimer's disease. Many efforts have been made on the development of effective inhibitors to prevent Aβ fibril formation or disassemble the preformed Aβ fibrils. Peptide inhibitors with sequences homologous to the hydrophobic segments of Aβ can alter the aggregation pathway of Aβ, together with decrease of the cell toxicity. In this study, the conjugate of ferrocenoyl (Fc) with pentapeptide KLVFF (Fc-KLVFF), was synthesized by HBTU/HOBt protocol in solution. The inhibitory effect of Fc-KLVFF on Aβ(1-42) fibril formation was evaluated by thioflavin T fluorescence assay, and confirmed by atomic force microscopy (AFM) and transmission electron microscopy (TEM) analyses. Fc-KLVFF shows high inhibitory effect towards the fibril formation of Aβ(1-42). Additionally, the attachment of ferrocenoyl moiety onto peptides allows us to investigate the interaction between the inhibitor and Aβ(1-42) in real-time by electrochemical method. As expected, tethering of ferrocenoyl moiety onto pentapeptide shows improved lipophilicity and significant resistance towards proteolytic degradation compared to its parent peptide.     

Design,solid phase synthesis and evaluation of cationic ferrocenoyl peptide bioconjugates as potential antioxidant enzyme mimics

Synthetic C-terminal amidated cationic ferrocenoyl peptide bioconjugates Fc-Orn-Orn-Orn (1) and Fc-Tyr-Orn-Orn-Orn (2) were rationally designed as superoxide dismutase (SOD) mimics based on the structure of the iron SOD from Escherichia coli. Ferrocenoyl peptide bioconjugates 1, 2 and ferrrocenecarboxylic acid (4) were subsequently evaluated as SOD mimics and as inhibitors of peroxynitrite-mediated tyrosine nitration. Due to their cationic character, ferrocenoyl peptide bioconjugates 1 and 2 exerted an acceptable SOD activity (EC₅₀ = 575 μM and 310 μM, respectively) in comparison with 4 (EC₅₀ = 1.4 mM). The C-terminal amidated cationic peptide Ac-Tyr-Orn-Orn-Orn (3), designed as marker of peroxynitrite, was used to evaluate the inhibitory activity of 1 and 4 towards peroxynitrite-mediated tyrosine nitration. Both compounds proved to inhibit the nitration especially the cationic ferrocenoyl peptide bioconjugates 1. The ferrocene moiety of conjugate 2 displayed a strong inhibitory activity of peroxynitrite-mediated nitration of the neighboring tyrosine.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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。     

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.     

Optimization of adsorption conditions of papain on dye affinity membrane using response surface methodology

The adsorption of papain on Reactive Blue 4 dye–ligand affinity membrane was investigated in a batch system. The combined effects of operating parameters such as initial pH, temperature, and initial papain concentration on the adsorption were analyzed using response surface methodology. The optimum adsorption conditions were determined as initial pH 7.05, temperature 39 °C, and initial papain concentration 11.0 mg/ml. At optimum conditions, the adsorption capacity of dye–ligand affinity membrane for papain was found to be 27.85 mg/g after 120 min adsorption. The papain was purified 34.6-fold in a single step determined by fast protein liquid chromatography. More than 85% of the adsorbed papain was desorbed using 1.0 M NaCl at pH 9.0 as the elution agent. The purification process showed that the dye–ligand immobilized composite membrane gave good separation of papain from aqueous solution.     

Mercaptan-induced fragmentation of a subunit-like proteolytic fragment of immunoglobulin M

Limited papain hydrolysis of immunoglobulin M (IgM) produces a subunit-like proteolytic fragment designated IgM(p) (Inman & Hazen, 1968). In the presence of mercaptans, IgM(p) partially dissociated into Fc(mu)-like and Fab(mu) fragments. Treatment of residual IgM (that remaining after a papain digestion) with 2mm-mercaptoethylamine resulted in fragmentation of the same type that occurs in a routine limited digestion of IgM with papain, although exogenous enzyme was not added to the mixture. When IgM was hydrolysed with (14)C-labelled papain, a small quantity of the enzyme was found to be associated with the residual IgM and IgM(p) fractions. IgM and IgM 7S subunit (IgM(s)) that had been exposed to papain in the absence of activating mercaptan and separated from the enzyme by gel filtration also fragmented when subsequently treated with 2mm-mercaptoethylamine. The fragments resembled those produced during a typical limited papain digestion of IgM. It was concluded that mercaptoethylamine induced fragmentation of IgM(p) by activating adsorbed papain.     

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.     

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.     

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.     

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.