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Purification of a novel cysteine protease, procerain B, from Calotropis procera with distinct characteristics compared to procerain
Abstract
Proteases have applications in food, detergent and pharmaceutical industries. A novel protease has been purified from the latex of Calotropis procera and characterized. As another cysteine protease, procerain, is reported from the same source, the newly purified enzyme was named as procerain B. The enzyme shows distinct properties compared to procerain, in terms of cleavage recognition site, immunological properties and other physical properties like molecular weight, isoelectric point, etc. The newly purified enzyme shows a broad optimum pH (6.5–8.5) as well as broad optimum temperature (40–60 °C). Additionally, the enzyme retains its activity where most of other proteases are not active. Moreover, the enzyme appeared to be very efficient in hydrolysis of blood stain and may have potential application in detergent industries. Simple and economic purification of procerain B, together with easy availability of latex, makes the large-scale production of procerain B possible, thus enables to explore various industrial as well as biotechnological applications.
... Since another cysteine protease (Procerain) is already known from the same source [24], we have named this protease as ''Procerain B''. Procerain B has wide functional pH range and retains more than 270% activity till 70uC where most of other enzymes became inactive [25]. We have tested its applicability in food, detergent and dairy industries and found it as a potential candidate for several industrial applications [21]. ...
... The enzyme was purified from the latex of Calotropis procera by the method of Singh et al., 2010 [25]. CM-Sepharose FF was purchased from GE Healthcare. ...
... Amberlite is a stable and comparatively robust matrix which can be used for immobilization purpose. We have purified a novel cysteine protease and proved its importance in several industries [21,25]. In present study we are focusing on immobilization of procerain B on stable and robust amberlite MB-150 beads and characterization of immobilized product for industrial use, which will further enhance the industrial importance of this enzyme. ...
Proteases are involved in several crucial biological processes and reported to have important physiological functions. They also have multifarious applications in different industries. The immobilized form of the enzyme further improves its industrial applicability. Here, we report covalent immobilization of a novel cysteine endopeptidase (procerain B) on amberlite MB-150 beads through glutaraldehyde by Schiff base linkage. The immobilized product was examined extensively by Fourier Transform Infrared Spectroscopy (FTIR), Scanning electron microscopy (SEM) and Energy Dispersive X-ray (EDX) analysis. The characterization of the immobilized product showed broader pH and thermal optima compared to the soluble form of the enzyme. The immobilized form of procerain B also showed lower Km (180.2766 mM) compared to the soluble enzyme using azocasein as substrate. Further, immobilized procerain B retains 38.6% activity till the 10 th use, which strongly represents its industrial candidature.
... 32 We have previously reported a novel cysteine protease from the latex of a medicinal plant Calotropis procera with a molecular mass of 25.7 kDa and broad pH and temperature optima. 33 Because of its broader functional range, high thermal stability, and compatibility with detergents, it may have several industrial applications in food, detergent, dairy, and leather industries. 34 In the present study, we aim to immobilize the procerain B on chitosan beads through covalent linkages and characterize the immobilized product. ...
... The temperature optimum of immobilized procerain B was 55°C ( Figure 4B), while the free enzyme shows maximum activity in the range of 40À60°C. 33 The conformational flexibility of procerain B was affected by immobilization. An increase in the temperature optima is a clear reflection of conformational rigidity, which makes it resistant to denaturation. ...
... The temperature stability of immobilized procerain B was investigated, and we found that it showed more than 80% activity up to 70°C ( Figure 4D), while soluble procerain B shows a similar activity up to 65°C only. 33 Thus, immobilization leads to a slight increase in thermal stability, which makes it industrially more valuable. ...
Proteases have several applications in the food industry. We report the immobilization of procerain B, a novel cysteine protease, on glutaraldehyde-activated chitosan beads through covalent attachment. Glutaraldehyde not only serves as a cross-linking agent but also links the procerain B on the surface of bead through primary amine group (either lysine side chain or N-terminal) by Schiff base linkage. Immobilized procerain B was characterized for optimum functional range and stability with respect to pH and temperature. The chitosan-immobilized procerain B has broad pH and thermal optima. The effects of substrate concentration and reusability of immobilized beads were also studied. It showed nearly 50% activity until the 10th use.
... However, the activity of both extracts was not strongly affected by the change in pH, which indicates their stability in a large pH range. According to the literature bibliography, the majority of proteases belonging to the milkweed family have an optimal pH between 5 and 8, as observed in papain [16], procerain B [29], and philibertain g I [30]where they marked an optimum pH of 7, 8, and 7, respectively. ...
... However, the activity of both extracts was not strongly affected by the change in pH, which indicates their stability in a large pH range. According to the literature bibliography, the majority of proteases belonging to the milkweed family have an optimal pH between 5 and 8, as observed in papain [16], procerain B [29], and philibertain g I [30] where they marked an optimum pH of 7, 8, and 7, respectively. bility observed between latex extract and leaf extract could be explained by the degree glycosidic bonds, which can be in a greater proportion in the latex protease than in t leaf protease. ...
... However, the activity of both extra was not strongly affected by the change in pH, which indicates their stability in a lar pH range. According to the literature bibliography, the majority of proteases belonging the milkweed family have an optimal pH between 5 and 8, as observed in papain[1 procerain B [29], and philibertain g I [30]where they marked an optimum pH of 7, 8, a 7, respectively. ...
The purpose of this study was to evaluate the caseinolytic and milk-clotting activities of aqueous crude extracts from leaves and latex of the Pergularia tomentosa, to determine their suitability as a rennet substitute. These extracts were subjected to a series of biochemical tests before being used in the production of cheese. The results showed that the enzymatic latex extract had a higher coagulant activity than the leaf extract. However, under different clotting conditions (pH, temperature, and CaCl2 concentration), both coagulants behaved similarly in the coagulation of Berridge substrate. The SDS-PAGE and zymographic analysis revealed identical protein bands with a single active zone in both extracts, corresponding to a molecular weight of 26.98 kDa and 26.03 kDa in the extract of leaf and latex, respectively. Both extracts were stable to different effectors but strongly inhibited by iodoacetamide and Hg, suggesting it to be a cysteine protease. Both extracts were able to hydrolyze casein and generate peptides of 14 kDa, with excessive hydrolysis of the other casein fractions. The physicochemical parameters of cheese made from latex and leaf extract evolved similarly to control cheese. According to the sensory evaluation, cheese made with latex had a mildly bitter flavor but showed a high acceptance rate (>80%).
... In different crops, allelopathic potential of many crop plants and weeds have been investigated (Kato-Noguchi & Tanaka, 2006). Reduction in dry weight of cotton has been observed due to the phyto-toxic effect of protease present in C. procera extract (Singh et al., 2010). Shoot dry biomass of Zea mays may also be reduced due to eucalyptus extracts (Singh et al., 2010). ...
... Reduction in dry weight of cotton has been observed due to the phyto-toxic effect of protease present in C. procera extract (Singh et al., 2010). Shoot dry biomass of Zea mays may also be reduced due to eucalyptus extracts (Singh et al., 2010). In brassica crop, all parts of the C. procera extract have significant effect on root length, shoot length, shoot dry weight, chlorophyll content and relative water content of the crop (Gulzar and Siddiqui, 2017). ...
... GC-MS analyses showed that essential oil The highest shoot dry mass seedling -1 of cotton was noticed in the jars where distilled water was given throughout the experiment period (0.85 g) and the lowest in 45 % fresh leaf extract applied in the jars (0.49 g) (Fig. 4). C. procera leaves extract likely contains several alkaloids which might be responsible to restrain the growth of cotton (Khan & Baloch, 1999;Hanna et al., 2002;Yarnia et al., 2009;Singh et al., 2010). ...
An experiment was conducted to explore the effects of C. procera fresh leaves extract on cotton growth and vigor during seedling and early establishment stage. The trial was carried out in randomized complete block designed (RCBD) with 3 replications and 4 treatments viz., distilled water, 15%, 30% and 45% solution of Calotropis procera fresh leaves extracts. Data regarding the cotton seedling vigor and growth was collected and mean value of each trait were statistically compared through HSD Tukey’s test (P ≤ 0.05). The highest shoot length seedling-1 (252.25 mm), root length seedling-1 (98.33 mm), stem diameter seedling-1 (0.35 mm) and dry weight of the shoot seedling-1 (0.85 g) was noticed in distilled water treatment. Leaf extract treatments caused significant reduction in seedling vigor and growth. The highest reduction was noticed when treated with 45% aqueous extract of C. procera. The above-mentioned treatment caused significant reduction in various seedling traits, which may hinder early establishment of cotton crop. Therefore, the farmers should remove the dense population of C. procera around cotton fields to avoid allelopathic effects of weed and to reduce damaging effects on early growing cotton plants.
... For understanding optimum temperature, CA of PE was evaluated across different temperatures with a parallel control (10-90 °C). Substrate solution was equilibrated prior to the assays at corresponding temperatures (Singh et al. 2010). ...
... Low recovery of protease activity (9.4%) experienced in the current study could be attributed to the availability of multiple proteases in CE which got separated during the process of purification. Similar experience has also been reported by earlier investigators (Yadav et al. 2012;Singh et al. 2010). Many latex proteases reported so far belong to cysteine family of proteases. ...
... Molecular weight of purified protease from T. divaricata is comparable to many cysteine proteases isolated from various plant lattices (Domsalla and Melzig 2008;Shivaprasad et al. 2016;Costa et al. 2010). Reported results of pH and temperature optima on purified cysteine proteases from latex of Araujia angustifolia fruits, Asclepias curassavica L., Calotropis procera and Ervatamia heyneana were in accordance with the present study (Obregon et al. 2006;Liggieri et al. 2009;Patel and Jagannadham 2003;Singh et al. 2010). Scientific validation of selected plants which have been used for inhibition/stoppage of bleeding on fresh cuts by tribal and rural people of India unveiled their protease components' potential behind these pharmacological effects (Richter et al. 2002;Thankamma 2003;Hosseinzadeh et al. 2015). ...
Protease was isolated and purified from Tabernaemontana divaricata latex and its hemostatic potential was analyzed. Crude latex enzyme was purified through ion exchange and gel filtration chromatography. Purified protease was characterized and its thrombin-like (coagulant assay, fibrinogen polymerizing, and fibrinogenolytic activity) and plasmin-like (blood and plasma clot lysis) activities were assessed accordingly. The homogeneous nature of protease was confirmed with the identification of a single band approximately at 25-kDa molecular weight position. The purified enzyme showed an enhancement of 77.32% in clot inducing ability and 89.86% improvement in blood clot lysis in comparison to that by the crude enzyme. All three subunits (Aα, Bβ and γ chains) of human fibrinogen were hydrolyzed by the purified enzyme. PAGE results of the fibrinolytic activity and blood clot lytic effect by the purified enzyme indicated the plasmin-like activity. The study lays a foundation for the development of enzyme-based approaches for pharmaceutical innovations, in which plant latex proteases can be utilized as a potential natural agent for wound healing applications.
... Casein has a random coil structure with several lysine, arginine, phenylalanine and alanine and thus is an easy target for such proteases [25]. Similarly, it was reported that procerain purified from Calotropis procera shows a similar cleavage pattern with BSA [38]. ...
... In contrast, it was reported proteases purified from the Moringa oleifera leaves show antimicrobial activity [61]. Proteases isolated from plant source like Similarly, Calotropis procera protease indicates a considerably enhanced cleaning effect in the removal of bloodstain and that can be a potential source for detergent industries [38], thus Cyamopsis protease could also be a potential alternative to proteases being used in detergent industries. ...
Proteases are essential enzymes with broad importance in biological, commercial, and therapeutic applications. A protease was isolated and purified from the seeds of Cyamopsis tetragonoloba. In the current study, the enzyme was further explored and characterized. The protease was found to exhibit significant cleavage towards synthetic substrates like N- succinyl phenyl alanine p-nitroanilide (82%) and N-succinyl Ala-Ala-Ala p-nitroanilide (93.2%) when compared to N-αBenzoyl-DL-arginine ϸ-nitroanilide. The protease also cleaved natural substrates and the cleavage pattern of BSA and casein indicated that the purified protease was an endopeptidase. The kinetic parameters Km and Vmax were determined as 97.2 mM and 0.71 mM/min respectively using N-αBenzoyl-DL-arginine ϸ-nitroanilide as a substrate. Activation energy, Enthalpy and temperature coefficient of the protease were determined to be 1.91 kcal/mol, 1.25 kcal/mol and 1.093 respectively. The protease activity was stable at up to 0.4 M NaCl. Furthermore, the protease activity increased in the presence of MgCl2 (110%) and was suppressed in the presence of CuCl2 (20%). The enzyme was also stable even in the presence of reducing agent 2-Mercaptoethanol (5mM). The proteolytic activity in non-germinated and germinated seeds of Cyamopsis tetragonoloba was estimated and found to be 1.1 mM/min and 1.8 mM/min respectively. The purified protease was resistant to fast auto-digestion as proteolytic activity remained nearly stable up to 80% at 30 °C as storage time increased from the 1st day to the 14th day after purification. The protease was investigated for its application as a cleansing additive and was efficient in blood stain removal.
Graphical Abstract
... The influence of ethylenediaminetetraacetic acid (EDTA) and different metal ions (Ca 2+ , Cu 2+ , Mg 2+ , Zn 2+ , Ni 2+ ) on the catalytic activity of MD2-SBro was performed according to the previous reports [1,[28][29][30], with some modifications. The catalytic activity was examined by adding EDTA or different metal ions with a final concentration of 10 mM at the optimum temperature and pH [1]. ...
... Notably, the catalytic efficiency of MD2-SBro was also much lower than the other bromelains, ranging from 17.86-52.53 µM −1 s −1 , depending on the type of bromelain and substrate used in the assay [19,29]. ...
Bromelain is a unique enzyme-based bioactive complex containing a mixture of cysteine proteases specifically found in the stems and fruits of pineapple (Ananas comosus) with a wide range of applications. MD2 pineapple harbors a gene encoding a small bromelain cysteine protease with the size of about 19 kDa, which might possess unique properties compared to the other cysteine protease bromelain. This study aims to determine the expressibility and catalytic properties of small-sized (19 kDa) bromelain from MD2 pineapple (MD2-SBro). Accordingly, the gene encoding MD2-SBro was firstly optimized in its codon profile, synthesized, and inserted into the pGS-21a vector. The insolubly expressed MD2-SBro was then resolubilized and refolded using urea treatment, followed by purification by glutathione S-transferase (GST) affinity chromatography, yielding 14 mg of pure MD2-SBro from 1 L of culture. The specific activity and catalytic efficiency (kcat/Km) of MD2-SBro were 3.56 ± 0.08 U mg−1 and 4.75 ± 0.23 × 10−3 µM−1 s−1, respectively, where optimally active at 50 °C and pH 8.0, and modulated by divalent ions. The MD2-SBro also exhibited the ability to scavenge the 2,2-diphenyl-1-picryl-hydrazyl-hydrate (DPPH) with an IC50 of 0.022 mg mL−1. Altogether, this study provides the production feasibility of active and functional MD2-Bro as a bioactive compound.
... The lactifier fluids of the Calotropis plants are rich in proteins [116][117][118][119][120][121][122][123][124][125][126][127][128]. In 2003, the cysteine protease procerain was obtained from C. procera [118] and its stability was investigated [119]. ...
... Subsequently, a second cysteine protease was purified and isolated from the latex of C. procera and named procerain B [118,119]. As proteases have several applications in the food, dairy, and detergent industries as well as in the pharmaceutical industry, procerain B has been prepared in an immobilized form on Amberlite MB-150 beads, using Schiff base linkage [120] to increase its potential Procegenin B (51) isolated from C. procera in Egypt [94]. IC 50 against tested cancer cell lines: A549 1.106 μM; HeLa 1.434 μM [94] Proceroside (2α,3β)-14-hydroxy-19-oxo-2,3-[(tetrahydro-3,4-dihydroxy-6-methyl-2H-pyran-3,2diyl)bis(oxy)]card-20(22)-enolide (52) isolated from the latex and the whole plant of C. procera [97,98] in Puerto Rico [98] Syriogenin (53) isolated from C. procera [97] Syriogenin 3,12-diacetate (54) isolated from C. procera [97] Uscharidin (11) isolated from the latex of C. procera) [97,99,108]; whole plant (C. ...
The traditional and current use of the latex of Calotropis procera and C. gigantea, two soft-wooded, xerophytic shrubs of the family Apocynaceae, are reviewed against the background of the plants’ chemical constituents and their biological activities. Due to their distinctive latex components, Calotropis plants have been considered a major resource in traditional medicine in many regions. The presence of high amounts of bioactive compounds, which include tannins, flavonoids, triterpenoids, and steroids in the latex of the Calotropis plants, has long been recognized. Peptides and proteins such as peroxidases, peptidases, protease inhibitors, osmotins, lysozymes, and chitinases are all well-studied enzymes associated with the defense system of Calotropis plants against herbivores and diseases. The chemistry and biological activities of the Calotropis plants are thought to be linked to external factors, including the geographic location and the developmental stage of the plant, the season of harvest as well as the storage of the harvested plant, which can lead to a considerable variation in the chemical constituents found.
... The lactifier fluids of the Calotropis plants are rich in proteins [116][117][118][119][120][121][122][123][124][125][126][127][128]. In 2003, the cysteine protease procerain was obtained from C. procera [118] and its stability was investigated [119]. ...
... Subsequently, a second cysteine protease was purified and isolated from the latex of C. procera and named procerain B [118,119]. As proteases have several applications in the food, dairy, and detergent industries as well as in the pharmaceutical industry, procerain B has been prepared in an immobilized form on Amberlite MB-150 beads, using Schiff base linkage [120] to increase its potential Procegenin B (51) isolated from C. procera in Egypt [94]. IC 50 against tested cancer cell lines: A549 1.106 μM; HeLa 1.434 μM [94] Proceroside (2α,3β)-14-hydroxy-19-oxo-2,3-[(tetrahydro-3,4-dihydroxy-6-methyl-2H-pyran-3,2diyl)bis(oxy)]card-20(22)-enolide (52) isolated from the latex and the whole plant of C. procera [97,98] in Puerto Rico [98] Syriogenin (53) isolated from C. procera [97] Syriogenin 3,12-diacetate (54) isolated from C. procera [97] Uscharidin (11) isolated from the latex of C. procera) [97,99,108]; whole plant (C. ...
The traditional and current use of the latex of Calotropis procera and C. gigantea, two soft-wooded, xerophytic shrubs of the family Apocynaceae, are reviewed against the background of the plants’ chemical constituents and their biological activities. Due to their distinctive latex components, Calotropis plants have been considered a major resource in traditional medicine in many regions. The presence of high amounts of bioactive compounds, which include tannins, flavonoids, triterpenoids, and steroids in the latex of the Calotropis plants, has long been recognized. Peptides and proteins such as peroxidases, peptidases, protease inhibitors, osmotins, lysozymes, and chitinases are all well-studied enzymes associated with the defense system of Calotropis plants against herbivores and diseases. The chemistry and biological activities of the Calotropis plants are thought to be linked to external factors, including the geographic location and the developmental stage of the plant, the season of harvest as well as the storage of the harvested plant, which can lead to a considerable variation in the chemical constituents found.
... The determination of ethylenediaminetetraacetic acid (EDTA) solution and metal ions effect on catalytic activity of Trx-MD2Bro was performed according to previous reports [30][31][32] with some modifications. The Trx-MD2Bro activity assayed with the addition of different metal ions (Ca 2+ , Cu 2+ , Mg 2+ , Zn 2+ , Ni 2+ ) or EDTA with a final concentration of 10 mM at the optimum temperature and pH. ...
... Meanwhile, another study on bromelain reported that EDTA did not significantly affect its activity [75]. Similar results were also reported on the activity of bromelain-like cysteine protease from Billbergia pyramidalis [76], Triticum aestivum [77], Calostropis procera [30], and Curcuma longa [78]. This indicated that the inhibitory effect of EDTA is apparently not the general characteristic for cysteine protease as found in other studies. ...
Bromelain, a member of cysteine proteases, is found abundantly in pineapple (Ananas comosus), and it has a myriad of versatile applications. However, attempts to produce recombinant bromelain for commercialization purposes are challenging due to its expressibility and solubility. This study aims to express recombinant fruit bromelain from MD2 pineapple (MD2Bro; accession no: OAY85858.1) in soluble and active forms using Escherichia coli host cell. The gene encoding MD2Bro was codon-optimized, synthesized, and subsequently ligated into pET-32b( +) for further transformation into Escherichia coli BL21-CodonPlus(DE3). Under this strategy, the expressed MD2Bro was in a fusion form with thioredoxin (Trx) tag at its N-terminal (Trx-MD2Bro). The result showed that Trx-MD2Bro was successfully expressed in fully soluble form. The protein was successfully purified using single-step Ni2+-NTA chromatography and confirmed to be in proper folds based on the circular dichroism spectroscopy analysis. The purified Trx-MD2Bro was confirmed to be catalytically active against N-carbobenzoxyglycine p-nitrophenyl ester (N-CBZ-Gly-pNP) with a specific activity of 6.13 ± 0.01 U mg−1 and inhibited by a cysteine protease inhibitor, E-64 (IC50 of 74.38 ± 1.65 nM). Furthermore, the catalytic efficiency (kcat/KM) Trx-MD2Bro was calculated to be at 5.64 ± 0.02 × 10–2 µM−1 s−1 while the optimum temperature and pH were at 50 °C and pH 6.0, respectively. Furthermore, the catalytic activity of Trx-MD2Bro was also affected by ethylenediaminetetraacetic acid (EDTA) or metal ions. Altogether it is proposed that the combination of codon optimization and the use of an appropriate vector are important in the production of a soluble and actively stable recombinant bromelain.
... To date, five cysteine peptidases have been purified and partially characterized from it (procerain, procerain B, CpCP 1, CpCP 2, and CpCP 3). However, their physiological functions have not been investigated ( Dubey and Jagannadham, 2003;Singh et al., 2010;Ramos et al., 2013). In addition, 20 cysteine peptidase genes from C. procera were sequenced by Kwon et al. (2015), but again their roles in plant defense were not examined. ...
... Other cysteine peptidases from C. procera latex, such as procerain and procerain B, exhibited optimum pH and temperature of 6.5-8.5 and 55-60 °C and 7.0 and 40-60 °C, respectively. How small differences in the amino acid sequences of each peptidase can influence their enzymatic kinetics is not clear and deserves further research (Dubey and Jagannadham, 2003;Singh et al., 2010;Ramos et al., 2013). ...
Cysteine peptidases (EC 3.4.22) are the most abundant enzymes in latex fluids. However, their physiological functions are still poorly understood, mainly related to defense against phytopathogens. The present study reports the cDNA cloning and sequencing of five undescribed cysteine peptidases from Calotropis procera (Aiton) Dryand (Apocynaceae) as well as some in silico analyses. Of these, three cysteine peptidases (CpCP1, CpCP2, and CpCP3) were purified. Their enzymatic kinetics were determined and they were assayed for their efficacy in inhibiting the hyphal growth of phytopathogenic fungi. The mechanism of action was investigated by fluorescence and atomic force microscopy as well as by induction of reactive oxygen species (ROS). The deduced amino acid sequences showed similar biochemical characteristics and high sequence homology with several other papain-like cysteine peptidases. Three-dimensional models showed two typical cysteine peptidase domains (L and R domains), forming a "V-shaped" active site containing the catalytic triad (Cys, His, and Asn). Proteolysis of CpCP1 was higher at pH 7.0, whereas for CpCP2 and CpCP3 it was higher at 7.5. All peptidases exhibited optimum activity at 35 °C and followed Michaelis-Menten kinetics. However, the major difference among them was that CpCP1 exhibited highest Vmax, Km, Kcat and catalytic efficiency. All peptidases were deleterious to the two fungi tested, with IC50 of around 50 μg/mL. The peptidases promoted membrane permeabilization, morphological changes with leakage of cellular content, and induction of ROS in F. oxysporum spores. These results corroborate the hypothesis that latex cysteine peptidases play a role in defense against fungi.
... A few studies have been accomplished on the cysteine proteases in Calotropis procera R. Br. [13][14][15][16] . Recently, we reported that Calotropis procera R. Br. can express at least 20 cysteine proteases, which are highly homologous to the cathepsin L 17 . ...
... Calotropis procera R. Br., a traditional medicinal plant in India, is a promising source of cysteine proteases and several proteases such as Procerain, Procerain B, CpCp-1-3 have been isolated and characterised [13][14][15] . Also, we have previously reported the cDNA sequences encoding the full open reading frame of these cysteine proteases from de novo transcriptome assembly using Trinitiy and Velvet-Oases 17 . ...
Cathepsin L of cancer cells has been shown to play an important role in degradation of extracellular matrix for metastasis. In order to reduce cell invasion, cathepsin L propeptide-like proteins which are classified as the I29 family in the MEROPS peptidase database were characterized from Calotropis procera R. Br., rich in cysteine protease. Of 19 candidates, the cloned and expressed recombinant SnuCalCp03-propeptide (rSnuCalCp03-propeptide) showed a low nanomolar Ki value of 2.3 ± 0.2 nM against cathepsin L. A significant inhibition of tumor cell invasion was observed with H1975, HT29, MDA-BM-231, PANC1, and PC3 with a 76, 67, 67, 63, and 79% reduction, respectively, in invasion observed in the presence of 400 nM of the rSnuCalCp03-propeptide. In addition, thermal and pH study showed rSnuCalCp03-propeptide consisting of secondary structures was stable at a broad range of temperatures (30–70 °C) and pH (2–10, except for 5 which is close to the isoelectric point of 5.2).
... This may be due to. Calotropis procera latex contains proteolytic enzymes that increase the rate of casein hydrolysis and these enzymes can cleave peptide bonds, destabilizing milk's micellar structure, speeding curdling and producing a firmer curd that traps more water, which leads to a reduced whey volume [30,31]. Similar research was done in [6,12] using Calotropis procera and lemon juice and found significant result on yield percentage, cheese converting time, cheese fresh and dry weight. ...
Milk coagulation, traditionally relied on Calf rennet, is a crucial enzymatic process in cheese production. Ethical and practical constraints in using Calf rennet have led to the exploration of alternative sources. This study aimed to identify plant-based substitutes for calf rennet by investigating the coagulating potential of extracts from three plants: Calotropis procera, lemon, and Solanum incanum. Each plant tested with three extract levels (15, 20, and 25 ml), and replicated three times. Juices were extracted from lemon and Solanum incanum fruits through incision and squeezing. Meanwhile, Calotropis procera aqueous filtrates were separated from its polymeric gum using centrifugation. Then after, all extracts were further purified through muslin cloth and Whatman filter paper. Extracts were added to a beaker containing 500 ml of milk heated at 50 °C, and allowed to coagulate, resulting in the separation of cheese, curds and whey. Then after whey production, fresh and dry weights of cheese, and cheese yield percentage were measured and recorded. Statistical analysis using SAS Studio showed that there is highly significant differences with P < 0.0001 among plant extracts in their effects on the cheese-making potential. For all extract levels, the highest cheese-dry weight and percentage of cheese yield were observed in Calotropis procera and the lowest cheese formation starting times observed from lemon juice. These findings offer insights into optimizing cheese-making processes using natural sources and suggest that Calotropis procera, along with lemon juice and Solanum incanum, could serve as alternative sources of rennet.
... Procerain B, a Cathepsin L like thermo-stable protease was previously isolated from the latex of Calotropis procera by our group [14]. It has been well characterized biochemically. ...
Keywords: Protease Full length cDNA Recombinant Procerain B RLM-RACE I29 inhibitory propeptide In vitro activation a b s t r a c t We have previously reported isolation and characterization of a novel plant cysteine protease, Procerain B, from the latex of Calotropis procera. Our initial attempts for active recombinant Procerain B in Esche-richia coli expression system was not successful. The reason for inactive enzyme production was attributed to the absence of 5 0 pro-region in the Procerain B cDNA that may be involved in proper folding and production of mature active protein. The current manuscript reports the cloning of full length Procerain B for the production of the active protein. The complete cDNA sequence of Procerain B with pro-region sequence was obtained by using RNA ligase mediated rapid amplification of 5 0 cDNA ends (RLM-RACE). The N-terminus pro-sequence region consists of 127 amino acids and characterized as the member of inhibitory I29 family. Further the three dimensional structure of full length Procerain B was modelled by homology modelling using X-ray crystal structure of procaricain (PDB ID: 1PCI). N-terminus pro-sequence of full length Procerain B runs along the active site cleft. Full length Procerain B was expressed in prokaryotic system and activated in vitro at pH 4.0. This is the first study reporting the production of active recombinant cysteine protease from C. procera.
... The fractions having SOD activity were pooled and dialyzed against 10 mM Tris-HCl buffer (pH 8.0) overnight at 4° C. After dialysis the sample was lyophilized and loaded on Sephacryl S-300 column (1.6 cm x 40 cm) for gel filtration chromatography [17]. The sample was eluted by 0.125 M NaCl in 10 mM Tris-HCl buffer (pH 8.0) with a flow rate of 1 mL/min and fractions of 3 mL were collected. ...
Superoxide dismutase is an important enzyme with various therapeutic applications. Search of a new source of superoxide dismutase with novel properties has significant importance. The current work reports purification of a novel superoxide dismutase enzyme with unique characteristics. A copper zinc superoxide dismutase (Cu-Zn SOD) was purified and characterized from Cicer arietinum L. seedlings germinated under aluminium (Al +3) stress. The specific activity of purified protein was 158 units/mg with 28 fold purification. The superoxide dismutase is a homodimeric protein with ap-prox subunit molecular weight of 33.27 kDa. The enzyme is identified as Cu-Zn category of superoxide dismutase, reflected by H 2 O 2 induced inhibition of in-gel activity and presence of quantifiable copper and zinc ions. The optimum pH range for purified Cu-Zn SOD activity was observed within 6.5-8.5 (highest at pH 8.0) and the pH stability was in the range of 6.0-8.5. The enzyme was more stable at low temperature (below 30°C) and the K m of purified Cu-Zn SOD for ri-boflavin as substrate was 10.16 ± 2.5 M. The N-terminal amino acid sequence showed homology at conserved residues with other plant Cu-Zn SODs.
... MDA concentration was determined with the help of extinction coefficient (155 mM -1 cm -1 ). Protein content determination in all the above enzymatic preparation was done by Bradford method (1976) taking bovine serum albumin (BSA) as standard as followed in our earlier publication Singh et al. (2010). ...
We explore physiological and biochemical response under increasing aluminium stress at different time interval in chickpea seedlings (Cicer arietinum L.). Germination percentage and root length were found to be highly reduced under increasing metal stress. Aluminium induced oxidative stress and led to fluctuations in antioxidative activity responses. Roots showed higher antioxidative activity compared to shoots. Low concentration of aluminium after a brief treatment induced higher superoxide dismutase (SOD; EC 1.15.1.1), ascorbate peroxidase (APX; EC 1.11.1.11) and guaiacol peroxidase (GPX; EC 1.11.1.7) activity whereas longer duration of treatment led to decrease in these activities. Malondialdehyde concentration indicated higher oxidative damage in roots compared to shoots. Taken together the data obtained indicated that high concentration and long exposition of aluminium increases oxidative stress and impairs antioxidative defense system, overall leading to poor growth and survival of seedlings.
... The abundance and variety of proteases in plant latex make it a promising source of plant coagulants. C. procera and C. gigantea, members of the Asclepiadaceae family, have traditionally been used to treat various health conditions (Singh et al., 2010) . Efforts have been made to purify and characterize the cysteine proteases in C. gigantea latex, such as Calotropin FI, FII, and Calotropin DI, DII (Abraham & Joshi, 1979;Pal & Sinha, 1980). ...
Cheesemaking has been relying solely on chymosin for so long. But factors like increased cheese production, diminishing supply of chymosin as well as religious and dietary limitations have been hindering the demand-production balance of cheese in the world. Hence, the primary objective of this study was to utilize the three-phase partitioning (TPP) purified Calotropis gigantea latex protease in cheesemaking. The cheese prepared using purified protease was compared with chymosin cheese for physicochemical, sensorial, textural, and microbiological evaluation. The optimum conditions for purified protease from response surface methodology (RSM) analysis were 6.25 milk pH and 45℃ milk temperature. The physicochemical parameters (moisture content, protein, ash, calcium, salt content, and pH) of cheeses prepared from latex protease and chymosin were significantly different (p<0.05). The yield of latex protease cheese was significantly higher (p<0.05) than chymosin cheese. In terms of texture and aftertaste, the cheese made with latex protease had significantly lower (p<0.05) mean scores than chymosin cheese. Compared to chymosin cheese, the latex protease cheese had a generally inferior textural character, with significantly (p<0.05) lower values for hardness, chewiness, gumminess, cohesiveness, and resilience. The total viable count and Lactobacilli count of the cheeses produced with chymosin and latex protease showed a significant (p<0.05) difference. Hence, this study highlighted that the TPP purified C. gigantea latex protease could be used as a plant coagulant for cheesemaking.
... Evaluation of the performance of procerain B in blood stain removal was also carried out by incubating stained cloth in 1% detergent solutions of commercially available brands (Surf Excel, Tide and Ariel) supplemented with 0.1 mg/ml of purified protease for 5 h. This particular investigation deduced its application as a cost-effective enzyme in detergent industry to remove blood stains and to improve cleaning efficiency 50 . Partially purified protease from latex of C. gigantea, was found to be compatible with three commercially available detergents. ...
Plant latex proteases from different species find applications in diverse industries ranging from food to medicine. Family Apocynaceae consists of species producing large quantities of milky latex. Latex is known to be a rich source of proteolytic enzymes or proteases. Proteases, mainly of the cysteine and occasionally serine type, have been characterized from the latices of different species of Apocynaceae. Many of these proteases have also demonstrated stability under a wide range of parameters including temperature, pH and presence of metal ions. The current review is an attempt to elucidate on latex proteases belonging to Apocynaceae family, highlighting those properties that offer potential for their large-scale applications in different industrial sectors.
... It is also reported in the literature that this protein catalyzes the gene MoPPG1, promoting several defects in fungi hyphal growth and do not allow the gene penetration on plants, which can cause diseases. 45 Then, the serine/threonine-protein kinase in the films may play an essential role in antifungal activity. As an exploratory analysis to determine the presence of proteins in the film, using the film with natural extract concentration of 25 µL·mL -1 (as the increase of conectration, increase the protein concetration), Bradford assay was performed. ...
The proliferation of diseases in plantations, control of fruit ripening, and damages in fruits during transportation are significant agricultural concerns. Thus, this research is based on the production of a sustainable packaging film based on starch conjugated to different concentrations of natural extract from Calotropis procera seeds for agricultural applications. The films were characterized by Fourier Transform Infrared (FTIR), Nuclear Magnetic Resonance (NMR) and Surface Contact Angle. In addition, antifungal activity against Colletotrichum musae were evaluated in vitro and in vivo, as the control of fruit fast maturing were figured using banana as a model of fruit. The cytotoxicity were also analyzed. The results showed that some compounds may have an important role in this protections, as proteins, calactin and calotoxin. Moreover, it demonstrated cytocompatibility using in vitro bioassays (> 80%). The analysis of fruit fast maturing showed that the film with 50 μL·mL-1 of natural extract presented the best results, controlling the fruit ripening and protecting against diseases. Thus, for the first time, a packaging film based on starch and natural extract from Calotropis procera seed demonstrated a versatile action towards agriculture applications.
... The C. procera extract was phytotoxic to proteases which may be responsible for decrease in the dry biomass. The Eucalyptus extracts reduces Zea mays shoot dry biomass (6,49), supporting our findings. On the other hand, several allelochemicals inhibits the macro and micronutrients absorption and IAA oxidase in plant root cells, these decreases the dry and fresh biomass (17). ...
The allelopathic potential of Calotropis procera (Aiton) W.T. Aiton was investigated on germination, growth and mineral uptake of five Agricultural cash crops, including gram (Cicer arietinum L.), tomato (Solanum lycopersicum L., mungbean [Vigna radiata (L.) Wilczek], fenugreek (Trigonella foenum-graecum L.) and lettuce (Lactuca sativa L.). For this purpose, we treated seeds with 0 %, 10 %, 20 % and 30 % concentrations of the entire plant aqueous extract. The effect on germination was examined for six days, while the root and shoot length of the seedling were measured after 10 days of growth. The germination parameters, including germination percentage and mean germination time (MGT), were recorded at each concentration, including control. The results showed that these parameters except MGT were significantly reduced at higher concentrations. Likewise, biomass, mineral uptake and chlorophyll contents were markedly reduced. With the extract treatment, the fungal species (i.e. Fusarium solani, Macrophomina phasiolina, Sclerotinia sclerotium, Fusarium semitectum, Phompsos sp., Lasiodiplodia theobromae and Rhizoctonia solani) exhibited prominent inhibition effects with the methanolic and to a much lesser extent by the aqueous extract respectively. Allelochemicals like p-hydroxybenzoic acid, coumaric acid, syringic acid, ferulic acid, quercetin and caffeic acid were identified using HPLC and showed their antifungal activities. These results suggested that C. procera is harmful to crops under field conditions, but its extract may be used as a fungicide to control pathogenic fungi.
... NEM and p-CMB have been used mostly in comparison to alkylating agents. Thiol-specific reagents like NEM, iodoacetamide, and p-CMB have been used for the identification of cysteine proteases (Kundu et al. 2000;Duarte et al. 2009;Singh et al. 2010). ...
The functional groups present in the active site of an enzyme are explored with the help of the determination of pKa values and group-specific reagents. Some of the commonly used group-specific reagents are listed along with the functional groups: iodoacetamide, iodoacetate, p-chloromercuribenzoate, N-ethylmaleimide (Cys); phenylmethylsulfonyl fluoride (Ser); tetranitromethane, iodine (Tyr); N-bromosuccinimide (Trp). The enzyme is incubated with group-specific reagent and its effect is tested on activity. The loss of activity indicates the presence of the functional group. The modification is also analyzed through UV-visible, fluorescence, and circular dichroism spectra analysis. The inactivation kinetics, kinetic parameters, protection in the presence of substrates, etc. have been elaborated.
... The LP of C. procera was composed of basic proteins, with molecular masses ranging from 5 to 95 kDa, and presented proteolytic potential (Freitas et al., 2007). Proteins, such as cysteine, chitinases, peroxidases and osmotin have been identified in C. procera latex, including proceraine, proceraine B, calotropin DI and DII, calotropaine, calotropaine FI and FII, and CpOsm (Freitas et al., 2011;Ranjit et al., 2012;Singh et al., 2010). ...
This study aimed to evaluate the anthelmintic and ultrastructural effects of Calotropis procera latex on Haemonchus contortus. C. procera latex was twice centrifuged at 10,000×g and dialyzed to obtain a fraction rich in proteins, named LP (latex protein), and at 3,000 rpm to obtain a fraction rich in secondary metabolites, named LNP (latex non-protein). Specimens of H. contortus exposed to LNP, LP and PBS in the Adult Worm Motility Test (AWMT) were submitted to scanning (SEM) and transmission (TEM) electron microscopy to verify changes in their ultrastructure. Phytochemical tests in the LNP indicated the presence of phenols, steroids, alkaloids and cardenolides. High-Performance Liquid Chromatography (HPLC) characterized the presence of the compounds gallic acid and quercetin in the LNP. The protein content in the LP was 43.1 ± 1.1 mg/mL and 7.7 ± 0.3 mg/mL in LNP. In AWMT, LNP and LP inhibited the motility of 100% of the nematodes, with LNP being more effective than LP and ivermectin more effective than both (p <0.05). Cuticle changes were observed by SEM and TEM in nematodes treated with LP and LNP. Calotropis procera latex has anthelmintic effects against H. contortus, causing damage to its cuticle and other alterations in its ultrastructure.
... Root growth is highly susceptible to the presence of allelochemicals in the rhizosphere (Baziramakenga et al., 1995) due to the fact that root tissues are more permeable to allelochemicals that do shoot tissues Nishida et al. (2005) which therefore may impair root metabolic activities and cell division in root tips. Dry weight reduction may be due to phytotoxic effect of proteases present in C. procera extract (Singh et al., 2010). These results are corroborated by the work of Sanginga and Swift (1992) and Khan et al. (1999) who reported reduction in Z. mays shoot dry biomass by eucalyptus extracts. ...
The present study intended to investigate the effect of aqueous extract from Calotropis procera on the growth of Brassica oleracea var botrytis. Seeds of brassica were soaked in solutions containing 20%, 40%, 60% and 80% concentrations of leaf, fruit and flower extract of C. procera. For control, distilled water was used. The effects of extracts on germination percentage, seedling growth, dry biomass, and relative water content were investigated. Higher concentrations of extract (60% and 80%) significantly reduced germination percentage, radicle length, plumule length, dry matter accumulation, and relative water content of the brassica seedlings as compared to control. The retardatory effect increases with the increase in the concentration of three types of extract used, with more pronounced effect noticed by leaf extract followed by fruit and flower extract. There were significant interactions among the different concentrations of extracts used, etype of extract with respect to gemination percentage, seedling length, dry biomass, and relative water content. The effect of pot based assay in relation to chlorophyll content was significantly reduced and antioxidant enzymes [superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) activities] show both significant and non-significant effect on antioxidant enzymes based on concentrations of extract and extract type used. The antioxidant enzymes show the significant decrease in its activity at low concentrations (20% and 40%) and non-significant increase at higher concentration (60% and 80%) of extracts in contrast to control. Based on the investigation, it could be speculated that the delayed germination and low germination rate of the test species after treatment by extracts could be due to the fact that extracts damaged the membrane system of the seeds and C. procera might release phe-nolics into the soil and these are probably involved in the growth inhibitory effect of test species.
... In a previous study, Freitas et al. (2016) showed that a proteolytic fraction obtained from Calotropis procera latex (CpLP) exhibited milkclotting activity and thus had potential for cheesemaking. CpLP was already characterized and five cysteine proteases were described: procerain, procerain B, CpCP1, CpCP2, and CpCP3 (Dubey & Jagannadham, 2003;Singh, Shukla, Jagannadham, & Dubey, 2010;Ramos et al., 2013). As pointed out before, the use of a mixture of peptidases can have several disadvantages for cheesemaking, such as extensive hydrolysis of caseins causing low yield and bitter taste. ...
... David et al., (2003) studied nickel induced changes in some aspects of protein metabolism in the tissues of Pila globosa. Singh et al., (2010) studied the effect of deltamethrin on protein, amino acid and nucleic acids levels in foot and nervous tissue of Mahajan (2012) studied changes in dissimilis on chronic exposure to copper sulphate. Shinde (2013), Bhangale and Mahajan (2015) reported that protein content gradually decreases with increase Hence, the estimation of protein is one of the most important parameter in toxicity testing to evaluate metal Proteins form the architecture of the cell. ...
... David et al., (2003) studied nickel induced changes in some aspects of protein metabolism in the tissues of Pila globosa. Singh et al., (2010) studied the effect of deltamethrin on protein, amino acid and nucleic acids levels in foot and nervous tissue of Mahajan (2012) studied changes in dissimilis on chronic exposure to copper sulphate. Shinde (2013), Bhangale and Mahajan (2015) reported that protein content gradually decreases with increase Hence, the estimation of protein is one of the most important parameter in toxicity testing to evaluate metal Proteins form the architecture of the cell. ...
... The standard Bromelain from A.comosus completely inhibited the protease activity using the cysteine protease inhibitors depicted in Table 1.These results endorse that the purified enzyme extracted from the leaves of B.pyramidalis belongs to cysteine protease family and hence termed as bromelain like cysteine protease. Very recently, similar results were reported in the enzyme inhibition profile of cysteine protease of Calotropis procera, Triticum aestivum and Curcuma longa by iodoacetamide [27][28][29][30] . From the MTT assay, the protease was found to highly inhibit the growth of Human Skin Carcinoma (A431) with an IC 50 value of 80.72µg/ml followed by Glioma cell line (Hs683), Cervical Cancer (HeLa) cell line and Human breast cancer (MCF-7) cell lines with IC 50 values 113.73µg/ml, 126.75µg/ml and138µg/ml, respectively, but trivial inhibitory effect on 3T3-L1 Pre adipocyte cell lines with an IC 50 value of 172.06µg/ml as shown in ( Fig 1).The lower IC 50 value indicating higher antiproliferative activity. ...
Billbergia pyramidalis (Bromeliaceae) a genus originated at south Brazil is a tropical and tender perennial. In
the present study investigations have been carried out on proteolytic activities of purified protease from
B.pyramidalis, using gelatin as substrate. The proteolytic activity of the purified enzyme was inhibited by thiol
blocking agents particularly iodoacetic acid and mercuric chloride, signifying that the enzyme belongs to the
cysteine protease family. The similar results with Bromelain a cysteine protease from Ananas comosus
(pineapple) of Bromeliaceae family was also observed. Bromelain is a proteolytic enzyme and has been
scientifically identified as a therapeutic agent. The objective of this study was to investigate the activity of a
protease identified as cysteine protease (PSA/BP-07), from the leaves of B.pyramidalis, in terms of
antiproliferation against four different human malignant cell lines namely Human skin carcinoma cell line
(A431),Human breast adenocarcinoma cell line (MCF-7), Human cervical cancer cell line (HeLa), Glioma cell
line Hs 683, and a non-cancerous 3T3- L1 Preadipocyte (HeLa) cell line from mouse, using MTT assay in
vitro. The protease was found to exhibit potent antiproliferative activity on A431 cell line with an IC50 value of
80.72 μg/ml but trivial inhibitory effect on 3T3- L1 cell line with an IC50 value of 172.06 μg/ml. In conclusion,
our findings indicate that the plant cysteine protease obtained from B. pyramidalis could further be explored
as a novel source of cancer therapy.
... The blood strain removal study was carried out as described by Singh et al. (2010). The venous blood was collected from healthy volunteers and transferred into the EDTA containing tube to avoid clot formation. ...
In the present study, one-dimensional gel electrophoresis (1-DE) and two-dimensional gel electrophoresis (2-DE) coupled zymography were performed for pupal gut proteins which revealed a single prominent proteolytic band/spot at molecular weight of 37 kDa and an isoelectric point (pI) at 5 to 6 approximately. The proteolytic spot was identified as 37 k-protease of B. mori using mass spectrometry. This 37k-Pupal gut serine protease (PGSP) was purified by anionic exchange and gel filtration chromatography. The biochemical characteristics of PGSP were evaluated using quantitative protease assay. PGSP has substrate specificity with gelatin and cannot be inhibited by various protease inhibitors. However, it was sensitive to reducing agents, dithiothreitol and β-mercaptoethanol. The proteolytic activity was not affected by metal ions (10 mM) except Cu²⁺ and Hg²⁺. The optimum pH and temperature of PGSP was determined as pH 9.0 and 60 °C. The purified protease revealed significant stability and compatibility towards surfactants, oxidizing agent and commercial detergents at 25% concentration. The PGSP (90 µg) with 1% detergent (Rin) showed complete destaining of blood stained cloth. It has significant clot lysis (> 40%) activity within 3 h. In addition, the PGSP shows stable activity in presence of 25% concentration of organic solvents. The overall results suggest that PGSP of B. mori could be utilized as an additive agent in detergent industry.
... These enzymes have been widely studied and their usefulness in different industrial processes has been highlighted [17,18]. Five closely related cysteine proteases were purified and characterized from the latex of Calotropis procera (Apocynaceae) and an extensive analysis of their biochemical and functional properties was performed [19][20][21]. Further, cysteine proteases have also been isolated and characterized from latex of Cryptostegia grandiflora, another species of the Apocynaceae family [21]. These enzymes can be readily obtained within a water-soluble latex proteolytic fraction according to the protocol established earlier [22]. ...
Dehairing of crude leather is a critical stage performed
at the beginning of its processing to obtain industrially
useful pieces. Tanneries traditionally apply a chemical
process based on sodium sulfide. Since this chemical reactive
is environmentally toxic and inefficiently recycled, innovative
protocols for reducing or eliminating its use in leather
depilation are welcomed. Therefore, latex peptidases from
Calotropis procera (CpLP) and Cryptostegia grandiflora
(CgLP) were assayed for this purpose. Enzyme activity on
substrates representative of skin such as hide powder azure
(UHPA), elastin (UE), azocollagen (UAZOCOL), keratin (UK),
and epidermis (UEP) was determined, while depilation
activity was assayed on cow hide. Only CpLP was active
against keratin (13.4 UK) and only CgLP was active against
elastin (0.12 UE). CpLP (93.0 UHPA, 403.6 UAZOCOL, 36.3
UEP) showed higher activity against the other substrates than
CgLP (47.6 UHPA, 261.5 UAZOCOL, 8.5 UEP). In pilot assays,
CpLP (0.05% w/v with sodium sulfite 0.6% w/v as activator)
released hairs from cow hide pieces. Macroscopic and
microscopic analyses of the hide revealed that the dehairing
process was complete and the leather structure was preserved.
The proteolytic system of C. procera is a suitable
bioresources to be exploited by tanneries.
... Root growth is highly susceptible to the presence of allelochemicals in the rhizosphere (Baziramakenga et al., 1995) due to the fact that root tissues are more permeable to allelochemicals that do shoot tissues Nishida et al. (2005) which therefore may impair root metabolic activities and cell division in root tips. Dry weight reduction may be due to phytotoxic effect of proteases present in C. procera extract (Singh et al., 2010). These results are corroborated by the work of Sanginga and Swift (1992) and Khan et al. (1999) who reported reduction in Z. mays shoot dry biomass by eucalyptus extracts. ...
This present study intended to investigate the effect of aqueous extract from Calotropis procera on the growth of Brassica oleracea var botrytis. Seeds of brassica were soaked in solutions containing 20%, 40%, 60% and 80% concentrations of leaf, fruit and flower extract of C. procera. For control, distilled water was used. The effects of extracts on germination percentage, seedling growth, Dry biomass, and relative water content were investigated. Higher concentrations of extract (60% and 80%) significantly reduced germination percentage, radicle length, plumule length, dry matter accumulation, and relative water content of the brassica seedlings as compared to control. The retardatory effect increases with the increase in the concentration of three types of extract used, with more pronounced effect noticed by leaf extract followed by fruit and flower extract. There were significant interactions among the different concentrations of extracts used, type of extract with respect to gemination percentage, seedling length, Dry biomass, and relative water content. The effect of pot based assay in relation to chlorophyll content was significantly reduced and antioxidant enzymes (superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) activities) show both significant and non-significant effects on antioxidant enzymes based on concentrations of extract and extract type used. The antioxidant enzymes show the significant decrease in its activity at low concentrations (20% and 40%) and non-significant increase at higher concentration (60% and 80%) of extracts in contrast to control. Based on the investigation, it could be speculated that the delayed germination and low germination rate of the test species after treatment by extracts could be due to the fact that extracts damaged the membrane system of the seeds and C. procera might release phenolics into the soil and these are probably involved in the growth inhibitory effect of test species.
... Procerain B is a plant cysteine protease that effectively promotes milk clotting and could be used as a substitute milk-clotting agent in the cheese industry. The enzyme may also be used as a dietary supplement to improve digestion (Singh and Shukla, 2010). The ratio of MCA and PA is an important indicator of cheese production efficiency (Arima et al., 1970). ...
Dregea sinensis Hemsl. is used as a milk coagulant to produce goat milk cakes in Yunnan. However, the composition of milk-clotting compounds and the related mechanism have not been reported. Crude protease was extracted from the stem, purified, and then separated with a Millipore ultrafiltration centrifuge tube. Cysteine protease (procerain B) was identified as the main milk-clotting protein through electrospray ionization mass spectrometry, and its molecular weight was 23.8 kDa. The protease can partially degrade α-casein (CN) and completely degrade β- and κ-CN, and κ-CN degradation resulted in milk clotting. The molecular weight and AA sequence of the peptide fractions were determined through matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and a peptide sequencer, respectively. The enzyme cleaved κ-CN at Ala90-Gln91 and produced deputy κ-CN and caseinomacropeptide with molecular weights of 12 and 6.9 kDa, respectively. This cleavage site differed from the majority of chymosins cleaved at Phe105-Met106.
... Procerain B is a novel cysteine protease of the latex of Calotropis procera, enzyme shows broad optimum pH and temperature range i.e pH 6.5 to 8.5 and 40 to 60 0 C temperature respectively. This enzyme had 25.70 kDa molecular weight and pI is 9.52 [126]. Crinumin is a chymotrypsin like serine protease isolated from the latex of Crinum asiaticum. ...
Protease plays an important role in the management of defense mechanism of living organisms. This review presents a comprehensive and exhaustive account of plant, animal and microbial enzymes with special reference to proteolytic properties. A large number of biological sources of proteases described in this review
clearly demonstrate the importance of plants and microbial proteases in applied and industrial uses. Proteases have potential applications in food, dairy, detergent, leather, alcoholic beverages, brewing, meat, pharmaceutical and photographic industries. The research work on protease has been going on since seven decades but no exhaustive review on protease from last eleven years is available in the literature. This review attempts to focus on some of the difficulties observed in the earlier work and tried to find out possible solutions and bridging up this gap by introducing recent information regarding proteinases. This review provides information on diverse groups of proteases with respect to scientific name, family, purification scheme, protease inhibitors, physiological functions, also industrial applications, sequence homology and the future scope of protease enzymes.
... Cysteine peptidases are particularly abundant in CpLP, and some of them have been studied more extensively than others (Ramos et al., 2013). A 25 kDa cysteine peptidase from the C. procera latex, termed Procerain B, was first reported and characterized by Singh et al. (2010). This protein was recognized by anti-CpOsm antibodies by Western blot, and it was then co-purified with CpOsm by immunoaffinity chromatography (Fig. 2C and Table 1). ...
Proteins that share similar primary sequences to the protein originally described in salt-stressed tobacco cells have been named osmotins. So far, only two osmotin-like proteins were purified and characterized of latex fluids. Osmotin from Carica papaya latex is an inducible protein lacking antifungal activity, whereas the Calotropis procera latex osmotin is a constitutive antifungal protein. To get additional insights into this subject, we investigated osmotins in latex fluids of five species. Two potential osmotin-like proteins in Cryptostegia grandiflora and Plumeria rubra latex were detected by immunological cross-reactivity with polyclonal antibodies produced against the C. procera latex osmotin (CpOsm) by ELISA, Dot Blot and Western Blot assays. Osmotin-like proteins were not detected in the latex of Thevetia peruviana, Himatanthus drasticus and healthy Carica papaya fruits. Later, the two new osmotin-like proteins were purified through immunoaffinity chromatography with anti-CpOsm immobilized antibodies. Worth noting the chromatographic efficiency allowed for the purification of the osmotin-like protein belonging to H. drasticus latex, which was not detectable by immunoassays. The identification of the purified proteins was confirmed after MS/MS analyses of their tryptic digests. It is concluded that the constitutive osmotin-like proteins reported here share structural similarities to CpOsm. However, unlike CpOsm, they did not exhibit antifungal activity against Fusarium solani and Colletotrichum gloeosporioides. These results suggest that osmotins of different latex sources may be involved in distinct physiological or defensive events.
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The response surface methodology (RSM) was utilized to optimize three-phase partitioning (TPP) parameters for the purification of Calotropis gigantea latex protease. The limits of purification parameters namely saturation of ammonium sulfate salt, volume ratio of tert-butanol to crude latex extract and pH value were selected from single-factor experiments. A Box-Behnken design was employed to determine the effects of TPP parameters on protease purification. From the optimization study, the degree of purification and recovery percentage reached maximum values of 6.7 and 126.12%, respectively, under optimized parameters of 52% saturation of (NH4)2SO4 (w/v), 1.0:1.5 ratio of crude latex extract to tert-butanol (v/v) and pH 6. The concentrated protease in the interfacial phase (IP) demonstrated higher enzyme activity when compared to the lower aqueous phase (AP). The molecular weight of recovered protease was 24 kDa from electrophoretic analysis, and the substrate zymography revealed the presence of protease in IP fraction. The findings of this study demonstrated that TPP is an effective and economic protocol for quick recovery of latex protease in comparison with expensive and time-consuming chromatographic techniques.
The protein extract of Moringa oleifera flowers is reported to have milk-clotting activity (MCA), but information regarding its protease is unclear. In this study, two milk-clotting proteases (MoFP 12 and MoFP 13) with molecular weights of 42.304 kDa and 31.741 kDa, respectively, were isolated and identified from M. oleifera flowers using liquid chromatography-mass spectrometry (LC-MS/MS). Bioinformatics analysis showed that these two milk-clotting proteases were primarily involved in hydrolase activity and catabolic process, and exhibited hydrophilicity. The secondary structure of MoFP 12 consisted of 43.65% helix, 13.23% strand, and 43.12% coil, and MoFP 13 consisted of 26.51% helix, 20.14% strand, and 53.35% coil. The proteases were stable in the pH range of 5.0 to 8.0 and showed their maximum MCA at 70℃. Additionally, by investigating the effect of proteases on caseins, κ-casein (CN) was observed to preferentially be hydrolyzed by the two proteases, followed by α-CN, and to a lesser extent β-CN. These findings revealed the main milk-clotting proteases in M. oleifera flowers and its milk-clotting properties, indicating its potential for application in the dairy and food sectors, especially in the cheese-making industry.
With insights gained from studies on plant proteases,
the current work aims in validating proteases from
selected medicinal plants for their milk coagulation
potential. Stem crude extracts (CE) from 30 plants
were screened for their milk clotting (MCA), their
caseinolytic activities (CA) and milk clotting index
(MCI). Plant proteases with high MCA and MCI were
partially purified using salt precipitation. Casein
hydrolytic pattern of the partially purified protease was
assayed through tricine- SDS PAGE. Commercial milk
coagulants, rennin and Enzeco® were used as positive
control. CE from thirty plants were screened and
Wrightia tinctoria, Rauvolfia tetraphylla, Bacopa
monnieri and Costus igneus were selected for partial
purification based on their high milk clotting ability.
High recovery of 76% and fold purification of ~3 was
observed in W. tinctoria salt precipitated fraction with
MCA. Comparable casein hydrolytic pattern was
observed between CE of R. tetraphylla and C. igneus,
salt precipitated extracts of W. tinctoria with
commercial coagulants. The results open new avenues
for researchers to explore these proteases as vegetable
coagulant alternative.
Calotropis procera cysteine peptidases (CpCPs) have been used for reducing cow’s milk allergenicity or as rennet in cheesemaking. Due to their residual presence in food products, the present study evaluated, for the first time, different protocols for their immobilization on different supports. Although the yield of immobilization on sulfopropyl-agarose (99%, at pH 7.0) was better than when using DEAE- and MANAE-agarose (40% and 15%, respectively), the derivatives had low recovered activity (4%). On the other hand, MANAE-agarose at pH 10.0 (200 mM buffer) exhibited the highest recovered enzymatic activity (~23%). Regarding the covalent immobilization, the peptidases immobilized on glyoxyl-agarose (glyoxyl-CpCPs) showed broader pH stability (pH 3.0-10.0), 60-fold more stable at 60 °C, and retained 70% of their initial activities after five reaction cycles, even though the immobilization has induced some structural changes analyzed by Fourier-Transform Infrared (FTIR) spectroscopy as well as altered some of enzyme kinetic parameters (Vmax, Km, Kcat, and catalytic efficiency). In addition, this biocatalyst (glyoxyl-CpCPs) hydrolyzed the major cow’s milk allergens (whey proteins) to a greater extent (65%) than the soluble enzymes (8%) and a commercial hypoallergenic formula (50%).
Procerain (Pc) and Procerain B (PcB) are two latex proteases from Calotropis procera having potential applications in food and other industries. However, autolytic degradation of these proteases limits their potential use in industry. Nevertheless, basic mechanism underlying the autoproteolysis has not been detailed. In order to understand the same, we subjected the enzymes to various denaturing and activating conditions. The results showed that structural changes induced by different denaturing conditions trigger their autoproteolysis. We also observed differential response of Pc, PcB and other papain-like proteases towards autocatalysis in presence of reducing agent in-spite of sharing the same structural fold, including the number of disulfide bonds. The possible reason underlying this intriguing observation is also discussed. Further, present work establishes that structural changes in the proteases lead to autoproteolysis and the enzymes are stable unless they experience structural perturbation. These findings could thus be useful for their practical applications in industries.
Understanding the relationship among the materials properties, characteristics, biosafety/ toxicity responses in human/animal life is imperative. This research focuses on extracted natural materials from Calotropis procera seeds based on two different routes (ethanol and water) to the first step on a comprehensive understanding of biosafety/toxicity using in vitro bioassays. The colloidal extracts were extensively characterized by infrared spectroscopy (FTIR), nuclear magnetic resonance (NMR), X-ray photoelectron spectroscopy (XPS), Bradford, and sodium-dodecyl sulfate polyacrylamide gel electrophoresis (SDS-page). In addition, the cytotoxicity against liver cancer cells (HEPG2), kidney cell lines of human embryos (HEK 293T), and Human ALK Knockout HeLa Cell Line (HeLa) were evaluated. The results showed that the natural extract presented different behavior depending on the cell line, probably because some proteins and compounds, i.e., serine/threonine-protein kinase, calactin, and calotoxin, mainly associated with anti-cancer properties of these compounds. Moreover, the different routes significantly affected the extraction of the compounds, as proteins, influencing the natural extract final composition due to the distinct solvents polarity character. Furthermore, the toxicity/biosafety difference among the cells (less 60 % for HEPG2 and over 80 % for HEK293T and HeLa) shows that these colloidal materials may be a key potential for sustainability applications of the natural extract.
Dregea sinensis (D. sinensis) stems have traditionally been used as milk coagulant in Dali of Yunnan Province, China. In this study, proteomics was used to investigate the bio-functions of D. sinensis stem proteins, leading to the purification and identification of the milk-clotting enzyme. A total of 205 proteins mainly involved in the catalytic and metabolic processes were identified, of which 28 proteins exhibited hydrolase activity. Among the 28 proteins, we focused on two enzymes (M9QMC9 and B7VF65). Based on proteomics, a cysteine protease (M9QMC9) with a molecular weight of 25.8 kDa and milk-clotting activity was purified from D. sinensis stems using double ammonium sulfate precipitation and was confirmed using liquid chromatography-mass spectrometry (LC-MS/MS). The milk-clotting temperature using the purified enzyme was around 80℃ (specific activity at 314.38 U/mg), and it was found to be stable in the pH range of 6–9 in NaCl concentration of less than 0.8 mol/L. These findings indicated that the enzyme isolated from D. sinensis stems has potential in the dairy and food sectors, especially in the cheese-making industry.
Background
Anti-inflammatory properties have been attributed to latex proteins of the medicinal plant Calotropis procera.
Purpose
A mixture of cysteine peptidases (LPp2) from C. procera latex was investigated for control of inflammatory mediators and inflammation in a mouse model of Salmonella infection.
Methods
LPp2 peptidase activity was confirmed by the BANA assay. Cytotoxicity assays were conducted with immortalized macrophages. Peritoneal macrophages (pMØ) from Swiss mice were stimulated with lipopolysaccharide (LPS) in 96-well plates and then cultured with nontoxic concentrations of LPp2. Swiss mice intravenously received LPp2 (10 mg/kg) and then were challenged intraperitoneally with virulent Salmonella enterica Ser. Typhimurium.
Results
LPp2 was not toxic at dosages lower than 62.2 μg/mL. LPp2 treatments of pMØ stimulated with LPS impaired mRNA expression of pro-inflammatory cytokines IL-1β, TNF-α, IL-6 and IL-10. LPp2 increased the intracellular bacterial killing in infected pMØ. Mice given LPp2 had a lower number of leukocytes in the peritoneal cavity in comparison to control groups 6 h after infection. The bacterial burden and histological damage were widespread in target organs of mice receiving LPp2.
Conclusion
We conclude that LPp2 contains peptidases with strong anti-inflammatory properties, which may render mice more susceptible to early disseminated infection caused by Salmonella.
Three Phase Partitioning (TPP) system as an elegant non-chromatographic and bulk separation method was successfully applied for the extraction and recovery of papain from the latex of Carica papaya. The optimized parameters of TPP allowed achieving a purification fold of 11.45 and activity recovery of 134% with 40% (NH4)2SO4, 1.0:0.75 ratio of crude extract: t-BuOH at pH and temperature of 6.0 and 25°C, respectively. The recovered papain had a molecular weight of 23.2 kDa and revealed maximum activity at pH 6.0 and temperature of 50°C. The maximum values of Km and Vmax parameters were 10.83 mg.mL-1 and 33.33 U.mL-1, respectively. The protease with 4 isoforms was stable at 40 – 80°C and a pH range of 6.0 – 7.5 against numerous metal ions and none of them inactivated its activity. Moreover, 10 mM Ca2+ improved 2-folds the activity and half-life of the protease at temperatures from 30 – 50°C. The milk-clotting activity tests revealed high stability of the protease at storage, namely at -20°C compared to 4°C and 25°C for up than 5 weeks. As a meat tenderizing agent, it showed promising role under different treatments by improving texture. The findings indicated that, one-step TPP system is a simple, quick, economical and very attractive process for fast recovery of latex papain compared to other proposed protocols.
This book offers an overview of the diverse fields application of proteases (also termed proteolytic enzymes or proteinases), including food science and technology, pharmaceutical industries, and detergent manufacturing, reviewing the advances in the biotechnological application plant proteolytic enzymes over the last decade. In recent years, they have been the focus of renewed attention from the pharmaceutical and biotechnology industries, not only because of their activity on a wide variety of proteins but also because they are active over a range of temperatures and pHs. The main audience of this book are researchers working with plant proteases but also professionals from several industry segments such as food production and pharmaceutical companies.
Some plants have enough amount of proteolytic enzymes in its tissues and can clot the milk under optimum conditions. Their use as coagulants in cheesemaking represents a traditional practice in many regions of the world. In the last years, the research and knowledge on plant proteases is increasing and crossing frontiers, expanding their use in biotechnological process and the production of novel exquisite cheeses. This chapter describes the milk-clotting properties of some plant coagulants used for cheese production at artisanal, experimental, and commercial scale. Some technological aspects for the formulation and standardization of enzymatic preparations from plant origin and its impact on physicochemical, technological, and sensorial properties of curds and cheeses are also considered. Cheese production is in constant growth and it is expected that in the near future the use of plant proteases in cheesemaking will increase, producing new specialty cheese favoring the cheese market.
Milk is often subjected to technological treatments which have impacts on the structure of milk constituents and the characteristics of rennet curds. In this paper, the influence of the dairy fat structure on the biochemical and textural characteristics of curds coagulated by an extract of Calotropis procera leaves was studied. Standardized milks were reconstituted with the same contents in protein (35 g·kg− 1) and fat (35 g·kg− 1) but with different structures of fat i.e. homogenized anhydrous milk fat (HAMF), homogenized cream (HC) and non-homogenized cream (NHC). As expected, the size distributions of fat globules in the different milks were different. After their coagulations by the plant extract, the physico-chemical characteristics of the curds and respective wheys were determined. No difference was observed in the coagulation time between the three milks but the whey removed more quickly from HAMF and HC curds than NHC-curd. The biochemical analyses of curds revealed a lower content in dry matter and fat in the NHC-curd compared to HAMF- and HC-curds. Otherwise, the NHC-whey exhibited the highest amount of fat. Observations by confocal microscopy showed that the fat globules were homogenously distributed and well trapped in the protein networks of HAMF- and HC-curds. In the NHC-curd, the fat globules were located in whey pockets, with less connectivity with the protein network. The textural analysis showed that the NHC-curd was more elastic, soft and adhesive than HAMF- and HC-curds. Homogenization significantly reduced the loss of fat during cheese manufacturing and conferred specific textural characteristics to the curds coagulated by an extract of Calotropis procera.
Chymosin is the major enzyme used in cheesemaking but latex enzymes are also used. The aim of this work was to characterize the composition and the structure of dairy gel obtained by an extract of Calotropis procera leaves in comparison with those obtained by chymosin. The biochemical and mineral compositions of the curds and the cheese yields obtained by using Calotropis procera extract or chymosin were relatively similar. Quantitative and qualitative evaluations of proteolysis after milk coagulation, determined by the non-protein nitrogen content and chromatography coupled to mass spectrometry, indicated that Calotropis procera extract was more proteolytic than chymosin and that κ-casein was proteolyzed. The main consequence of proteolysis by Calotropis procera extract or chymosin was the formation of a similar and regular network with the presence of aggregates of casein micelles. These results support that Calotropis procera extract can be used as effective coagulant in cheesemaking.
Superoxide dismutase is an important enzyme with various therapeutic applications. Search of a new source of superoxide dismutase with novel properties has significant importance. The current work reports purification of a novel superoxide dismutase enzyme with unique characteristics. A copper zinc superoxide dismutase (Cu-Zn SOD) was purified and characterized from Cicer arietinum L. seedlings germinated under aluminium (Al+3) stress. The specific activity of purified protein was 158 units/mg with 28 fold purification. The superoxide dismutase is a homodimeric protein with approx subunit molecular weight of 33.27 kDa. The enzyme is identified as Cu-Zn category of superoxide dismutase, reflected by H2O2 induced inhibition of in-gel activity and presence of quantifiable copper and zinc ions. The optimum pH range for purified Cu-Zn SOD activity was observed within 6.5-8.5 (highest at pH 8.0) and the pH stability was in the range of 6.0-8.5. The enzyme was more stable at low temperature (below 30 degrees C) and the K-m of purified Cu-Zn SOD for riboflavin as substrate was 10.16 +/- 2.5 M. The N-terminal amino acid sequence showed homology at conserved residues with other plant Cu-Zn SODs.
The latex from the patagonic plant Philibertia gilliesii Hook. et Arn. (Apocynaceae) is a milky-white suspension containing a proteolytic system constituted by several cysteine endopeptidases. A proteolytic preparation (philibertain g) from the latex of P. gilliesii fruits was obtained and characterized to evaluate its potential use in bioprocesses. Philibertain g contained 1.2 g/L protein and a specific (caseinolytic) activity of 7.0 Ucas/mg protein. It reached 80 % of its maximum caseinolytic activity in the pH 7-10 range, retained 80 % of the original activity after 2 h of incubation at temperatures ranging from 25 to 45 °C and could be fully inactivated after 5 min at 75 °C. Philibertain g retained 60 % of the initial activity even at 1 M NaCl and was able to hydrolyze proteins from stickwater one, of the main waste effluents generated during fishmeal production. Furthermore, as a contribution to the knowledge of the proteolytic system of P. gilliesii, we are reporting the purification of a new peptidase, named philibertain g II (pI 9.4, molecular mass 23,977 Da, N-terminus LPESVDWREKGVVFPXRNQ) isolated from philibertain g through a purification scheme including acetone fractionation, cation exchange, molecular exclusion chromatography, and ultrafiltration.
The first analysis of glycoconjugates that often needs to be carried out is to see if they indeed contain sugar. For glycoproteins
in gels or oligosaccharides in solution, this can be readily achieved by periodate oxidation at two concentrations, the first
to detect sialic acids, and the second, any monosaccharide that has two free vicinal hydroxyl groups (1). Periodate cleaves between the hydroxyl groups to yield reactive aldehydes, which can be detected by reduction with NaB3H4 or coupled to high sensitivity probes available in commercial kits, e.g., from Boeringher Mannheim (Mannheim, Germany) or
Oxford Glycosystems (Abingdon, UK). In solution, a quick spot assay can be carried out for the presence of any monosaccharide
or oligosaccharide having a C-2 hydroxyl group by visualization with charring by phenol/sulfuric acid reagent (2). These methods are relatively specific for mono/oligosaccharides (3).
Proteases are one of the most important classes of enzyme and expressed throughout the animal and plant kingdoms as well as in viruses and bacteria. The protease family has drawn special attention for drug target for cure of several diseases such as osteoporosis, arthritis and cancer. Many proteases from various sources are being studied extensively with respect to activity, inhibition and structure. In this review, we hope to bring together the information available about the proteases with particular emphasis on papain like plant cysteine proteases. Besides, protease inhibitors and their potential utilities are also discussed.
Most of the species belonging to Asclepiadaceae family usually secrete an endogenous milk-like fluid in a network of laticifer cells in which sub-cellular organelles intensively synthesize proteins and secondary metabolites. A new papain-like endopeptidase (asclepain c-II) has been isolated and characterized from the latex extracted from petioles of Asclepias curassavica L. (Asclepiadaceae). Asclepain c-II was the minor proteolytic component in the latex, but showed higher specific activity than asclepain c-I, the main active fraction previously studied. Both enzymes displayed quite distinct biochemical characteristics, confirming that they are different enzymes. Crude extract was purified by cation exchange chromatography (FPLC). Two active fractions, homogeneous by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and mass spectrometry, were isolated. Asclepain c-II displayed a molecular mass of 23,590 Da, a pI higher than 9.3, maximum proteolytic activity at pH 9.4-10.2, and showed poor thermostability. The activity of asclepain c-II is inhibited by cysteine proteases inhibitors like E-64, but not by any other protease inhibitors such as 1,10-phenantroline, phenylmethanesulfonyl fluoride, and pepstatine. The Nterminal sequence (LPSFVDWRQKGVVFPIRNQGQCGSCWTFSA) showed a high similarity with those of other plant cysteine proteinases. When assayed on N-alpha-CBZ-amino acid-p-nitrophenyl esters, the enzyme exhibited higher preference for the glutamine derivative. Determinations of kinetic parameters were performed with N-alpha-CBZ-L-Gln-p-nitrophenyl ester as substrate: K(m)=0.1634 mM, k(cat)=121.48 s(-1), and k(cat)/K(m)=7.4 x 10(5) s(-1)/mM.
A highly stable cysteine protease was purified to homogeneity from the latex of Ervatamia coronaria by a simple purification procedure involving ammonium sulfate precipitation and ion-exchange chromatography. The molecular mass was estimated to be approximately 25,000 Da by SDS-PAGE and gel filtration. The extinction coefficient (epsilon 280 nm 1%) of the enzyme was 24.6. The enzyme hydrolyzed denatured natural substrates like casein, hemoglobin, azoalbumin, and azocasein with a high specific activity but showed low specific activity towards synthetic substrates. The pH and temperature optima were 7.5-8.0 and 50 degrees C respectively. The activity of the enzyme was strongly inhibited by thiol-specific inhibitors like leupeptin, iodoacetamide, PCMB, NEM, and mercuric chloride. The striking property of this enzyme was its stability over a wide pH range (2-12) and other extreme conditions of temperature, denaturants, and organic solvents. The N-terminal sequence showed marked similarity to known cysteine proteases.
The availability of the human and mouse genome sequences has allowed the identification and comparison of their respective degradomes--the complete repertoire of proteases that are produced by these organisms. Because of the essential roles of proteolytic enzymes in the control of cell behaviour, survival and death, degradome analysis provides a useful framework for the global exploration of these protease-mediated functions in normal and pathological conditions.
The first analysis of glycoconjugates that often needs to be carried out is to see if they indeed contain sugar. For glycoproteins in gels or oligosaccharides in solution, this can be readily achieved by periodate oxidation at two concentrations, the first to detect sialic acids, and the second, any monosaccharide that has two free vicinal hydroxyl groups (1). Periodate cleaves between the hydroxyl groups to yield reactive aldehydes, which can be detected by reduction with NaB3H4 or coupled to high sensitivity probes available in comMercial kits, e.g., from Boeringher Mannheim (Mannheim, Germany) or Oxford GlycoSciences (Abingdon, UK). In solution, a quick spot assay can be carried out for the presence of any monosaccharide or oligosaccharide having a C-2 hydroxyl group by visualization with charring by phenol/sulfuric acid reagent (2). These methods are relatively specific for mono/oligosaccharides (3).
The ginger proteases (GP-I and GP-II), isolated from the ginger rhizome Zingiber officinale, have an unusual substrate specificity preference for cleaving peptides with a proline residue at the P2 position. The complete amino-acid sequence of GP-II, a glycoprotein containing 221 amino acids, and about 98% that of GP-I have been determined. Both proteases, which are 82% similar, have cysteine residues at positions 27 and histidines at position 161, corresponding to the essential cysteine–histidine diads found in the papain family of cysteine proteases, and six corresponding cysteine residues that form the three invariant disulfide linkages seen in this family of proteins. The sequence homology with other members (papain, bromelain, actinidin, protease omega, etc.) of this family is ≈ 50%. GP-II has two predicted glycosylation sites at Asn99 and Asn156. Analyisis by electrospray and collision-induced dissociation MS showed that both sites were occupied by the glycans (Man)3(Xyl)1(Fuc)1(GlcNAc)2 and (Man)3(Xyl)1(Fuc)1(GlcNAc)3, in a ratio of ≈ 7 : 1. Both glycans are xylose containing biantennary complex types that share the common core structural unit, Man1→6(Man1→3) (Xyl1→2)Man1→4GlcNAc1→4(Fuc1→3)GlcNAc for the major form, with an additional N-acetylglucosamine residue being linked, in the minor form, to one of the terminal mannose units of the core structure.
A proteinase from the sarcocarp of Benincasa cerifera was purified. ItsMW was estimated by two different methods to be about 50000. The maximum activity was found in the alkaline pH region against casein as a substrate. The enzyme was strongly inhibited by di-isopropyl fluorophosphate and not inhibited by EDTA and p-chloromercuribenzoic acid.
Quantitation of the amount of protein in a solution is possible in a simple spectrometer. Absorption of radiation in the near
UV by proteins depends on the Tyr and Trp content (and to a very small extent on the amount of Phe and disulfide bonds). Therefore
the A
280 varies greatly between different proteins (for a 1 mg/mL solution, from 0 up to 4 for some tyrosine-rich wool proteins, although
most values are in the range 0.5–1.5 [1]). The advantages of this method are that it is simple, and the sample is recoverable. The method has some disadvantages,
including interference from other chromophores, and the specific absorption value for a given protein must be determined.
The extinction of nucleic acid in the 280-nm region may be as much as 10 times that of protein at their same wavelength, and
hence, a few percent of nucleic acid can greatly influence the absorption.
The latex of madar plants, Calotropis gigantea, contained at least five proteases, two of which designated as calotropins DI and DII were isolated and crystallized. The crystalline preparations were homogeneous by ion exchange chromatography, polyacrylamide gel and sodium dodecyl sulfate-gel electrophoresis, and sedimentation in the ultracentrifuge. They contained a single sulfhydryl group and the enzymic activity depended on it. The physicochemical properties of both calotropins were similar to those of other plant cysteine proteases. As in the case of papain, their sedimentation coefficients were independent of protein concentration. The average molecular weights calculated from sedimentation and diffusion coefficient values, Archibald experiment, amino acid composition, and sodium dodecyl sulfate-gel electrophoresis were 23,800 for calotropin DI and 24,200 for calotropin DII. Their amino acid compositions were very similar with minor differences in individual amino acids. Like papain they were devoid of carbohydrate moieties. In the enzymic properties both calotropins were found to be indistinguishable. Hydrolysis of azoalbumin by each enzyme was optimal in the pH range 7.5–8.0 at 55 °C. Their caseinolytic activities were about one-third as active as papain or ficin. They, unlike papain and ficin, showed no measurable activity toward synthetic substrates such as benzoyl-l-arginine ethyl ester, p-tosyl-l-arginine methyl ester, carbobenzoxy glycine p-nitrophenyl ester, benzoyl-dl-arginine p-nitroanilide, and benzoyl-l-arginine amide.
The present study was carried out to evaluate the effect of dry latex (DL) of Calotropis procera, a plant of the family Asclepiadaceae, on the functions of liver and kidney in normal rats.
Aqueous suspension of DL was orally administered to rats at doses of 10, 100 and 400 mg/kg for a period of 45 days and the effect on various parameters reflecting liver and kidney functions was compared with that of normal controls.
Treatment with DL did not alter the serum levels of aspartate transaminase (AST), alanine transaminase (ALT), alkaline phosphatase (ALP), creatinine, urea and urinary levels of glucose and protein as compared to the normal rats. It exhibited a modulatory role in maintaining the levels of blood glucose and serum insulin. The liver and kidney of DL treated and normal rats were also comparable with regard to the tissue levels of oxidative stress markers and histology. Further, no signs of toxicity were observed in the DL treated rats over the study period.
Our study reveals that aqueous suspension of Calotropis procera latex does not produce any toxicity and could be safely used for therapeutic purpose at the doses studied.
A novel protease was purified to homogeneity from the latex of Pedilanthus tithymaloids by a simple purification procedure involving ammonium sulfate precipitation and cation-exchange chromatography. The molecular weight of the protease was estimated to be approximately 63.1 kDa and the extinction coefficient (epsilon(1%)(280nm)) was 28.4. The enzyme hydrolyzes denatured natural substrates like casein, azoalbumin and azocasein with a high specific activity but little activity towards synthetic substrates. The pH and temperature optima were pH 8.0-9.5 and 65-70 degrees C, respectively. The proteolytic activity of the enzyme was inhibited by different protease-specific inhibitors (e.g., thiol, serine, metallo, etc.) up to a certain extent but not completely by any class of inhibitors. The enzyme was relatively stable towards pH change, temperature, denaturants and organic solvents. The enzyme consists of five disulfide bridges compared to three observed in most plant cysteine proteases. Overall, the striking features of this protease are its high molecular weight, high cysteine content and only partial inhibition of activity by different classes of protease inhibitors contrary to known proteases from other plant sources. The enzyme is named as pedilanthin as per the protease nomenclature.
Two groups of asclepains have been isolated from Asclepias syriaca L. (milk-weed) latex and a representative of each has been purified. Asclepains A3 and B5 are homogeneous proteins with molecular weights of 23 000 and 21 000, respectively. Both require a reducing and chelating agent for maximum activity and hydrolyze ester, amide and peptide bonds. The optimum pH for hydrolysis of casein is 7.5 to 8.5 for asclepain A3 and 7.0 to 7.5 for asclepain B5. Both enzymes are autolytic when active and are inhibited by p-chloromercuribenzoate, iodoacetic acid and sodium tetrathionate. Asclepains A3 and B5 each contain one titratable SH group per molecule and no bound carbohydrate. Each of the two enzymes has leucine as the N-terminal amino acid. There are notable differences in their amino acid compositions.
A protein determination method which involves the binding of Coomassie Brilliant Blue G-250 to protein is described. The binding of the dye to protein causes a shift in the absorption maximum of the dye from 465 to 595 nm, and it is the increase in absorption at 595 nm which is monitored. This assay is very reproducible and rapid with the dye binding process virtually complete in approximately 2 min with good color stability for 1 hr. There is little or no interference from cations such as sodium or potassium nor from carbohydrates such as sucrose. A small amount of color is developed in the presence of strongly alkaline buffering agents, but the assay may be run accurately by the use of proper buffer controls. The only components found to give excessive interfering color in the assay are relatively large amounts of detergents such as sodium dodecyl sulfate, Triton X-100, and commercial glassware detergents. Interference by small amounts of detergent may be eliminated by the use of proper controls.
Using an improved method of gel electrophoresis, many hitherto unknown
proteins have been found in bacteriophage T4 and some of these have been
identified with specific gene products. Four major components of the
head are cleaved during the process of assembly, apparently after the
precursor proteins have assembled into some large intermediate
structure.
1A method is described for the preparation from Actinidia chinensis (Chinese gooseberry) fruit of a crystalline proteinase which catalyzes the hydrolysis of N2-benzoyl-l-arginine ethylester, N2-p-toluene-sulphonyl-l-glutamine p-nitrophenyl ester (used in the routine assay) and at least 15% of the peptide bonds of gelatin. The synthesis of the tosyl-glutamine p-nitrophenyl ester is described. The enzyme resembles papain in its action against benzoyl-l-arginine ethyl ester, having a broad pH-optimum from pH 5–7, with Km(app) of 89 mM and kcat of about 2.6 sec−1 at pH 5.6, 25° in 0.3 M KCl.2The enzymic activity elutes from Sephadex G-50 as a protein of molecular weight 12800 ± 700, and is partially resolved from inactive protein of apparent molecular wight 15400 ± 800. The preparation has minimum solubility near pH 3.1 and migrates at both pH 8.3 and 5.0 on polyacrylamide gel electrophoresis as two closely-spaced anionic bands. These two electrophoretic components are partially resolved by chromatography on DEAE-cellulose and both are enzymically active against tosyl-l-glutamine p-nitrophenyl ester.3The inhibitions of enzymic activity by 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB) and 4,4′-dithiodipyridine, both reversible by 1,4-dithiothreitol, show that enzymic activity is dependent on an intact sulphhydryl group assaying about 0.23 moles per 12800 g protein. The enzyme is rapidly inactivated by iodoacetate and thereafter gives a low sulphhydryl assay with dithiodipyridine. Conversely, pretreatment with DTNB gives substantial protection against iodoacetate.
Ginger proteases in ginger rhizome (Zingiber officinale roscoe) were extracted from the ginger acetone powder and purified on DEAE-Sepharose and Sephadex G-75 columns. Before the purification, excess p-chloromercuribenzoate was added to the enzymes to prevent their autodigestion. The mercuribenzoate-proteases were further purified and fractionated by isoelectric focusing in Ampholine of pH 3-10 or pH 4-6. The proteases were fractionated into three components by the isoelectric focusing, having pI value of 4.5, 4.6 and 4.8 respectively. All these proteases had a molecular mass of 29,000 as measured by SDS-polyacrylamide gel electrophoresis and by TSK G2000SW XL gel chromatography. The Ampholine in the purified enzymes can quickly be removed by the gel chromatography of TSK G2000SW. Some divalent metal ions, such as Hg2+, Cu2+, Cd2+, and Zn2+, strongly inhibited these purified enzymes.
Thermal denaturation of four Carica papaya cysteine proteinases (papain, chymopapain, papaya proteinases 3 and 4) was studied as a function of pH using high‐sensitivity differential scanning calorimetry.
The ratios of calorimetric enthalpy to Van't Hoff enthalpy suggest that, for all these proteins, denaturation occurs as a non two state process, via an intermediate structure.
Differences in the thermal stabilities of the proteinases; chymopapain > papaya proteinase 3 > papain > papaya proteinase 4, were correlated to their amino acid sequence to explain the observations in terms of mobility and specific residue mutation.
Three‐dimensional structures of papain and papaya proteinase 3 were similarly used to illustrate the influence of atomic mobility on stability.
Calotropis procera (Ait.) R.Br. commonly known, as 'Arka' is a popular medicinal plant found throughout the tropics of Asia and Africa and is used in many traditional systems of medicine. Important factors of the various parts of this plant have been widely reported. Good record keeping of subjective and objectively recorded cures by practitioners of traditional medicinal system will help in the establishment of the use of C. procera as an antimalarial plant. It has been attempted to see the effect of crude fractions of its flower, bud and roof against a chloroquine sensitive strain, MRC 20 and a chloroquine resistant strain, MRC 76 of Plasmodium falciparum using the Desjardins method and the effectiveness of its fractions compare better with the CQ sensitive strain than the CQ resistant strain in vitro.
The ginger proteases (GP-I and GP-II), isolated from the ginger rhizome Zingiber officinale, have an unusual substrate specificity preference for cleaving peptides with a proline residue at the P2 position. The complete amino-acid sequence of GP-II, a glycoprotein containing 221 amino acids, and about 98% that of GP-I have been determined. Both proteases, which are 82% similar, have cysteine residues at positions 27 and histidines at position 161, corresponding to the essential cysteine-histidine diads found in the papain family of cysteine proteases, and six corresponding cysteine residues that form the three invariant disulfide linkages seen in this family of proteins. The sequence homology with other members (papain, bromelain, actinidin, protease omega, etc.) of this family is approximately 50%. GP-II has two predicted glycosylation sites at Asn99 and Asn156. Analyisis by electrospray and collision-induced dissociation MS showed that both sites were occupied by the glycans (Man)3(Xyl)1(Fuc)1(GlcNAc)2 and (Man)3(Xyl)1(Fuc)1(GlcNAc)3, in a ratio of approximately 7 : 1. Both glycans are xylose containing biantennary complex types that share the common core structural unit, Man1-->6(Man1-->3) (Xyl1-->2)Man1-->4GlcNAc1-->4(Fuc1-->3)GlcNAc for the major form, with an additional N-acetylglucosamine residue being linked, in the minor form, to one of the terminal mannose units of the core structure.
Human erythrocytes were exposed in a dose dependent manner to various ethanolic plant extracts, and fractions obtained from plant parts of Calotropis procera (Ait.) R. Br. and the gum--oleo resin of Commiphora wightii (Arnott.) Bhand. These have been screened for in vitro schizontocidal activity and graded with respect to their 50% inhibitory concentration (IC(50)) derived from the twofold serial dilution of the dose range 0.0625--2 mg/ml. An attempt had been made to relate their antiplasmodial activity with their cytotoxicity as represented by the in vitro rate of hemolysis. Intact erythrocytes were found to respond with a dose--time-integral and fitted to models of pseudo first-order reaction, Michaelis--Menten equation and Hill equation with k(1), k(2) and k(3) as their rate constants, respectively. Hemolysis isotherms of flower and root of C. procera and gum--oleo resin of C. wightii extracts were representative. Erythrocytic membrane instability is possibly a major factor as has been earlier reported with ethanol and chloroquine for the cytotoxicity of these plant extracts.
A protease was purified to homogeneity from the latex of medicinal plant Calotropis procera (Family-Asclepiadaceae). The molecular mass and isoelectric point of the enzyme are 28.8 kDa and 9.32, respectively. Hydrolysis of azoalbumin by the enzyme was optimal in the range of pH 7.0-9.0 and temperature 55-60 degree C. The enzyme hydrolyses denatured natural substrates like casein, azoalbumin, and azocasein with high specific activity. Proteolytic and amidolytic activities of the enzyme were activated by thiol protease activators and inhibited by thiol protease inhibitors, indicating the enzyme to be a cysteine protease. The enzyme named as procerain, cleaves N-succinyl-Ala-Ala-Ala-p-nitroanilide but not -Ala-Ala-p-nitroanilide, -Ala p-nitroanilide and N-d-Benzoyl--Arg-p-nitroanilide and appears to be peptide length dependent. The extinction coefficient (epsilon 1% 280 nm) of the enzyme was 24.9 and it had no detectable carbohydrate moiety. Procerain contains eight tryptophan, 20 tyrosine and seven cysteine residues forming three disulfide bridges, and the remaining one being free. Procerain retains full activity over a broad range of pH 3.0-12.0 and temperatures up to 70 degree C, besides being stable at very high concentrations of chemical denaturants and organic solvents. Polyclonal antibodies against procerain do not cross-react with other related proteases. Procerain unlike most of the plant cysteine proteases has blocked N-terminal residue.
The crystal structure of a cysteine protease ervatamin B, isolated from the medicinal plant Ervatamia coronaria, has been determined at 1.63 A. The unknown primary structure of the enzyme could also be traced from the high-quality electron density map. The final refined model, consisting of 215 amino acid residues, 208 water molecules, and a thiosulfate ligand molecule, has a crystallographic R-factor of 15.9% and a free R-factor of 18.2% for F > 2sigma(F). The protein belongs to the papain superfamily of cysteine proteases and has some unique properties compared to other members of the family. Though the overall fold of the structure, comprising two domains, is similar to the others, a few natural substitutions of conserved amino acid residues at the interdomain cleft of ervatamin B are expected to increase the stability of the protein. The substitution of a lysine residue by an arginine (residue 177) in this region of the protein may be important, because Lys --> Arg substitution is reported to increase the stability of proteins. Another substitution in this cleft region that helps to hold the domains together through hydrogen bonds is Ser36, replacing a conserved glycine residue in the others. There are also some substitutions in and around the active site cleft. Residues Tyr67, Pro68, Val157, and Ser205 in papain are replaced by Trp67, Met68, Gln156, and Leu208, respectively, in ervatamin B, which reduces the volume of the S2 subsite to almost one-fourth that of papain, and this in turn alters the substrate specificity of the enzyme.
A water-soluble (at pH 8) aromatic disulfide [5,5′-dithiobis(2-nitrobenzoic acid)] has been synthesized and shown to be useful for determination of sulfhydryl groups.Several applications have been made to show its usefulness for biological materials.A study of the reaction of this disulfide with blood has produced some evidence for the splitting of disulfide bonds by reduced heme.
A cysteine protease, with a high cysteine content and a high degree of amino terminal sequence homology with ervatamins B and C, has been purified from the latex of Ervatamia heyneana (Family Apocynaceae). The enzyme designated as heynein (M(r) = 23 kDa) has a comparatively high cysteine content (11), high isoelectric point (10.8), and high stability against pH (2.5-11.5), temperature (63 degrees C, 15 min), strong denaturants, and organic solvents. The enzyme has high specific activities for natural substrates such as casein and azoalbumin. The pH and temperature optima are pH 8.0-8.5 and 52 +/- 2 degrees C, respectively. Hydrolysis of synthetic substrates and digestion of bovine serum albumin confirm a distinct specificity of heynein as compared to ervatamins and papain. Also, heynein has distinct immunogenicity as monitored by enzyme-linked immunosorbent assay and Ouchterlony's double immunodiffusion. Strong enzyme activation by reducing agents such as beta-mercaptoethanol, dithiothreitol, and strong enzyme inhibition by thiol proteinase inhibitors such as E-64 and iodoacetic acid have evidenced heynein to be a cysteine protease. High stability, specific activity, and easy purification may make heynein a potential protease for food and biotechnology applications.
The diverse roles of plant proteases in defence responses that are triggered by pathogens or pests are becoming clearer. Some proteases, such as papain in latex, execute the attack on the invading organism. Other proteases seem to be part of a signalling cascade, as indicated by protease inhibitor studies. Such a role has also been suggested for the recently discovered metacaspases and CDR1. Some proteases, such as RCR3, even act in perceiving the invader. These exciting recent reports are probably just the first examples of what lies beneath. More roles for plant proteases in defence, as well as the regulation and substrates of these enzymes, are waiting to be discovered.
Chemical glycosylation of proteins occurs in vivo spontaneously, especially under stress conditions, and has been linked in a number of cases to diseases related to protein denaturation and aggregation. It is the aim of this work to study the origin of the change in thermodynamic properties due to glucosylation of the folded beta-lactoglobulin A. Under mild conditions Maillard products can be formed by reaction of epsilon-amino groups of lysines with the reducing group of, in this case, glucose. The formed conjugates described here have an average degree of glycosylation of 82%. No impact of the glucosylation on the protein structure is detected, except that the Stokes radius was increased by approximately 3%. Although at ambient temperatures the change in Gibbs energy of unfolding is reduced by 20%, the denaturation temperature is increased by 5 degrees C. Using a combination of circular dichroism, fluorescence, and calorimetric approaches, it is shown that the change in heat capacity upon denaturation is reduced by 60% due to the glucosylation. Since in the denatured state the Stokes radius of the protein is not significantly smaller for the glucosylated protein, it is suggested that the nonpolar residues associate to the covalently linked sugar moiety in the unfolded state, thereby preventing their solvent exposure. In this way coupling of small reducing sugar moieties to solvent exposed groups of proteins offers an efficient and unique tool to deal with protein stability issues, relevant not only in nature but also for technological applications.
Until fairly recently, proteases were considered primarily to be protein-degrading enzymes. However, this view has dramatically changed and proteases are now seen as extremely important signalling molecules that are involved in numerous vital processes. Protease signalling pathways are strictly regulated, and the dysregulation of protease activity can lead to pathologies such as cardiovascular and inflammatory diseases, cancer, osteoporosis and neurological disorders. Several small-molecule drugs targeting proteases are already on the market and many more are in development. The status of human protease research and prospects for future protease-targeted drugs are reviewed here, with reference to some key examples where protease drugs have succeeded or failed.
A novel protease is purified to homogeneity from the latex of a medicinally important plant Cryptolepis buchanani of family Apocynaceae (formerly Asclepiadaceae). The enzyme named cryptolepain has a molecular mass of 50.5 kDa. The isoelectric point and extinction coefficient (epsilon280nm1%) are 6.0 and 26.4, respectively. Cryptolepain contains 15 tryptophans, 41 tyrosines, and eight cysteine residues forming four disulfide bridges. The detectable carbohydrate moiety in the enzyme was found to be 6-7%. Cryptolepain hydrolyzes denatured natural substrates like casein, azocasein, and azoalbumin with high specific activity. The protease is exclusively inhibited by serine protease inhibitors phenylmethansulfonyl fluoride and diisopropyl fluorophosphate. Hydrolysis of azoalbumin by the cryptolepain is optimal in the pH range of 8-10 and temperatures of 65-75 degrees C. The enzyme shows high stability against pH (2.5-11.5), temperature (up to 80 degrees C), and chemical denaturants. The Km value of the enzyme was found to be 10 microM with azocasein as the substrate. The N-terminal sequence of cryptolepain is unique and shows only little homology to other known serine proteases, which makes this enzyme an ideal candidate for our ongoing biochemical and structure-function investigations of proteases. Easy availability of the latex and simple purification procedures make the enzyme a good system for exploring the biophysical chemistry of serine proteases as well as applications in the food industry.
Immuno-chemical analysis of protein concentration Protein Structure—A Practical Approach
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- Goldberg
Friguet B, Djavadi-Ohaniance L, Goldberg ME. Immuno-chemical analysis of protein concentration. In: Crieghton TE, editor. Protein Structure—A Practical Approach. Oxford: IRL Press; 1989. p. 287–310.
Guide to Protein Purification in Gel-staining Techniques
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Merril CR. In: Deutscher MP, editor. Guide to Protein Purification in Gel-staining Techniques. San diego, CA: Academic Press, Inc.; 1990. p. 477–88.
To evaluate the stain removal, clean cotton cloth pieces were soiled with blood and dried. (A) Soaked only in 1% detergent (without proteases) acts as standard for comparison
- Fig
Fig. 8. To evaluate the stain removal, clean cotton cloth pieces were soiled with blood and dried. (A) Soaked only in 1% detergent (without proteases) acts as standard for comparison. (B) Incubated with 1% detergent with 0.1 mg/ml of purified protease for 5 h.
Papain-like proteases: applications of their inhibitors
- V K Dubey
- M Pande
- B K Singh
- M V Jagannadham
Dubey VK, Pande M, Singh BK, Jagannadham MV. Papain-like proteases:
applications of their inhibitors. Afr J Biotechnol 2007;6:1077-86.
Protein determination by UV absorption
- A Aitken
- M Learmoth
Aitken A, Learmoth M. Protein determination by UV absorption. In: Walker
JM, editor. The Protein Protocol Handbook. Totowa, NJ: Humana Press; 1997.
p. 3-6.
Immuno-chemical analysis of protein concentration
- B Friguet
- L Djavadi-Ohaniance
- M E Goldberg
Friguet B, Djavadi-Ohaniance L, Goldberg ME. Immuno-chemical analysis of
protein concentration. In: Crieghton TE, editor. Protein Structure-A Practical
Approach. Oxford: IRL Press; 1989. p. 287-310.
Papain-like proteases: applications of their inhibitors
- Dubey
Immuno-chemical analysis of protein concentration
- Friguet