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V Prakash - One of the best experts on this subject based on the ideXlab platform.
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comparison of activity and conformational changes of Ficin during denaturation by urea and guanidine hydrochloride
Process Biochemistry, 2011Co-Authors: K B Devaraj, Parigi Ramesh Kumar, V PrakashAbstract:The activity and conformational changes of Ficin (EC 3.4.22.3), a cysteine protease from Ficus carica have been investigated during the denaturation by urea and guanidine hydrochloride (GuHCl). The denaturation of Ficin was followed by activity measurements, fluorescence and circular dichroism (CD) spectroscopic studies. The enzyme activity decreased significantly at low concentration of both urea and GuHCl before unfolding of the enzyme molecule. The enzyme molecule was resistant for unfolding by urea under neutral conditions even at higher concentrations. However, the protein is susceptible to unfolding by urea at lower pH and transition follows a cooperative two-state rule with increasing concentration of urea. On the other hand, Ficin molecule loses its complete structure in presence of 4 M GuHCl under neutral conditions. The GuHCl-induced unfolding occurs in a simple two-state cooperative process. These results indicate the differential structural stability and fragility of active site of the enzyme towards denaturation by urea and GuHCl.
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characterization of acid induced molten globule like state of Ficin
International Journal of Biological Macromolecules, 2009Co-Authors: K B Devaraj, Parigi Ramesh Kumar, V PrakashAbstract:Effect of pH on the conformational behaviour of Ficin (EC 3.4.22.3), a cysteine protease from the latex of Ficus carica was monitored by circular dichroism, fluorescence spectroscopy, ANS binding and hydrodynamic studies. The results obtained from near- and far-UV CD, intrinsic fluorescence and ANS binding studies demonstrate that Ficin exhibits the characteristic properties of molten globule at acidic conditions between pH 1.4 and 2.0. Ficin at pH 1.4 retained about ∼74% secondary structure with a substantial loss of tertiary structure. The acid-induced state was found to have a compact shape as measured by Stokes radius on size exclusion chromatography.
Parigi Ramesh Kumar - One of the best experts on this subject based on the ideXlab platform.
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comparison of activity and conformational changes of Ficin during denaturation by urea and guanidine hydrochloride
Process Biochemistry, 2011Co-Authors: K B Devaraj, Parigi Ramesh Kumar, V PrakashAbstract:The activity and conformational changes of Ficin (EC 3.4.22.3), a cysteine protease from Ficus carica have been investigated during the denaturation by urea and guanidine hydrochloride (GuHCl). The denaturation of Ficin was followed by activity measurements, fluorescence and circular dichroism (CD) spectroscopic studies. The enzyme activity decreased significantly at low concentration of both urea and GuHCl before unfolding of the enzyme molecule. The enzyme molecule was resistant for unfolding by urea under neutral conditions even at higher concentrations. However, the protein is susceptible to unfolding by urea at lower pH and transition follows a cooperative two-state rule with increasing concentration of urea. On the other hand, Ficin molecule loses its complete structure in presence of 4 M GuHCl under neutral conditions. The GuHCl-induced unfolding occurs in a simple two-state cooperative process. These results indicate the differential structural stability and fragility of active site of the enzyme towards denaturation by urea and GuHCl.
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characterization of acid induced molten globule like state of Ficin
International Journal of Biological Macromolecules, 2009Co-Authors: K B Devaraj, Parigi Ramesh Kumar, V PrakashAbstract:Effect of pH on the conformational behaviour of Ficin (EC 3.4.22.3), a cysteine protease from the latex of Ficus carica was monitored by circular dichroism, fluorescence spectroscopy, ANS binding and hydrodynamic studies. The results obtained from near- and far-UV CD, intrinsic fluorescence and ANS binding studies demonstrate that Ficin exhibits the characteristic properties of molten globule at acidic conditions between pH 1.4 and 2.0. Ficin at pH 1.4 retained about ∼74% secondary structure with a substantial loss of tertiary structure. The acid-induced state was found to have a compact shape as measured by Stokes radius on size exclusion chromatography.
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purification characterization and solvent induced thermal stabilization of Ficin from ficus carica
Journal of Agricultural and Food Chemistry, 2008Co-Authors: Kamsagara Basavarajappa Devaraj, Parigi Ramesh Kumar, Vishweshwaraiah PrakashAbstract:Ficin (EC 3.4.22.3), a cysteine proteinase isolated from the latex of a Ficus tree, is known to occur in multiple forms. Although crude Ficin is of considerable commercial importance, Ficin as such has not been fully characterized. A major Ficin from the commercial crude proteinase mixture preparation of Ficus carica was purified and characterized. The purified enzyme was homogeneous in both sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and gel-filtration chromatography and is a single polypeptide chain protein with a molecular mass of 23 100 ( 300 Da as determined by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF). The enzyme was active in the pH range of 6.5-8.5, and maximum activity was observed at pH 7.0. The N-terminal core sequence of Ficin has homology with N-terminal sequences of plant cysteine proteinases. The enzyme contains three disulfide bonds and a single free cysteine residue at the active site. The effect of co-solvents, such as sorbitol, trehalose, sucrose, and xylitol, on the thermal stability of Ficin was determined by activity measurements, fluorescence, and thermal denaturation studies. The apparent thermal denaturation temperature (Tm) of Ficin was significantly increased from the control value of 72 ( 1 °C in the presence of all co-solvents. However, the maximum stabilization effect was observed in terms of thermal stabilization by the co-solvent trehalose.
Huzhi Zheng - One of the best experts on this subject based on the ideXlab platform.
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Ficin encapsulated in mesoporous metal organic frameworks with enhanced peroxidase like activity and colorimetric detection of glucose
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy, 2020Co-Authors: Wen Zheng, Danyang Yi, Yijuan Long, Huzhi ZhengAbstract:Abstract Ficin has been reported to possess peroxidase activity, but its applications in some respects have been limited because of its relatively low activity. Herein, a mesoporous metal-organic framework, PCN-333(Fe), was synthesized, which was selected to encapsulate Ficin to form Ficin@PCN-333(Fe). Compared with Ficin, the peroxidase-like activity of Ficin@PCN-333(Fe) toward 3,3′,5,5′-tetramethylbenzidine (TMB) oxidation was about 3 times increase in the presence of H2O2, and followed classical Michaelis-Menten model. The kinetic parameters showed that stronger affinity and higher catalytic constant (Kcat) of Ficin@PCN-333(Fe) to both TMB and H2O2 compared with Ficin, and Kcat of Ficin@PCN-333(Fe) was increased by 3.65 folds and 3.59 folds for TMB and H2O2, respectively. Taking advantages of higher catalytic property of Ficin@PCN-333(Fe), we developed a colorimetric method with high sensitivity and selectivity to detect glucose, which displayed a good linear response toward glucose in the range of 0.5–180 μM with a limit of detection of 97 nM. Furthermore, Ficin@PCN-333(Fe) has been proven to successfully detect glucose in human serum, implying its great potentialities and wide applications as peroxidase mimics.
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enhancing the peroxidase like activity of Ficin by rational blocking thiol groups for colorimetric detection of biothiols
Talanta, 2019Co-Authors: Wen Zheng, Dongjun Shen, Danyang Yi, Yijuan Long, Huzhi ZhengAbstract:Abstract The peroxidase-like activity of Ficin is relatively low, which limits its application. It was found that thoil groups of Ficin could inhibit its peroxidase-like activity. So, two procedures, i.e., direct blocking with N -ethylmaleimide (NEM), or using tris (2-carboxyethyl) phosphine hydrochloride (TCEP) to interrupt disulfide bonds then blocking thiol groups with NEM, were applied to block thiol groups of Ficin, Ficin-NEM (Ficin-N) and Ficin-TCEM-NEM (Ficin-TN) were produced, respectively. The blocking of thiol groups accelerated the peroxidase activity dramatically. The peroxidase catalytic activity of Ficin-N and Ficin-TN toward the peroxidase substrate 3,3′,5,5′-tetramethylbenzidine (TMB) oxidation by H 2 O 2 was about 2.5-fold and 5-fold increase compared with Ficin, respectively, which accompanied a color change from colorless to blue and followed classic Michaelis-Menten model. The kinetic parameters indicated that higher affinity of Ficin-N ( K m = 0.31) and Ficin-TN ( K m = 0.39) to H 2 O 2 compared with Ficin ( K m = 0.58), and Ficin-TN had the highest K cat which increased by 6.5 times and 4.5 times for TMB and H 2 O 2 , respectively. According to these findings, a colorimetric method with high sensitivity for the detection of biothiols was developed due to sulfhydryl compounds inhibited the peroxidase activity of Ficin. Comparing with Ficin and Ficin-N, Ficin-TN had the widest detection range (0.01–16 μM) and the lowest detection limit (3 nM). The practical applications of Ficin-TN for biothiol determination in human serum samples have been demonstrated with satisfactory results. Ficin-N and Ficin-TN are promising to apply to the bioanalysis.
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one pot synthesis of a composite consisting of the enzyme Ficin and a zinc ii 2 methylimidazole metal organic framework with enhanced peroxidase activity for colorimetric detection for glucose
Mikrochimica Acta, 2019Co-Authors: Yanjiao Pang, Wen Zheng, Yijuan Long, Yuming Huang, Huzhi ZhengAbstract:: An enzyme-metal organic framework (MOF) composite with saucer-like structure was prepared via one-pot synthesis from Ficin (a cysteine proteolytic enzyme with POx activity), zinc(II) ions and 2-methylimidazole. The composites exhibit a 2.5-fold higher catalytic activity and stronger affinity for substrates compared to free Ficin. This was exploited to design a colorimetric assay for the determination of glucose. The addition of glucose oxidase causes the formation of H2O2 which is catalytically oxidized by Ficin to form a blue coloration that can be measured at 652 nm. The assay has a 0.12 μM detection limit and excellent selectivity. It was successfully applied to the determination of glucose in diluted serum samples. Graphical abstract Schematic presentation of the synthesis process and the enhanced peroxidase activity of Ficin@MOF composites. Ficin was immobilized in a new saucer-like shape of Ficin@MOF composites, which showed higher peroxidase activity than free Ficin. Scheme and graphical abstract contains poor quality of text.We have attach a new picture with 600 dpi as scheme and graphical abstract.
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enhancing the peroxidase like activity of Ficin via heme binding and colorimetric detection for uric acid
Talanta, 2018Co-Authors: Yufang Yang, Yijuan Long, Yanjiao Pang, Huzhi ZhengAbstract:Abstract Ficin, a classical sulfhydryl protease, was found to possess intrinsic peroxidase-like activity. In this paper, we have put forward a novel strategy to improving the peroxidase-like activity of Ficin through binding heme. Heme-Ficin complexes were successfully obtained by simple one-step syntheticism. The results demonstrated that the catalytic activity and efficiency of heme-Ficin complexes were about 1.7 times and 3 times higher than those of native Ficin, respectively. Taking advantages of the high peroxidase-like activity, the heme-Ficin complexes were used for colorimetric determination of uric acid with a low detection limit of 0.25 μM. Based on the excellent selectivity and sensitivity, we detected the concentration of uric acid in human serum successfully. On the basis of these findings, the heme-Ficin complexes are promising for wide applications in various fields. Thus we not only optimized the peroxidase-like activity of the Ficin, but also established a new strategy for development of artificial enzyme mimics by mimicking the architecture of the active site in horseradish peroxidase.
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colorimetric detection of glucose based on Ficin with peroxidase like activity
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy, 2018Co-Authors: Yanjiao Pang, Yijuan Long, Yufang Yang, Zili Huang, Huzhi ZhengAbstract:Abstract In this work, we developed a colorimetric biosensing system for glucose detection by coupling the peroxidase-like of Ficin and the glucose oxidase (GOx). GOx can catalyze the oxidation of glucose to produce H2O2, then, Ficin catalyzes the oxidation of peroxidase substrate 3,3′,5,5′-tetramethylbenzidine (TMB) by H2O2 to produce a blue color reaction. The present sensing system showed a linear response toward glucose detection over range of 2.0–100 μM with a detection limit of 0.5 μM. This system is simple, low cost, highly sensitive and selective for glucose detection, and was also applied to measuring glucose in human serum. Furthermore, in order to expand the application of Ficin in biological sensing, we immobilized Ficin onto the SiO2@Fe3O4 NPs, which exhibited the merits of recycling as well as allowing the repeated detection of glucose. Thus it may provide great potential applications in biomedicine, biotechnology and environmental chemistry.
K B Devaraj - One of the best experts on this subject based on the ideXlab platform.
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comparison of activity and conformational changes of Ficin during denaturation by urea and guanidine hydrochloride
Process Biochemistry, 2011Co-Authors: K B Devaraj, Parigi Ramesh Kumar, V PrakashAbstract:The activity and conformational changes of Ficin (EC 3.4.22.3), a cysteine protease from Ficus carica have been investigated during the denaturation by urea and guanidine hydrochloride (GuHCl). The denaturation of Ficin was followed by activity measurements, fluorescence and circular dichroism (CD) spectroscopic studies. The enzyme activity decreased significantly at low concentration of both urea and GuHCl before unfolding of the enzyme molecule. The enzyme molecule was resistant for unfolding by urea under neutral conditions even at higher concentrations. However, the protein is susceptible to unfolding by urea at lower pH and transition follows a cooperative two-state rule with increasing concentration of urea. On the other hand, Ficin molecule loses its complete structure in presence of 4 M GuHCl under neutral conditions. The GuHCl-induced unfolding occurs in a simple two-state cooperative process. These results indicate the differential structural stability and fragility of active site of the enzyme towards denaturation by urea and GuHCl.
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characterization of acid induced molten globule like state of Ficin
International Journal of Biological Macromolecules, 2009Co-Authors: K B Devaraj, Parigi Ramesh Kumar, V PrakashAbstract:Effect of pH on the conformational behaviour of Ficin (EC 3.4.22.3), a cysteine protease from the latex of Ficus carica was monitored by circular dichroism, fluorescence spectroscopy, ANS binding and hydrodynamic studies. The results obtained from near- and far-UV CD, intrinsic fluorescence and ANS binding studies demonstrate that Ficin exhibits the characteristic properties of molten globule at acidic conditions between pH 1.4 and 2.0. Ficin at pH 1.4 retained about ∼74% secondary structure with a substantial loss of tertiary structure. The acid-induced state was found to have a compact shape as measured by Stokes radius on size exclusion chromatography.
Nader Sheibani - One of the best experts on this subject based on the ideXlab platform.
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autolysis control and structural changes of purified Ficin from iranian fig latex with synthetic inhibitors
International Journal of Biological Macromolecules, 2016Co-Authors: Hamid R Zare, Ali Akbar Moosavimovahedi, Maryam Salami, Nader Sheibani, Khosro Khajeh, Mehran HabibirezaeiAbstract:The fig's Ficin is a cysteine endoproteolytic enzyme, which plays fundamental roles in many plant physiological processes, and has many applications in different industries such as pharmaceutical and food. In this work, we report the inhibition and activation of autolysis and structural changes associated with reaction of Ficin with iodoacetamide and tetrathionate using high-performance liquid chromatography (HPLC), ultra filtration membrane, and dynamic light scattering (DLS) methods. The Ficin structural changes were also determined using UV-absorption, circular dichroism (CD), fluorescence spectroscopy, and differential scanning calorimetry (DSC) techniques. These techniques demonstrated that iodoacetamide completely inhibited Ficin autolysis, which was irreversible. However, tetrathionate partially and reversibility inhibited its autolysis. The Ficin structural changes with two synthetic inhibitors were associated with secondary structural changes related to decreased alpha-helix and increased beta sheet and random coil conformations, contributing to its aggregation.
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purification and autolysis of the Ficin isoforms from fig ficus carica cv sabz latex
Phytochemistry, 2013Co-Authors: Hamid R Zare, Ali Akbar Moosavimovahedi, Maryam Salami, Morteza Mirzaei, Ali Akbar Saboury, Nader SheibaniAbstract:Ficin (EC 3.4.22.3), a cysteine endoproteolytic protease in fig trees’ latex, has multiple isoforms. Until now, no data on autolysis of individual Ficins (Ficin isoforms) are available. Following purification, Ficins’ autolysis was determined by HPLC chromatogram changes and ultrafiltrations at different temperatures and storage times. These results showed that the number of HPLC peaks in latex proteins purification of Ficus carica cv. Sabz varied from previous fig varieties or cultivars. Proteolytic activity of Ficins was inhibited by specific cysteine protease inhibitors, confirming the participation of the cysteine residue in the active site. The zeta potential of the first two eluted peaks (I and II) was negative, while that of other peaks were positive. All Ficins were susceptible to autolysis when stored at high temperatures. In contrast, only the last two Ficins (B, C) were prone to autolysis at cold temperature after long storage period. The rate of degradation of the Ficins was significantly increased with the increased storage time. The Ficin (A) related to peak (III) had the highest and the lowest surface hydrophobic patches and ratio of autolytic to proteolytic activity, respectively.