Search

A novel heme-peroxidase has been extracted from the latex of the medicinal plantArtocarpus lakoocha (A. lakoocha), known for its potential anti-inflammatory and wound healing properties. To study its stability, structure, and dynamics, this protein was analyzed using far-UV circular dichroism, fluorescence spectroscopy, and activity measurements. The results showed the existence of six different folding states, including the native state (N) at neutral pH, the molten globule state (MG) at pH 2, the acid-unfolded state (UA) at pH 1.5 or lower, acid-refolded (A) at pH 0.5, the alkaline denatured state (UB) at pH 8-12, and the complete denatured state (D) at a GuHCl concentration beyond 5 M. Absorbance studies indicated the presence of free heme in the pH range of 1-2. The protein showed stability and structural integrity across a wide pH range (3-10), temperature (70 °C), and high concentrations of GuHCl (5 M) and urea (8 M). This study is the first to report multiple ‘partially folded intermediate states’ ofA. lakoochaperoxidase, with varying amounts of secondary structure, stability, and compactness. These results demonstrate the high stability ofA. lakoochaperoxidase and its potential for biotechnological and industrial applications, making it a valuable model system for further studies on its structure-function relationship.

The use of a microbial supernatant has become a potential source for the eco-friendly and rapid synthesis of nanoparticles. In this study, we have targeted to synthesize silver nanoparticles (AgNPs) using extracellular secretion of Streptomyces sp. The strain was successfully isolated from the soil samples and identified as Streptomyces sp. BHUMBU-80 on the basis of morphological, biochemical and Phylogenetic analysis. The synthesized AgNPs was characterized using several modern characterizing techniques such as UV-vis Spectroscopy, FTIR spectroscopy, SEM, EDX spectroscopy, TEM, XRD and XPS. The presence of characteristics SPR band at 450 nm confirmed the synthesis of AgNPs. The FTIR spectra confirmed the involvement of various functional groups present in culture supernatant responsible for the reduction of Ag + ions into Ag 0 . SEM and TEM analysis confirmed the presence of spherical AgNPs with an average size of 21 ± 1 nm. The XPS analysis confirmed the presence of two individual peaks which attributed to the Ag 3d 3/2 and Ag 3d 5/2 binding energies corresponding to the presence of metallic silver. The electrochemical studies of the green synthesized AgNPs using a glassy carbon electrode showed the superior electrocatalytic activity towards the reduction of H 2 O 2 . The calibration plot was established for H 2 O 2 in the concentration range from 50 μM to 1000 μM with 50 μM detection limit, at a signal-to-noise ratio of 3 in a three-electrode cell with a Pt plate as the counter electrode. Additionally, the AgNPs also showed potent antibacterial activity against pathogenic bacteria like E. coli, and S. aureus .

Euphorbia tirucalli peroxidase (ETP) was immobilized on chitosan beads having magnetic properties for the ease of separation and increasing the reusability of ETP for cost effective assay conditions. The present work reports immobilization of ETP on polymeric support chitosan-cobalt oxide beads subsequently activated with 0.05% cynuric chloride. The magnetic immobilized enzyme was characterized by Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD) analysis and scanning electron microscopy (SEM). The immobilized ETP can be reused up to 10 cycles with retention of more than 60% activity. The optimum pH was shifted from 6.0 to 5.5 for soluble ETP to immobilized ETP and optimum temperature from 50°C and 55°C for the immobilized ETP. Based on response surface methodology, the optimal immobilization conditions obtained were: enzyme concentration, 2mg/286mg beads; optimal pH, 4.93; temperature, 28.88; cynuric chloride concentration, 0.17%; reaction time, 14.4h, which resulted 74.51% maximum immobilization. The enzyme magnetic nanoparticles could be separated magnetically for easy reuse. Immobilization of ETP onto the magnetic nanoparticles could be useful for biotechnological applications and bioassay due to its reusability and improved stability.

A novel peroxidase from the latex of medicinal plant Euphorbia tirucalli (Pencil tree) belonging to the Euphorbiaceae family is purified to homogeneity using cation exchange chromatography. The enz...

In this study, an eco-friendly and sustainable green route was employed for the synthesis of stable silver nanoparticles (AgNPs) using aqueous leaf extract of Euphorbia hirta (AEE) as both reducing as well as a stabilizing agent. The synthesis of AgNPs was confirmed by UV-visible spectroscopy which produced a prominent SPR band at λmax 425nm after 25min of sunlight exposure. The AgNPs thus synthesized were optimized using one factor at a time approach, and these optimized conditions were 25min of sunlight exposure time, 5.0% (v/v) of AEE inoculum dose and 3.0mM of AgNO3 concentration. The Field Emission Scanning Electron Microscopy (FE-SEM) and High Resolution Transmission Electron Microscopy (HRTEM) analysis confirmed the presence of spherical AgNPs with average size 15.5nm. The crystallinity was determined by X-ray Diffractometer (XRD) and Selected Area Electron Diffraction (SAED) pattern. Chemical and elemental compositions were determined by Fourier Transformed Infrared Spectroscopy (FTIR) and Energy Dispersive X-ray Spectroscopy (EDX) respectively. The Atomic Force Microscopy (AFM) images with average roughness 1.15nm represented the lateral and 3D topological characteristic of AgNPs. The AgNPs thus synthesized showed effective antibacterial activity against gram negative and gram positive bacteria as well as hydrogen peroxide sensing property with a minimum detection limit of 10(-7)M.

The crystal structure of peroxidase from Barley Grain Peroxidase 1 (1BGP) provides a good template for modeling the three dimensional structure of Euphorbia tirucalli peroxidase (ETP). Homologous three dimensional structure of ETP was built on the basis of the crystal structure of 1BGP. The reliability of the model was assessed by Ramachandran plot, Profile-3D, PROCHECK, ERRAT and PROSA analysis. The overall structure of the resulting ETP model is similar to those of the template structure. The three dimensional structure of peroxidase was docked with heme and the peroxidase-heme complex was further docked with substrates of peroxidase as well as thiol inhibitors (β-mercaptoethanol and l -cysteine) using Autodock 4.2. software. The modeled peroxidase exhibited substrate specificity in the order of pyrogallol ˂ guaiacol ˂ O-dianisidine. The inhibition kinetics of peroxidase in presence of these inhibitors were investigated. IC50 values for these inhibitors were calculated and used for kinetic studies. These studies revealed that β-mercaptoethanol is more potent inhibitor on peroxidase activity as compared to l -cysteine. To explore the binding model of the substrates with the ETP model was docked into the active site of the model and hydrophobic interaction and hydrogen bonding were found to play an important role in substrate recognition and orientation.

The current work describes the biosynthesis of stable AgNPs using aqueous extract of Xanthium strumarium (AEX) which act as both reducing as well as a stabilizing agent. The biosynthesis was confirmed by UV–visible spectroscopy where the presence of SPR band at λmax 436 nm corresponded to the existence of AgNPs in reaction mixture. The optimum conditions for biosynthesis of AgNPs were 30 min of sunlight exposure time, 3.0% (v/v) of AEX inoculum dose and 3.5 mM AgNO3 concentration. The synthesized AgNPs was characterized by HRTEM, SAED, FESEM, EDX, XRD, AFM, and FTIR which showed potent antibacterial and antileishmanial activity.

A series of ABA type well-defined amphiphilic poly(N-vinylpyrrolidone) (PNVP)-b-poly(D,L-lactide)-b-PNVP triblock copolymers have been synthesized via the combination of ring opening polymerization and xanthate-mediated reversible addition–fragmentation chain transfer polymerization, and analyzed by 1H NMR spectroscopy and gel permeation chromatography. Aggregation properties of these amphiphilic triblock copolymers have been revealed by fluorescence spectroscopy, transmission electron microscopy and dynamic light scattering, and supported by 1H NMR spectroscopy. Doxorubicin (DOX) has successfully been loaded into the block copolymer micelles with a loading efficiency of 37.5%. DOX-loaded PNVP51-b-PDLLA48-b-PNVP51 block copolymer showed sustained release within 36 h. Antibacterial properties of DOX-loaded micelles have been found to be significantly effective with respect to free DOX in terms of minimum inhibitory concentration, disk diffusion assay, growth curve, bacterial reduction and enzymatic assay based on in vitro studies.

Artocarpus lakoocha is a medicinal plant used in cosmetics and traditional remedies of many diseases. We have purified a protein from its latex using anion exchange chromatography and gel filtration, which resulted in 28.82% yield of protein, with specific activity of 10.8 units/mg. FPLC spectroscopy and isoelectrofocusing demonstrated that purified protein has molecular mass of 53 kDa with isoelectric point of pH 7.4. Isolated peroxidase consists of 17 tryptophan, 14 tyrosine, and 8 cysteine residues per molecule with extinction coefficient of 16.3 M −1 cm −1 . The pH and temperature optima for this peroxidase are 6.0 and 55 °C respectively. MALDI-TOF/TOF was done and MASCOT analysis specified that isolated protein is a peroxidase. Isolated peroxidase shows oxidizing activity with various phenolic substrates like guaiacol, o -phenylenediamine, aminoantipyrine and pyrogallol with K cat 1.90, 0.74, 3.22, 1.49 s −1 respectively. Stability experiments with chemical denaturants, metal ions and organic solvents indicate that isolated protein is very stable under studied conditions. Spectroscopic studies, like absorbance, fluorescence, and circular dichroism reveal that the peroxidase has α / β type secondary structure with approximately 42.6% α -helix, 9.7% β -sheet. In vitro scratch wound healing assay and oxidative damage protection experiments via MTT assay demonstrate that purified peroxidase have wound healing properties. In conclusion, this novel peroxidase is stable at various conditions and helps in healing which hints it׳s potential to be used in inflammation prevention, wound healing, and other biotechnological and industrial applications.

Poly(N-vinyl pyrrolidone) (PNVP)-grafted graphene oxide (GO) (GO-PNVP) has been synthesized using a GO-based macro-RAFT agent prepared via click reaction of alkyne-terminated RAFT agent (S)-2-(propynyl propionate)-(o-ethyl xanthate) and azide-functionalized GO (GO-N3). FTIR, XPS, Raman, TGA and DSC studies confirmed its formation. Silver nanoparticles are then immobilized on GO-PNVP and GO via in situ reduction of silver nitrate in the presence and absence of glucose at 40 and 95 °C, respectively. FT-IR, UV-Vis, XRD, SEM and TGA studies supported the incorporation of silver (Ag) nanoparticles. Ag nanoparticles immobilized on GO-PNVP are small, spherical and narrowly distributed (homogenous, monodisperse) compared to GO. These nanocomposites are explored as catalysts for the reduction of p-nitrophenol into p-aminophenol and also as antibacterial agents towards Gram(+) S. aureus and Gram(−) E. coli bacteria. Ag nanoparticle immobilized GO-PNVP showed efficient catalytic activity and excellent reusability along with an excellent antibacterial activity. Hence, grafting of PNVP enhances the catalytic and antibacterial properties of GO.

A folding pattern, conformational stability and therapeutic role of a protein helps in developing a suitable drug. Crinumin, a thrombolytic and anti platelet agent, has been studied for its functional and conformational properties by equilibrium unfolding methods. The crinumin belongs to α+β class of protein and exhibits a non native structure and two molten globule states at different conditions. Two domains in the molecular structure of the protein with altered stability are present that unfold sequentially. The enzyme maintains activity as well as structural integrity even in adverse conditions. These observations provide an understanding of protein folding as well as facilitate the development of a potential drug.

Inhibition of the Tryparedoxin peroxidase interaction has been becomes a new therapeutic strategy in leishmaniasis. Docking analysis was carried out to study the effects of quercetin and taxifolin on Tryparedoxin Peroxidase (TryP). Tryparedoxin peroxidase of Trypanosomatidae functions as antioxidants through their Peroxidase and peroxynitrite reductase activities. The 3D models of Tryparedoxin Peroxidase of Leishmania braziliensis (L. braziliensis TryP) was modeled using the template Tryparedoxin Peroxidase I from Leishmania Major (L. Major TryPI) (PDB ID: 3TUE). Further, we evaluated for TryP inhibitory activity of flavonoids such as quercetin and taxifolin using in silico docking studies. Docking results showed the binding energies of - 11.8601and -8.0851 for that quercetin and taxifolin respectively. Flavonoids contributed better L. braziliensis TryP inhibitory activity because of its structural parameters. Thus, from our in silico studies we identify that quercetin and taxifolin posses anti-leishmanial acitivities mediated through TryP inhibition mechanism.

An extracellular L-asparaginase produced by a protease-deficient isolate, Bacillus aryabhattai ITBHU02, was purified to homogeneity using ammonium sulfate fractionation and subsequent column chromatography on diethylaminoethyl-Sepharose fast flow and Seralose CL-6B. The enzyme was purified 68.9-fold with specific activity of 680.47 U mg(-1). The molecular weight of the purified enzyme was approximately 38.8 kDa on SDS-PAGE and 155 kDa on native PAGE gel as well as gel filtration column revealing that the enzyme was a homotetramer. The optimum activity of purified L-asparaginase was achieved at pH 8.5 and temperature 40 °C. Kinetic studies depicted that the K m, V max, and k cat values of the enzyme were 0.257 mM, 1.537 U μg(-1), and 993.93 s(-1), respectively. Circular dichroism spectroscopy has showed that the enzyme belonged to α + β class of proteins with approximately 74 % α-helices and 12 % β-sheets. BLASTP analysis of N-terminal sequence K-T-I-I-E-A-V-P-E-L-K-K-I-A of purified L-asparaginase had shown maximum similarity with Bacillus megaterium DSM 319. In vitro cytotoxicity assays with HL60 and MOLT-4 cell lines indicated that the L-asparaginase has significant antineoplastic properties.

In order to understand the mechanism of molecular i nteractions at the active site of Tryparedoxin Pero xidase (Try P), homology modeling and docking studies were performed. We generated a Three-Dimensional (3D) model of target protein based on the Crystal struct ure of Leishmania Major Try PI (PDB ID: 3TUE) using modeler software. Docking analysis was carried out to study the effects of methotrexate on Tryparedoxi n Peroxidase (Try P). Inhibition of the Tryparedoxin peroxidase interaction has become a new therapeutic strategy in treating leishmaniasis. Docking analysi s was carried out to study the effects of methotrex ate on Tryparedoxin Peroxidase (TryP). Tryparedoxin peroxidase of Trypanosomatidae family functions as antioxidant through their peroxidase and peroxynitr ite reductase activities. The theoretical docking s tudy, conducted on a sample previously reported for anti- cancer properties of Methotrexate at the binding si te of 3D models of Tryparedoxin Peroxidase of Leishmania braziliensis (L. braziliensis Try P) examine interaction energy. Our studies indicate that Methotrexate disp lays potent activity against Try P with lowest bind ing energy and RMSD values to be -14.5879 Kcal/Mol and 2.0 A. The results of the present study clearly demonstrated the Tryparedoxin Peroxidase inhibitory activity by methotrexate in in silico docking analysis and in vitro assay which contributes towards understanding the mechanism of antileishmanial activity.

A.niger Rnase was designed from ACTBIND (PDB ID: 3D3Z). Yeast actin-human gelsolin segment 1 complex (PDB ID: 1YAG) was used as template for L. donovani actin protein for 3D model in Modeller9v8. These models were testified by PROCHECK, ERRAT, WHAT-IF, PROSA2003 and VERIFY-3D. All evidences suggest that the geometric quality of the backbone conformation, energy profile, residue interaction and contact of the str uctures were well within the limits of reliable str uctures. The interaction energy of docking was calculated us ing the HEX server. Etotal and calculated RMSD values were -1.902, -9.323 kcal moL -1 and 0.402 A, respectively. The study presented her e has an advantage to design molecules that may have antileishmanial acti vity.

Interaction of sodium dodecyl sulfate (SDS) with a globular protein cryptolepain to induce stabilization of the pre-molten globule state of the protein under acidic condition was studied. At pH 2.0, cryptolepain is in partially unfolded state with native like secondary structure, exposed hydrophobic surfaces without any rigid tertiary structure. Unfolding of cryptolepain in the pre-molten globule state (state at pH 2.0) is co-operative suggesting that the protein unfolds as a single entity. Addition of SDS below its critical micelle concentration of 2.0 mM at acidic pH drives the protein to a non-native state similar to molten globule state seen with other proteins. Temperature induced unfolding of such SDS-induced state of the cryptolepain is biphasic suggesting that the molecular structure of the protein is constituted of two structural entities which are stabilized differentially in the presence of SDS and unfold sequentially. Whereas the chemical induced unfolding of SDS-induced state of cryptolepain is co-operative with higher transition midpoints indicating that the protein is stabilized as a whole. Unfolding of SDS-induced state of cryptolepain is very similar to the unfolding of the cryptolepain at very low pH (0.5), where the protein exists in molten globule state. Thus, SDS interactions result in the refolding of the acid unfolded state of the protein and drive the protein into a non-native state similar to the molten-globule state seen at very low pH.

Plant peroxidases are extensively used in a wide range of biotechnological applications owing to their high environmental and thermal stability. A new peroxidase, named banyan peroxidase, was purified from the latex of Ficus benghalensis and crystallized. X-ray diffraction data were collected from native crystals and from bromide and xenon derivatives to resolutions of up to 1.66 Å in the trigonal space group P3(2)21, with unit-cell parameters a = b = 73.1, c = 164.6 Å. The anomalous signal of the intrinsic iron and calcium ions was sufficient for structure solution by SAD, although the sequence is not yet known.

A dimeric serine protease Neriifolin S of molecular mass 94kDa with milk clotting activity has been purified from the latex of Euphorbia neriifolia by anion exchange and size-exclusion chromatography. It hydrolyses peptidyl substrates l-Ala-pNA with highest affinity (Km of 0.195mM) and physiological efficiency (Kcat/Km of 144.5mMs). Enzyme belongs to the class of neutral proteases with pI value of 6.8, optimal proteolytic activity displayed at pH 9.5 and temperature 45°C. Its proteolytic activity is strongly stimulated in the presence of Ca+2 ions and exclusively inhibited by serine protease inhibitors. Enzyme is fairly stable toward chemical denaturants, pH and temperature. The apparent Tm, was found to be 65°C. Thermal inactivation follow first order kinetics with activation energy (Ea), activation enthalpy (ΔH∗), free energy change (ΔG∗) and entropy (ΔS∗) of 27.54kJmol-1, 24.89kJmol-1, -82.34kJmol-1 and 337.20Jmol-1K-1.

A serine protease was purified to homogeneity from the latex of Ficus religiosa. The enzyme, named religiosin C is a monomer with molecular mass of 80 kDa. The enzymatic activity of the protein was inhibited by serine protease inhibitors. Isoelectric point of the enzyme is pH 4.6 with optimum pH and temperature of pH 6–8 and 45–50 °C, respectively. The specific extinction coefficient ( e 280 1 % ) of the enzyme is 14.68 with 16 tryptophan, 20 tyrosine and 7 cysteine residues in its molecular structure. The enzyme shows broad substrate specificity and hydrolyzes both natural and synthetic substrates. The enzyme is highly stable over a broad range of pH and temperature as well as in the presence of high concentration of chemical denaturants, organic solvents and metal ions. The N-terminal residues of religiosin C exhibited considerable homology with cucumisin and other cucumisin/subtilisin-like serine proteases. The high milk-clotting ability of religiosin C supports its probable use in the food and other biotechnological industries.

A novel milk-clotting serine protease, named religiosin B, is purified from Ficus religiosa . The molecular mass of the protein is 63,000 with p I value of pH 7.6. The proteolytic activity of the enzyme is strongly inhibited by phenylmethanesulfonyl fluoride (PMSF) and chymostatin. Religiosin B acts optimally at pH 8.0–8.5 and temperature 55 °C. The molar absorption coefficient of the enzyme is 149,725 M −1 cm −1 with 23 tryptophan, 15 tyrosine and 7cysteine residues per molecule of the enzyme. The enzyme shows broad substrate specificity with natural as well as synthetic substrates. Religiosin B is highly stable against denaturants and metal ions as well as over a wide range of pH and temperature. The de novo sequencing confirms the novelty of the enzyme. In addition to its high milk-clotting ability, it could be used in the cheese industry, as well as other food and biotechnological industries.

Neriifolin, a chymotrypsin-like serine protease, has been purified from the latex of Euphorbia neriifolia Linn. by ammonium sulfate precipitation, cation exchange chromatography and gel filtration. The molecular mass of the enzyme is 35.24 kDa, with an isoelectric point of pH 5.7. The enzyme consists of 18 tryptophan, 25 tyrosine and 9 cysteine residues with 4 disulfide bridges. The extinction coefficient ( e 280 nm 1 % ) is 38.28. The Km values are 1.39 ± 0.08 mM and 1.94 ± 0.17 mM, with N-succinyl- l -Phe-p-nitroanilide and α-leucine-p-nitroanilide as substrates, respectively. Neriifolin retains proteolytic activity over a wide range of pH and temperature value, with pH optima of 8.5 and an optimal temperature of 55 °C. Inhibition of enzyme activity by chymostatin and amidolytic activity against synthetic substrates specific to chymotrypsin indicates that the enzyme belongs to chymotrypsin-like serine protease class. Polyclonal antibodies specific to neriifolin and immunodiffusion reveal that the enzyme has unique antigenic determinants. The amino terminal sequence of the first 14 residues of neriifolin is D–F–P–P–N–T–H–I–G–I–P–N–G–Y. A high ratio of milk-clotting activity to proteolytic activity as well as stability against variations in pH and temperature, surfactants, oxidizing agents and compatibility with detergent additives make neriifolin an excellent candidate for industrial applications.

A high molecular mass novel metalloprotease, cotinifolin is purified from the latex of Euphorbia cotinifolia by a combination of anion exchange and hydrophobic interaction chromatography. The nonglycosylated enzyme has a molecular mass of 79.76 kDa (ESI-MS) and the isoelectric point of the enzyme is pH 7.7. Cotinifolin hydrolyzes denatured natural substrates such as casein, azoalbumin, and hemoglobin with high specific activity. The K m value of the enzyme was found to be 20 μM with azocasein. The enzyme is not prone to autolysis even at very low concentrations. Polyclonal antibodies specific to enzyme was raised and immunodiffusion reveals that the enzyme has unique antigenic determinants. Maximum caseinolytic activity of cotinifolin is observed in the range of pH 7.0–8.0 and temperature of 50 °C. Using 0.2 mL of 1 mM solution of each metal ion, the purified protease was inhibited slightly by Ba 2+ and Mn 2+ , moderately by Mg 2+ , Ca 2+ and Cs 2+ and significantly by Zn 2+ , Cu 2+ and Co 2+ . On the other hand, substantial activation in caseinolytic activity was achieved by Ni 2+ . The enzyme activity was also inhibited by EDTA and o-phenanthroline but not by any other protease inhibitors. Perturbation studies by temperature, pH, and chaotrophs of the enzyme also reveal its high stability as seen by CD, fluorescence and proteolytic activity. Spectroscopic studies reveal that cotinifolin has secondary structural features with α/β type with approximately 9% of α-helicity. Easy availability and simple purification procedure makes the enzyme a good system for biophysical study, biotechnological and industrial applications.

The 3D models of human actin protein and A.niger RNase were designed using the templates ACTBIND (PDB ID: 3D3Z) and crystalline profilin-beta-actin (PDB ID: 2BTF), respectively in Modeller9v5. These models are testified using several validation methods including PROCHECK, ERRAT, WHAT-IF, PROSA2003 and VERIFY-3D. The stereo-chemical quality of the models was judged by Ramachandran plot with PROCHECK. The total quality G-factor -0.2, shows a good quality model. The ERRAT score for the human actin and A.niger RNase models are 86.104 and 84.615, respectively, fit well within the range of a high quality model. The ERRAT score for the templates 2BTF and 3D3Z are 91.111 and 97.391, respectively. The WHAT-IF evaluation justifies a reasonable homology model structure as none of the scores for each residue in the homology model is lower than -5.0. The energy-minimized model of human actin with PROSA reveals the Z-score value -10.52 between native conformations of the crystal structures. The VERIFY 3D average score is 0.36. All evidence suggests that the geometric quality of the backbone conformation, the residue interaction, the residue contact and the energy profile of the structures were well within the limits of reliable structures. The interaction energy of docking was calculated using the HEX server. The Etotal, lowest docked energy, and calculated RMSD values were -1.608 kcal mol(-1), -8.369 kcal mol(-1) and 0.617 Å, respectively. The study presented in the current project may be useful to design molecules that may have anticancer activity.

A serine protease, streblin, was purified 4.6-fold with 75% recovery from the latex of a plant, Streblus asper. This is the first report of identification and purification of a serine protease from the genus Streblus of the Moraceae family. Streblin, has a molecular mass of 64 kDa and the extinction coefficient ( ϵ 280 1 % ) is 5.29. Streblin is a basic protein with pI value of 9.2 that acts optimally at pH 9.0; such optimum activity at high pH has not been reported for most of the isolated plant serine proteases and the enzyme is stable over a wide range of pH (3.0–12.5). The enzyme is also thermostable, retaining complete activity at 15–85 °C and acts optimally at 65 °C. Furthermore, it is highly stable in the presence of various denaturants in which SDS resistance is the most striking property of the purified enzyme. Streblin strongly coagulated skimmed milk. Easy availability of the latex, simple purification procedures, high stability of streblin against pH, or autodigestion, and under various conditions make the enzyme a good system for exploring the biophysical chemistry of proteases. In addition to its high milk-clotting ability, it could be used in the cheese industry, as well as other food and biotechnological industries.

Cysteine proteases are vital to cell physiology and many plants secrete these proteases for defense purposes. Many recent studies have reported unusually high stabilities for several plant cysteine proteases which possibly enable these proteases to function under adverse environmental conditions. Here, we have examined the conformational features of a new plant cysteine protease heynein using spectroscopic tools to understand the basis for its robust functional stability. The studies revealed structural integrity over a wide range of pH (2.5-12.0), temperature (65 oC) and urea (8M). However, at pH 2.0, the protein gets acid-unfolded (UA -state) with exposed hydrophobic patches, which upon addition of more protons (pH 0.5) or anions (0.5 M KCl and 0.2 M Na2 SO4 ) yields conformationally distinct refolded intermediates respectively termed: A-, I 1 - and I 2 -states. Strikingly, a high methanol level drives the UA -state into a predominantly -sheet rich conformation (O-state). We observed three-state unfolding kinetics of the I 2 -state by urea, possibly suggesting presence of two domains in the heynein molecule.

Although β-galactosidases are physiologically a very important enzyme and have may therapeutics applications, very little is known about the stability and the folding aspects of the enzyme. We have used β-galactosidase from Pisum sativum (PsBGAL) as model system to investigate stability, folding, and function relationship of β-galactosidases. PsBGAL is a vacuolar protein which has a tendency to multimerize at acidic pH with protein concentration ≥100 μg mL⁻¹ and dissociates into its subunits above neutral pH. It exhibits maximum activity as well as stability under acidic conditions. Further, it has different conformational orientations and core secondary structures at different pH. Substantial predominance of β-content and interfacial interactions through Trp residues play crucial role in pH-dependent multimerization of enzyme. Equilibrium unfolding of PsBGAL at acidic pH follows four-state model when monitored by changes in the secondary structure with two intermediates: one resembling to molten globule-like state while unfolding seen from activity and tertiary structure of PsBGAL fits to two-state model. Unfolding of PsBGAL at higher pH always follows two-state model. Furthermore, unfolding of PsBGAL reveals that it has at least two domains: α/β barrel containing catalytic site and the other is rich in β-content responsible for enzyme multimerization.

The effect of deglycosylation on the physiological and functional organization of milin was studied under different denaturizing conditions. Trifluoromethanesulfonic acid mediated deglycosylation resulted in insoluble milin, which was found to be soluble only in 1.5 M GuHCl with native-like folded structure. Kinetic stability, proteolytic activity, and dimeric association were lost in deglycosylated milin. Urea-induced unfolding revealed two inactive, highly stable equilibrium intermediates at pH 7.0 and pH 2.0. These intermediates were stable between 5.5-6.5 and 5.0-6.0 M total chaotropes (urea + 1.5 M GuHCl) at pH 7.0 and pH 2.0, respectively. GuHCl-induced unfolding was cooperative and noncoincidental with a broad transition range (2.0-5.0 M) at pH 7.0 and pH 2.0. Equilibrium unfolding of deglycosylated milin by urea and GuHCl substantiates the involvement of various inactive monomeric intermediates. This study provides a way to understand the role of glycosylation in the unfolding mechanism, stability, and functional activity of the serine protease milin.

A novel enzyme with endochitinase/lysozyme activity was purified to homogeneity from latex of Ipomoea carnea subsp. fistulosa using latex collection, gum removal, ammonium sulphate precipitation, hydrophobic interaction, and anion exchange chromatography. The enzyme was glycosylated (5–6%) and homogeneous on SDS-PAGE; has a molecular mass of 30.06 kDa (MALDI-TOF) and an isoelectric point of pH 4.6. The enzyme exhibited chitinase activity for hydrolysis of glycol chitin and the chitinolytic activity was significantly inhibited by allosamidin and mercuric chloride. The enzyme is stable in the pH range of 4.0–9.5, 80 °C and the optimal activity was observed at pH 5.5 and 50 °C. The enzyme consists of 8 tryptophan, 14 tyrosine, 6 cysteine residues forming three disulfide bridges and the extinction coefficient was estimated as 21.35 M −1 cm −1 . The polyclonal antibody was raised in rabbit and immunodiffusion suggests that the antigenic determinants are unique. The first 15 N-terminal residues G-E-I-T-I-Y-W-G-Q-N-G-F-E-G-S exhibited high identity to other known plant chitinases. Owing to the economic purification, high yield, unique and extraordinary features, stability and behavior; the enzyme ICChII can be widely employed in agriculture, industry, environmental protection, and in recycling chitinous waste from arthropod shellfish and for chito-oligosaccharide production.

Plant latex could be a potential source of novel proteases usable in the food and feed industries because of broad substrate specificity with high stability in extreme conditions. Crinumin, a glycosylated serine protease with chymotrypsin-like activity was purified from the latex of Crinum asiaticum using cation-exchange column chromatography. Crinumin shows activity over a wide range of pH (4.5–11.5 and optimum at 8.5), temperature (75 °C and optimum at 70 °C) and is also functional against chaotrophs, organic solvents, and detergents, even after prolonged exposure. The molecular mass (67.7 kDa), extinction coefficient (17.7), isoelectric point (6.9), and numbers of tryptophan (13), tyrosine (24) and cysteine (15 with 7 disulphide bridges) residues were estimated. K m of the enzyme was 31.7 μM with casein and 5 × 10 4 μM with N -succinyl- l -phenylalanine- p -nitroanilide. Easy availability of the aqueous latex, simple purification procedure, high yield (33%), stability and activity in adverse conditions makes it applicable for the pharmaceutical and food industries.

Kinetically stable homodimeric serine protease milin reveals high conformational stability against temperature, pH and chaotrope [urea, guanidine hydrochloride (GuHCl) and guanidine isothiocynate (GuSCN)] denaturation as probed by circular dichroism, fluorescence, differential scanning calorimetry and activity measurements. GuSCN induces complete unfolding in milin, whereas temperature, urea and GuHCl induce only partial unfolding even at low pH, through several intermediates with distinct characteristics. Some of these intermediates are partially active (viz. in urea and 2 M GuHCl at pH 7.0), and some exhibited strong ANS binding as well. All three tryptophans in the protein seem to be buried in a rigid, compact core as evident from intrinsic fluorescence measurements coupled to equilibrium unfolding experiments. The protein unfolds as a dimer, where the unfolding event precedes dimer dissociation as confirmed by hydrodynamic studies. The solution studies performed here along with previous biochemical characterization indicate that the protein has alpha-helix and beta-sheet rich regions or structural domains that unfold independently, and the monomer association is isologous. The complex unfolding pathway of milin and the intermediates has been characterized. The physical, physiological and probable therapeutic importance of the results has been discussed.

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.

A serine protease was purified to homogeneity from the latex of medicinal plant Ficus benghalensis by a single step procedure using anion exchange chromatography. The enzyme, named benghalensin, has a molecular mass of 47 kDa (MALDI-TOF and SDS-PAGE). The purified protein is a glycoprotein, and the enzymatic activity is solely inhibited by PMSF and chymostatin, indicating that the enzyme belongs to the serine protease class. The isoelectric point of the enzyme is pH 4.4 with optimum pH and temperature of pH 8.0 and 55 degrees C respectively. The extinction coefficient (epsilon(1%)(280)) of the enzyme is 29.25, and the molecular structure consists of 17 tryptophan, 31 tyrosine and 09 cysteine residues. Peptide mass fingerprinting and de novo sequencing of tryptic-digested fragments of the protein did not find any putative conserved domains in BLAST analysis. The enzyme is stable and retains full activity over a broad range of pH and temperature or prolonged storage at 4 degrees C. Simple purification, high yield and stability enable exploration of the protein for structure-function relationship studies as well as other applications.

Extraordinary optical properties, biocompatibility, and chemical stability make silver nanoparticles ideal candidate for application in bio-medical field. They are stabilized by virtue of their protein conjugation property. Here we report that in addition to protein conjugation nano silver also has excellent protein stabilization property. Formation of bioconjugate with nano silver was coupled with enhanced stabilization of protein secondary and tertiary structures, thereby retaining native conformation and preventing denaturation which is a rare phenomenon with other nanomaterials. This would be of immense significance for applications of conjugated peptides where the protein functions can be utilized. Using nano silver-BSA complex as model we have characterized optimal conditions for bioconjugation and mapped protein conformational changes ensued upon formation of conjugate. Conjugated BSA showed more resistance towards thermal denaturation as compared to its native counterpart. Our approach offers promise for designing of biologically functional, stable and controllable protein-silver nanoparticle composites.

Pepsin exists as alkaline denatured state (Ip) in pH range 8–10, where the N-terminal domain of the protein is mostly unfolded while the C-terminal domain is intact. The effects of fluorinated (TFE) and non-fluorinated (methanol) organic solvents on this partially unfolded state (Ip) of pepsin were investigated using various spectroscopic methods. Both, fluorinated (TFE) and non-fluorinated (methanol) organic solvents induce secondary structure (α-helix) after a critical concentration. The Ip state of pepsin unfolds in cooperative manner but the transition was found to be non-cooperative in the presence of 40% methanol or TFE. The differences in the unfolding of the protein in the presence and the absence of these organic solvents were interpreted. Our results indicate that unfolding transitions in Ip state are mostly dominated by unfolding of C-terminal domain because the N-terminal domain is largely unstructured in this state. The organic solvents (TFE and methanol) induce more secondary structure in N-terminal domain and make it another unfolding entity with different stability compare to C-terminal resulting into sequential unfolding of the domain.

A multi-functional enzyme ICChI with chitinase/lysozyme/exochitinase activity from the latex of Ipomoea carnea subsp. fistulosa was purified to homogeneity using ammonium sulphate precipitation, hydrophobic interaction and size exclusion chromatography. The enzyme is glycosylated (14-15%), has a molecular mass of 34.94 kDa (MALDI-TOF) and an isoelectric point of pH 5.3. The enzyme is stable in pH range 5.0-9.0, 80 degrees C and the optimal activity is observed at pH 6.0 and 60 degrees C. Using p-nitrophenyl-N-acetyl-beta-D-glucosaminide, the kinetic parameters K(m), V(max), K(cat) and specificity constant of the enzyme were calculated as 0.5mM, 2.5 x 10(-8)mol min(-1)microg enzyme(-1), 29.0 s(-1) and 58.0mM(-1)s(-1) respectively. The extinction coefficient was estimated as 20.56 M(-1)cm(-1). The protein contains eight tryptophan, 20 tyrosine and six cysteine residues forming three disulfide bridges. The polyclonal antibodies raised and immunodiffusion suggests that the antigenic determinants of ICChI are unique. The first fifteen N-terminal residues G-E-I-A-I-Y-W-G-Q-N-G-G-E-G-S exhibited considerable similarity to other known chitinases. Owing to these unique properties the reported enzyme would find applications in agricultural, pharmaceutical, biomedical and biotechnological fields.

Spectroscopic, calorimetric, and proteolytic methods were utilized to evaluate the stability of the kinetically stable, differentially glycosylated, dimeric serine protease milin as a function of pH (1.0–11.0), temperature, urea, and GuHCl denaturation in presence of 8 M urea at pH 2.0. The stability of milin remains equivalent to that of native at pH 1.0–11.0. However, negligible and reversible alteration in structure upon temperature transition has been observed at pH 2.0 and with 1.6 M GuHCl. Irreversible and incomplete calorimetric transition with apparent T m > 100°C was observed at basic pH (9.0 and 10.0). Urea-induced unfolding at pH 4.0, and at pH 2.0 with GuHCl, in presence of 8 M urea also reveals incomplete unfolding. Milin has been found to exhibit proteolytic resistant in either native or denatured state against various commercial proteases. These results imply that the high conformational stability of milin against various denaturating conditions enable its potential use in protease-based industries.

Wrightin, a serine protease from Wrightia tinctoria, has been used as model system to examine structure-function and stability. Our studies show high stability of the enzyme with major elements of secondary structure being beta-sheets. Under neutral conditions the enzyme is stable in 8 M urea and high temperature. GuHCl induced unfolding of wrightin at lower pH cannot be satisfactorily fit to a two state model for unfolding. Multiple intermediates were identified during unfolding of wrightin. Further, two intermediates, early and late are identified in the urea induced unfolding pathway at pH 3.0. Spectroscopic properties of intermediate states are analyzed and interpreted.

Milin, a potent molluscicide from the latex of Euphorbia milii, holds promise in medicinal biochemistry. Electrophoresis, size exclusion chromatography, mass spectrometry and other biochemical characteristics identify milin as a homodimeric, plant subtilisin-like serine protease, the first of its kind. The subunits of milin are differentially glycosylated affecting dimer association, solubility and proteolytic activity. The dimeric dissociation is SDS-insensitive and strongly temperature dependent but does not appear to be linked by disulfide bridges. N-terminal sequence of acid hydrolyzed peptide fragments shows no homology to known serine protease. Peptide mass fingerprinting and de novo sequencing of the tryptic fragments also did not identify putative domains in the protein. Milin seems to be a novel plant enzyme with subunit association partly similar to human herpes virus serine proteases and partly to penicillin binding proteins. Its behaviour on SDS-PAGE gels and other properties is like "kinetically stable" proteins. Such subunit association and properties might play a critical role in its physiological function and in controlling Schistosomiasis.

A novel heme peroxidase MGP from the latex of Ipomoea carnea subsp. fistulosa (morning glory) belonging to the Convolvulaceae family was purified to homogeneity using ammonium sulfate precipitation, anion exchange, hydrophobic interaction, and gel filtration chromatography. The enzyme is glycosylated and has a molecular mass of 42.06 kDa (MALDI-TOF) and an isoelectric point of pH 4.3. The enzyme has high yield, broad substrate specificity, and a high stability toward pH, temperature, chaotrophs, and organic solvents. The extinction coefficient (epsilon 280 (1%)) of the enzyme was estimated as 20.56 and it consists of 13 tryptophan, 9 tyrosine, and 8 cysteine residues forming 4 disulfide bridges. There is significant effect of inhibitors targeting S-S bridges (mercaptoethanol, l-cysteine, glutathione), as well as of inhibitors targeting heme (sodium azide and hydroxylamine) on peroxidase activity, whereas inhibition was not observed with ethylmaleinimide due to the absence of reduced cysteine in the enzyme. Polyclonal antibodies against the enzyme have been raised in rabbit, and immunodiffusion suggests that the antigenic determinants of MGP are unique. The N-terminal sequence of MGP (D-E-A-C-I-F-S-A-V-K-E-V-V-D-A) exhibited considerable similarity to the sequence of other known plant peroxidases. Spectroscopic studies (absorbance, fluorescence, and circular dichroism) reveal that MGP has secondary structural features with alpha/beta type with approximately 20% alpha-helicity.

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.

Euphorbian latex is commonly used as molluscicides and the Euphorbia milii latex was reported as most powerful molluscicidal agents. The physiological and lethal effects of the latex components of Euphorbia milii, on the intermediate host Biomphalaria spp., of the human liver parasite Schistosoma mansoni were described in this study. The standard methodologies for testing plant derived molluscicides formulated by World Health Organisation (WHO) were followed with some modifications. The young specimen of fresh water snails showed altered physiological and physical response towards latex components. The working concentration of non-proteinaceous fraction (up to 0.1%) of the latex reduced the active physiological behaviour but was non-lethal to young specimen of snails. However, proteinaceous fractions (0.1 mg/l) of the latex were found lethal to snail population, and lethality was enhanced with small amount of the non-proteinaceous fraction (0.01%) of the latex. Milin, a serine protease(up to 0.1 mg/l), isolated from the latex of Euphorbia milii significantly reduced the growth and feeding activity but was not lethal to young specimen of snails. With an addition of 0.01% of non-proteinaceous fractions to Milin, lethality result was similar to that of crude latex. Milin is likely to be responsible for alteration of normal physiological functions and lethality of snails, thus it may be used as a molluscicide to control transmission of the endemic disease schistosomiasis.

Ervatamin A, a cysteine proteases from Ervatamia coronaria, has been used as model system to examine structure-function relationship by equilibrium unfolding methods. Ervatamin A belongs to alpha+beta class of proteins and exhibit stability towards temperature and chemical denaturants. Acid induced unfolding of ervatamin A was incomplete with respect to the structural content of the enzyme. Between pH 0.5 and 2.0, the enzyme is predominantly in beta-sheet conformation and shows a strong ANS binding suggesting the existence of a partially unfolded intermediate state (I(A) state). Surprisingly, high concentrations of GuHCl required to unfold this state and the transition mid points GuHCl induced unfolding curves are significantly higher. GuHCl induced unfolding of ervatamin A at pH 3.0 as well as at pH 4.0 is complex and cannot be satisfactorily fit to a two-state model for unfolding. Besides, a strong ANS binding to the protein is observed at low concentration of GuHCl, indicating the presence of intermediate in the unfolding pathway. On the other hand, even in the presence of urea (8M) the enzyme retains all the activity as well as structural parameters at neutral pH. However, the protein is susceptible to urea unfolding at pH 3.0 and below. Urea induced unfolding of ervatamin A at pH 3.0 is cooperative and the transitions curves obtained by different probes are and non-coincidental. Temperature denaturation of ervatamin A in I(A) state is non-cooperative, contrary to the cooperativity seen with native protein, suggesting the presence of two parts in the molecular structure of ervatamin A may be domains, with different stability that unfolds in steps. Careful inspection of biophysical properties of intermediate states populated in urea and GuHCl (I(UG) state) induced unfolding suggests all these three intermediates are identical and populated in different conditions. However, the properties of the intermediate (I(A) state) identified at pH approximately 1.5 are different from those of the I(UG) state.

pH and chemical denaturant dependent conformational changes of a serine protease cryptolepain from Cryptolepis buchanani are presented in this paper. Activity measurements, near UV, far UV CD, fluorescence emission spectroscopy, and ANS binding studies have been carried out to understand the folding mechanism of the protein in the presence of denaturants. pH and chemical denaturants have a marked effect on the stability, structure, and function of many globular proteins due to their ability to influence the electrostatic interactions. The preliminary biophysical study on cryptolepain shows that major elements of secondary structure are beta-sheets. Under neutral conditions the enzyme was stable in urea while GuHCl-induced equilibrium unfolding was cooperative. Cryptolepain shows little ANS binding even under neutral conditions due to more hydrophobicity of beta-sheets. Multiple intermediates were populated during the pH-induced unfolding of cryptolepain. Temperature-induced denaturation of cryptolepain in the molten globule like state is non-cooperative, contrary to the cooperativity seen with the native protein, suggesting the presence of two parts, possibly domains, in the molecular structure of cryptolepain, with different stability that unfolds in steps. Interestingly, the GuHCl-induced unfolding of A state (molten globule state) of cryptolepain is unique, as lower concentration of denaturant, not only induces structure but also facilitate transition from one molten globule like state (MG(1)) into another (MG(2)). The increase of pH drives the protein into alkaline denatured state characterized by the absence of any ANS binding. GuHCl- and urea-induced unfolding transition curves at pH 12.0 were non-coincidental indicating the presence of an intermediate in the unfolding pathway.

Cryptolepain is a stable glycosylated novel serine protease purified from the latex of the medicinally important plant Cryptolepis buchanani. The molecular weight of the enzyme is 50.5 kDa, as determined by mass spectrometry. The sequence of the first 15 N-terminal resides of the protease showed little homology with those of other plant serine proteases, suggesting it to be structurally unique. Thus, it is of interest to solve the structure of the enzyme in order to better understand its structure-function relationship. X-ray diffraction data were collected from a crystal of cryptolepain and processed to 2.25 A with acceptable statistics. The crystals belong to the orthorhombic space group C222(1), with unit-cell parameters a = 81.78, b = 108.15, c = 119.86 A. The Matthews coefficient was 2.62 A(3) Da(-1) with one molecule in the asymmetric unit. The solvent content was found to be 53%. Structure determination of the enzyme is under way.

Structural and functional characteristics of jack bean urease (JBU), a hexameric enzyme having identical subunits, were investigated under neutral as well as acidic conditions by using CD, fluorescence, ANS binding and enzyme activity measurements. At low pH and low ionic strength, JBU exists in a partially unfolded state (U(A)-state), having predominantly beta structure and no tertiary interactions along with a strong ANS binding. Addition of salts like NaCl, KCl and Na(2)SO(4) to the U(A)-state induces refolding resulting in structural propensities similar to that of native hexamer. Moreover, at low concentrations, GuHCl behaves like an anion by inducing refolding of the U(A)-state. The anion-induced refolded state (I(A)-state) is more stable than U(A)-state and the stability is nearly equal to that of the native protein against chemical-induced and thermal denaturation. Overall, these observations support a model of protein folding for a multimeric protein where certain conformations (ensembles of substates) of low energy prevail and populated under non-native conditions with different stability.

Deciphering the code that determines the three-dimensional structure of proteins and the ability to predict the final folded form of a protein is still elusive to molecular biophysists. In the case of several proteins a similar tertiary structure is not accompanied by any significant sequence similarity. The question now remains whether a code beyond the genetic code that describes the arrangement of the amino acid within a three dimensional protein structure. The available data undoubtedly demonstrates that the redundancy of this code must be tremendous. Several techniques such as nuclear magnetic resonance spectroscopy and laser detection techniques, coupled with fast initiation of the folding reaction, can now probe the folding events in milliseconds or even faster and provide highly relevant information. The thermodynamic analysis of the folding process and of kinetic intermediates opens whole new avenue of understanding. Breaking the protein folding code would enable scientists to look at a gene whose function is unknown and predict the three-dimensional structure of the protein it encodes. This would give them a very good idea of what the gene does. In this review we hope to bring together the information available about protein folding with particular emphasis on folding intermediate(s). Additionally, the practical consequences of the solution of the protein folding problem in medicine and biotechnology are also discussed.

Ervatamin C is an unusually stable cysteine protease from the medicinal plant Ervatamia coronaria belonging to the papain family. Though it cleaves denatured natural proteins with high specific activity, its activity toward some small synthetic substrates is found to be insignificant. The three-dimensional structure and amino acid sequence of the protein have been determined from X-ray diffraction data at 1.9 A (R = 17.7% and R(free) = 19.0%). The overall structure of ervatamin C is similar to those of other homologous cysteine proteases of the family, folding into two distinct left and right domains separated by an active site cleft. However, substitution of a few amino acid residues, which are conserved in the other members of the family, has been observed in both the domains and also at the region of the interdomain cleft. Consequently, the number of intra- and interdomain hydrogen-bonding interactions is enhanced in the structure of ervatamin C. Moreover, a unique disulfide bond has been identified in the right domain of the structure, in addition to the three conserved disulfide bridges present in the papain family. All these factors contribute to an increase in the stability of ervatamin C. In this enzyme, the nature of the S2 subsite, which is the primary determinant of specificity of these proteases, is similar to that of papain, but at the S3 subsite, Ala67 replaces an aromatic residue, and has the effect of eliminating sufficient hydrophobic interactions required for S3-P3 stabilization. This provides the possible explanation for the lower activity of ervatamin C toward the small substrate/inhibitor. This substitution, however, does not affect the binding of denatured natural protein substrates to the enzyme significantly, as there exist a number of additional interactions at the enzyme-substrate interface outside the active site cleft.

The sodium dodecyl sulfate (SDS)-induced conformational and functional changes of ervatamin B were monitored by optical methods (circular dichroism (CD), fluorescence), 8-anilino-1-napthalenesulfonic acid (ANS) binding and proteolytic activity. An enhancement in the α-helicity of protein molecule is observed as a function of increasing concentration of SDS along with a blue shift in the fluorescence emission maximum and a decrease in fluorescence intensity. At a certain concentration of SDS (≈2 mM) ervatamin B loses all the proteolytic activity and rigid tertiary structure but possesses considerable amount of secondary structure along with strong ANS binding, indicating the presence of an intermediate state with different conformation from the native and unfolded states. This state may be the SDS-induced molten globule state seen in the case of ervatamin B. The SDS-induced state is more stable then the native state towards chemical and thermal denaturation and shows biphasic transition curves as compared with the native state in the absence of the detergent where the transitions are cooperative. Further, the cooperative unfolding transition curve of ervatamin B in the absence of SDS intersects at the point where the second transition of SDS-induced state starts suggesting that the molecule of ervatamin B consist of at least two structural domains which are stabilized differentially in presence of SDS and thus unfolds sequentially. As the α-helicity of ervatamin B enhances in the presence of SDS, it can be postulated that the α-rich domain of the protein was stabilized due to interaction with SDS, and therefore, it may unfold later as compared with β-rich domain during temperature and chemical induced denaturation. Thus, the study of equilibrium unfolding pathway of ervatamin B in the presence of SDS provides a better understanding about the stability of this plant cysteine protease at the domain level.

The structural and functional aspects along with equilibrium unfolding of procerain, a cysteine protease from Calotropis procera, were studied in solution. The energetic parameters and conformational stability of procerain in different states were also estimated and interpreted. Procerain belongs to the alpha + beta class of proteins. At pH 2.0, procerain exists in a partially unfolded state with characteristics of a molten globule-like state, and the protein is predominantly a beta-sheet conformation and exhibits strong ANS binding. GuHCl and temperature denaturation of procerain in the molten globule-like state is noncooperative, contrary to the cooperativity seen with the native protein, suggesting the presence of two parts in the molecular structure of procerain, possibly domains, with different stability that unfolds in steps. Moreover, tryptophan quenching studies suggested the exposure of aromatic residues to solvent in this state. At lower pH, procerain unfolds to the acid-unfolded state, and a further decrease in the pH drives the protein to the A state. The presence of 0.5 M salt in the solvent composition directs the transition to the A state while bypassing the acid-unfolded state. GuHCl-induced unfolding of procerain at pH 3.0 seen by various methods is cooperative, but the transitions are noncoincidental. Besides, a strong ANS binding to the protein is observed at low concentrations of GuHCl, indicating the presence of an intermediate in the unfolding pathway. On the other hand, even in the presence of urea (8 M), procerain retains all the activity as well as structural parameters at neutral pH. However, the protein is susceptible to unfolding by urea at lower pH, and the transitions are cooperative and coincidental. Further, the properties of the molten globule-like state and the intermediate state are different, but both states have the same conformational stability. This indicates that these intermediates may be located on parallel folding routes of procerain.