Browsing by Author "M.V. Jagannadham"

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A kinetically stable plant subtilase with unique peptide mass fingerprints and dimerization properties
(2009) Subhash Chandra Yadav; M.V. Jagannadham; Suman Kundu; Medicherla V. Jagannadham
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. © 2008 Elsevier B.V. All rights reserved.
A stable serine protease, wrightin, from the latex of the plant Wrightia tinctoria (Roxb.) R. Br.: Purification and biochemical properties
(2008) Ritu Tomar; Reetesh Kumar; M.V. Jagannadham
Today proteases have become an integral part of the food and feed industry, and plant latex could be a potential source of novel proteases with unique substrate specificities and biochemical properties. A new protease named "wrightin" is purified from the latex of the plant Wrightia tinctoria (Family Apocynaceae) by cation-exchange chromatography. The enzyme is a monomer having a molecular mass of 57.9 kDa (MALDI-TOF), an isoelectric point of 6.0, and an extinction coefficient (ε1%280) of 36.4. Optimum activity is achieved at a pH of 7.5-10 and a temperature of 70°C. Wrightin hydrolyzes denatured natural substrates such as casein, azoalbumin, and hemoglobin with high specific activity; for example, the Km value is 50 μM for casein as substrate. Wrightin showed weak amidolytic activity toward L-Ala-Ala-p-nitroanilide but completely failed to hydrolyze N-α-benzoyl-DL-arginine-p-nitroanilide (BAPNA), a preferred substrate for trypsin-like enzymes. Complete inhibition of enzyme activity by serine protease inhibitors such as PMSF and DFP indicates that the enzyme belongs to the serine protease class. The enzyme was not inhibited by SBTI and resists autodigestion. Wrightin is remarkably thermostable, retaining complete activity at 70°C after 60 min of incubation and 74% of activity after 30 min of incubation at 80°. Besides, the enzyme is very stable over a broad range of pH from 5.0 to 11.5 and remains active in the presence of various denaturants, surfactants, organic solvents, and metal ions. Thus, wrightin might be a potential candidate for various applications in the food and biotechnological industries, especially in operations requiring high temperatures. © 2008 American Chemical Society.
Accumulation of partly folded states in the equilibrium unfolding of ervatamin A: Spectroscopic description of the native, intermediate, and unfolded states
(2007) Sreedevi Nallamsetty; Vikash K. Dubey; Monu Pande; P.K. Ambasht; M.V. Jagannadham
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 α+β 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 β-sheet conformation and shows a strong ANS binding suggesting the existence of a partially unfolded intermediate state (IA 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 (8 M) 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 IA 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 (IUG state) induced unfolding suggests all these three intermediates are identical and populated in different conditions. However, the properties of the intermediate (IA state) identified at pH ∼1.5 are different from those of the IUG state. © 2007 Elsevier Masson SAS. All rights reserved.
Anion-induced folding of rabbit muscle pyruvate kinase: Existence of multiple intermediate conformations at low pH
(Academic Press Inc., 2000) F. Edwin; M.V. Jagannadham
Structural and functional characteristics of rabbit muscle pyruvate kinase (PK), a tetrameric enzyme having identical subunits, were investigated under neutral as well as acidic conditions by using enzymatic activity measurements and a combination of optical methods, such as circular dichroism, fluorescence, and ANS binding. At low pH and low ionic strength, pyruvate kinase exists in a partially unfolded state (U(A) state) retaining half of the secondary structure and no tertiary interactions along with a strong binding to the hydrophobic dye, ANS. Addition of anions, like NaCl, KCl, and Na2SO4, to the acid-unfolded state induces refolding, resulting structural propensities similar to that of native tetramer. When anion concentration exceeds a critical limit (0.7 M KCl), a sudden loss of secondary structure and decrease in fluorescence intensity with a redshift in the emission maximum are seen which may be due to the aggregation of the protein, probably due to the intermolecular association. The anion-refolded state is more stable titan the U(A) state, and its stability is nearly equal to that of native protein toward chemical-induced unfolding by Gu-HCl and urea. Moreover, at low concentrations, Gu-HCl behaves like an anion, by inducing refolding of the acid-unfolded state with structural features equivalent to that of native molecule. These observations support a model of protein folding where certain conformations of low free energy prevail and are populated under non-native conditions with different stability. (C) 2000 Academic Press.
Benghalensin, a highly stable serine protease from the latex of medicinal plant Ficus benghalensis
(2009) Anurag Sharma; Moni Kumari; M.V. Jagannadham
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 °C respectively. The extinction coefficient (ε1% 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°C. Simple purification, high yield and stability enable exploration of the protein for structure-function relationship studies as well as other applications. ©2009 American Chemical Society.
Biophysical characterization and folding studies of plant protease, wrightin: Identification of folding intermediate under different conditions
(Springer New York, 2009) Ritu Tomar; Vikash Kumar Dubey; M.V. Jagannadham
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 β-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. © 2009 Springer Science+Business Media, LLC.
Crystallization and preliminary X-ray analysis of crinumin, a chymotrypsin-like glycosylated serine protease with thrombolytic and antiplatelet activity
(2011) Kunwar Awaneesh Singh; M.V. Jagannadham; G.R.K. Rao; Patrick H. N. Celie
Crinumin, a novel glycosylated serine protease with chymotrypsin-like catalytic specificity, was purified from the medicinally important plant Crinum asiaticum. Crinumin is a 67.7 kDa protease with an extraordinary stability and activity over a wide range of pH and temperature and is functional in aqueous, organic and chaotropic solutions. The purified protease has thrombolytic and antiplatelet activity. The use of C. asiaticum extracts has also been reported for the treatment of a variety of disorders such as injury, joint inflammation and arthritis. In order to understand its structure-function relationship, the enzyme was purified from the plant latex and crystallized by the hanging-drop vapour-diffusion method. X - ray diffraction data were collected from a single crystal and processed to 2.8 Å resolution. The crystal belonged to the monoclinic space group C2, with unit-cell parameters a = 121.61, b = 95.00, c = 72.10 Å, = = 90, β = 114.19°. The Matthews coefficient was 2.81 Å 3 Da -1, corresponding to a solvent content of 56%, assuming one molecule in the asymmetric unit. Structure determination of the enzyme is in progress. © 2011 International Union of Crystallography. All rights reserved.
Deciphering the molecular structure of cryptolepain in organic solvents
(2012) N.K. Prasanna Kumari; M.V. Jagannadham
Solvent composition plays a major role in stabilizing/destabilizing the forces that are responsible for the native structure of a protein. Often, the solvent composition drives the protein into non-native conformations. Elucidation of such non-native structures provides valuable information about the molecular structure of the protein, which is unavailable otherwise. Inclusion of methanol (non-fluorinated alcohol) or TFE (fluorinated alcohol) in the solvent composition drove cryptolepain, a serine protease and an all-β-protein, into a non-native structure with an enhanced β-sheet or induction of α-helix. These solvents did not much affect cryptolepain under neutral conditions, even at higher concentrations, but the effects were predominant at lower pH, when the protein molecule is under stress. The organic solvent-induced state is partially unfolded with similar characteristics to the molten globule state seen with protein under a variety of conditions. Chemical- or temperature-induced unfolding of cryptolepain in the presence of organic solvent is distinctly different from that in the absence of organic solvent. Such different unfolding provided evidence of two structural variants in the molecular structure of the protein as well as the differential stabilization/destabilization of such structural variants and their sequential unfolding. © 2011 Elsevier Masson SAS. All rights reserved.
Decolorization of crude latex by activated charcoal, purification and physico-chemical characterization of religiosin, a milk-clotting serine protease from the latex of ficus religiosa
(2010) Moni Kumari; Anurag Sharma; M.V. Jagannadham
The crude latex of Flcus religiosa is decolorized by activated charcoal. Decolorization follows the Freundlich and Langmuir equations. A serine protease, named religiosin, has been purified to homogeneity from the decolorized latex using anion exchange chromatography. Religiosin is a glycoprotein with a molecular mass of 43.4 kDa by MALDI-TOF. Religiosin is an acidic protein with a p/value of 3.8 and acts optimally at pH 8.0-8.5 and temperature 50 °C. The proteolytic activity of religiosin is strongly inhibited by PMSF and chymostatin indicating that the enzyme is a serine protease. The extinction coefficient (ε1%280) of religiosin is 29.47 M-1 cm -1with 16 tryptophan, 26 tyrosine, and 11 cysteine residues per molecule. The enzyme shows broad substrate specificity against natural as well as synthetic substrates with an apparent Km of 0.066 mM and 6.25 mM using casein and Leu-pNA, respectively. MS/MS analysis confirms the novelty of the enzyme. Religiosin is highly stable against denaturants, metal ions, and detergents as well as over a wide range of pH and temperature. In addition, the enzyme exhibits milk-clotting as well as detergent activity. © 2010 American Chemical Society.
Differences in the Unfolding of Procerain Induced by pH, Guanidine Hydrochloride, Urea, and Temperature
(2003) Vikash Kumar Dubey; M.V. Jagannadham
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 α + β 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 β-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.
Effect of alkyl alcohols on partially unfolded state of Proteinase K: Differential stability of α-helix and β-sheet rich regions of the enzyme
(2009) Ritu Tomar; Vikash Kumar Dubey; M.V. Jagannadham
Proteinase K (E.C. 3.4.21.64), a serine proteinase from fungus Tritirachium album, has been used as a model system to investigate the conformational changes induced by monohydric alcohols at low pH. Proteinase K belongs to α/β class of proteins and maintains structural integrity in the range of pH 7.0-3.0. Enzyme acquires partially unfolded conformation (UP) at pH 2.5 with lower activity, partial loss of tertiary structure and exposure of some hydrophobic patches. Proteinase K in stressed state at pH 2.5 is chosen and the conformational changes induced by alkyl alcohols (methanol/ethanol/isopropanol) are studied. At critical concentration of alcohol, conformational switch occurs in the protein structure from α/β to β-sheet driving the protein into O-state. Complete loss of tertiary contacts and proteolytic activity in O-sate emphasize the involvement of alpha regions in maintaining the active site of the enzyme. Moreover, isopropanol induced unfolding of proteinase K in UP state occurred in two steps with the formation of β state at low alcohol concentration followed by stabilization of β state at high alcohol concentration. GuHCl and temperature induced unfolding of proteinase K in O-state (in 50% isopropanol) is non-cooperative as the transition curves are biphasic. This suggests that the structure of proteinase K in O-state has melted alpha regions and stabilized beta regions and that these differentially stabilized regions unfold sequentially. Further, the O-state of proteinase K can be attained from complete unfolded protein by the addition of 50% isopropanol. Hence the alcohol-induced O-state is different from native state or completely unfolded state and shows characteristics of the molten globule-like state. Thus, this state may be functioning as an intermediary in the folding pathway of proteinase K. © 2009 Elsevier Masson SAS. All rights reserved.
Evidence for a molten globule state in Cicer α-galactosidase induced by pH, temperature, and guanidine hydrochloride
(2013) Neelesh Singh; Reetesh Kumar; M.V. Jagannadham; Arvind M. Kayastha
Physiologically as well as industrially, α-galactosidases are very important enzymes, but very little is known about the stability and folding aspect of enzyme. In the present study, we have investigated the temperature, pH, and guanidine hydrochloride (GuHCl) induced unfolding of Cicer α-galactosidase using circular dichroism and fluorescence spectroscopy. Strong negative ellipticities at 208, 215, and 222 nm indicate the presence of both α and β structures in Cicer α-galactosidase and showed that its secondary structure belongs to α+β class of proteins with 31 % α-helicity. For Cicer α-galactosidase the emission maximum was found to be 345 nm which suggests that tryptophan residues are less exposed to solvent. However, at pH2.0, protein showed blue-shift. This state of protein lacked activity but it retained significant secondary structure. Enhanced binding of ANS at pH2.0 indicated significant unfolding and exposure of hydrophobic regions. The unfolded state of Cicer α-galactosidase showed a red-shift of 15 nm with a concomitant decrease in the fluorescence intensity. The enzyme maintained its native structure and full activity up to 40°C; however, above this temperature, denaturation was observed. © 2013 Springer Science+Business Media.
Highly stable glycosylated serine protease from the medicinal plant Euphorbia milii
(2006) Subhash C. Yadav; Monu Pande; M.V. Jagannadham
A serine protease, named as "Milin" was purified to homogeneity from the latex of Euphorbia milii, a medicinal plant of Euphorbiaceae family. The molecular mass (SDS-PAGE), optimum pH and temperature of the enzyme were 51 kDa, pH 8.0 and 60 °C, respectively. Milin retains full proteolytic activity over a wide range of pH (5.5-12) and temperature (up to 65 °C) with casein and azoalbumin as substrates. The activity of milin is inhibited by serine proteases inhibitors like PMSF, APMSF and DFP, but not by any other protease inhibitors such as E-64 and PCMB. Like the other serine proteases from the genus Euphorbia, the activity of milin was not inhibited by the proteinaceous inhibitor soyabean trypsin inhibitor (SBTI) even at very high concentrations that is naturally present in plants. The specific extinction coefficient (ε280 nm1 %), molar extinction coefficient (am) and isoelectric point of the enzyme were found to be 29, 152,500 M-1 cm-1 and pH 7.2, respectively. The enzyme is a glycoprotein with detectable carbohydrate moiety (7-8%) in its constitution, which is essential for the activity. The numbers of tryptophan, tyrosine and cysteine residues in the sequence of milin were estimated chemically and are 23, 14 and 14, respectively. Of the 14-cysteine residues, 12 constituted 6-disulfide linkages while two are free cysteines. The N-terminal sequence (first 12 amino acid residues) was determined and does not match with any sequence of known plant serine proteases. Perturbation studies by temperature, pH and chaotropes of the enzyme also reveal its high stability as seen by CD, fluorescence and proteolytic activity. Thus, this serine protease may have potential applications in food industry. © 2006 Elsevier Ltd. All rights reserved.
Identification of a novel pulmonary oedema producing toxin from Indian red scorpion (Mesobuthus tamulus) venom
(Elsevier Ltd, 2005) S.B. Deshpande; A.B. Alex; M.V. Jagannadham; G.R.K. Rao; A.K. Tiwari
The experiments were conducted to identify the toxin that produces pulmonary oedema in Mesobuthus tamulus (BT) envenomed animals. Crude BT venom was subjected to Sephadex gel filtration (G-75) and the fractions were screened for optical density (OD), neurotoxicity (prolongation of compound action potential in frog sciatic nerve) and lethality. All these parameters exhibited a peak between 54-94 ml eluates. Fractions of this peak were pooled (SP) and loaded on to carboxymethyl cellulose column. The column was then eluted with increasing buffer concentrations at constant pH and temperature. Eluates were screened for neurotoxicity and OD. Four peaks of neurotoxic activity (T1-T4) were detected. T2 and T3 were lethal whereas T1 and T4 were non-lethal. T2 exhibited mainly neurotoxicity and failed to augment phenyldiguanide (PDG)-induced reflex response or to produce pulmonary oedema. T3 was having minimal neurotoxic actions but augmented PDG-reflex and produced pulmonary oedema. The effects of T3 persisted even after dialysis with 8 kDa cut-off filter but not those of T2. The T3 effects resembled toxic manifestations of BT venom and were blocked by aprotinin pre-treatment. T3 demonstrated a band at ∼100 kDa in SDS-PAGE. The results demonstrate the presence of a lethal, high molecular weight, pulmonary oedema producing toxin in BT venom. © 2005 Elsevier Ltd. All rights reserved.
Modeled structure of trypanothione reductase of Leishmania infantum
(The Biochemical Society of the Republic of Korea, 2008) Bishal K. Singh; Nandini Sarkar; M.V. Jagannadham; Vikash K. Dubey
Trypanothione reductase is an important target enzyme for structure-based drug design against Leishmania. We used homology modeling to construct a three-dimensional structure of the trypanothione reductase (TR) of Leishmania infantum. The structure shows acceptable Ramachandran statistics and a remarkably different active site from glutathione reductase(GR). Thus, a specific inhibitor against TR can be designed without interfering with host (human) GR activity.
N-terminal domain unfolds first in the sequential unfolding of papain
(2003) Yagya Valkya Sharma; M.V. Jagannadham
Temperature and Guanidine hydrochloride induced unfolding transitions of papain at pH 2.0 are biphasic implying independent and sequential unfolding of its two domains. To determine the order of unfolding, the active site located in the interface of the domains was labeled with an environment specific fluorescent probe (1,8-IAEDANS). Unfolding of this complex relative to the free protein followed by intrinsic and extrinsic fluorescence measurements suggests that the N domain unfolds initially in the sequential unfolding of domains.
Neriifolin S, a dimeric serine protease from Euphorbia neriifolia Linn.: Purification and biochemical characterisation
(2012) Ravi Prakash Yadav; Ashok Kumar Patel; M.V. Jagannadham
A dimeric serine protease Neriifolin S of molecular mass 94 kDa 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 (K m of 0.195 mM) and physiological efficiency (K cat/K m of 144.5 mM s). 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 T m, 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 27.54 kJ mol -1, 24.89 kJ mol -1, -82.34 kJ mol -1 and 337.20 J mol -1 K -1. © 2011 Elsevier Ltd. All rights reserved.
Physiological changes and molluscicidal effects of crude latex and Milin on Biomphalaria glabrata
(2008) Subhash C. Yadav; M.V. Jagannadham
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. © 2007 Elsevier Ltd. All rights reserved.
Procerain, a stable cysteine protease from the latex of Calotropis procera
(Elsevier Ltd, 2003) Vikash Kumar Dubey; M.V. Jagannadham
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 °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 L-Ala-Ala-p-nitroanilide, L-Ala p-nitroanilide and N-d-Benzoyl-DL-Arg-p-nitroanilide and appears to be peptide length dependent. The extinction coefficient (ε280nm1%) 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 °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. © 2003 Elsevier Science Ltd. All rights reserved.
Purification and biochemical characterization of a chymotrypsin-like serine protease from Euphorbia neriifolia Linn.
(2011) Ravi Prakash Yadav; Ashok Kumar Patel; M.V. Jagannadham
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 (280nm1%) 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. © 2011 Elsevier Ltd.