Debasish Bhattacharyya

Haemorrhagic protein of Russell's viper venom with fibrinolytic and esterolytic activities.

A haemorrhagic toxin specifically active on skin and muscle at the site of introduction in mice has been purified from Vipera russelli russelli (Indian subspecies of Russells viper) venom by CM-Sephadex C-50 ion exchange chromatography and size exclusion (SE)-HPLC. This toxic protein also has strong fibrinolytic and arginine esterolytic activities. The purified preparation was a single polypeptide chain of molecular weight 73,000, as revealed by SDS-PAGE and SE-HPLC under native and denatured conditions. It has been named as VRR-73. Atomic absorption spectrometry indicated the existence of Mg(2+) in a mol per mol ratio. Antiserum was effective in neutralizing haemorrhage when administered immediately following VRR-73 but was ineffective in inhibiting fibrinolytic and esterolytic activities. On the other hand phenylmethyl sulphonyl fluoride and EDTA inhibited fibrinolysis and esterolysis but did not affect haemorrhage. Thermal denaturation of VRR-73, after exposure at 100 degrees C for 10 min, led to inactivation of all of its activities. Fibrinolytic and esterolytic activities, but not the haemorrhagic activity, were slowly regained after cooling at 25 degrees C. Thus the two pathological activities of VRR-73 appear to be associated with two different regions of the molecule.

Spectroscopic and chromatographic evidences of NADPH in human placental extract used as wound healer.

An aqueous extract of human placenta, which is used as wound healer, has been investigated in terms of fluorescence properties. When excited at 340 nm, it results in fluorescence emission having maxima around 436 nm, which is fairly specific for nicotinamide adenine dinucleotide, reduced form (NADH) and nicotinamide adenine dinucleotide phosphate, reduced form (NADPH). The excitation spectra, having emission at 440 nm, show patterns comparable to these nucleotides. Thin layer chromatography and reversed-phase (RP) HPLC confirm presence of only NADPH in the extract. The emission and excitation patterns of NADPH purified after HPLC resemble exactly with the reference compound. Its content has been estimated to be 0.018 +/- 0.003 mg/ml based on fluorescence emission with respect to a standard calibration curve (n=6). Biological functionality of NADPH in the extract has been confirmed by glutathione reductase assay (n=5).

Purification and partial characterization of a haemorrhagin (VRH-1) from Vipera russelli russelli venom

A haemorrhagic toxin (VRH-1) has been purified to homogeneity from Vipera russelli russelli venom by subjecting it to chromatography twice successively on CM-Sephadex C-50. It is a protein of mol. wt 22,000 and contains one mole of Mg2+. Intradermal administration of this haemorrhagin in mice resulted in severe lung haemorrhage but produced little haemorrhage in skin. This apparent organ preference led us to develop a new haemorrhage assay method utilizing dye diffusion from lung in vitro. Proteolytic activity of VRH-1 was demonstrated using dimethylcasein as substrate following quantitation by reaction with trinitrobenzoyl sulfonic acid. Both haemorrhagic and proteolytic activities of VRH-1 were inhibited by serine protease inhibitors like phenylmethyl sulfonyl fluoride and chymostatin, but metal chelators had no effect. Lung haemorrhage is unlikely to be a direct reflection of a high local concentration of VRH-1. The administration of supernatant generated by incubation of chopped liver from untreated mouse and VRH-1 (in subhaemorrhagic dose) results in severe lung haemorrhage. This raises the possibility that VRH-1 leads to the formation of intermediate(s) which causes the haemorrhage.

Two L-amino acid oxidase isoenzymes from Russell’s viper (Daboia russelli russelli) venom with different mechanisms of inhibition by substrate analogs

Two isoforms, L1 and L2, of l‐amino acid oxidase have been isolated from Russell’s viper venom by Sephadex G‐100 gel filtration followed by CM‐Sephadex C‐50 ion exchange chromatography. The enzymes, with different isoelectric points, are monomers of 60–63 kDa as observed from size exclusion HPLC and SDS/PAGE. Partial N‐terminal amino acid sequencing of L1 and L2 showed significant homology with other snake venom l‐amino acid oxidases. Both the enzymes exhibit marked substrate preference for hydrophobic amino acids, maximum catalytic efficiency being observed with l‐Phe. Inhibition of L1 and L2 by the substrate analogs N‐acetyltryptophan and N‐acetyl‐l‐tryptophan amide has been followed. The initial uncompetitive inhibition of L1 followed by mixed inhibition at higher concentrations suggested the existence of two different inhibitor‐binding sites distinct from the substrate‐binding site. In the case of L2, initial linear competitive inhibition followed by mixed inhibition suggested the existence of two nonoverlapping inhibitor‐binding sites, one of which is the substrate‐binding site. An inhibition kinetic study with O‐aminobenzoic acid, a mimicking substrate with amino, carboxylate and hydrophobic parts, indicated the presence of three and two binding sites in L1 and L2, respectively, including one at the substrate‐binding site. An inhibitor cross‐competition kinetic study indicated mutually excluding binding between N‐acetyltryptophan, N‐acetyl‐l‐tryptophan amide and O‐aminobenzoic acid in both the isoforms, except at the substrate‐binding site of L1. Binding of substrate analogs with different electrostatic and hydrophobic properties provides useful insights into the environment of the catalytic sites. Furthermore, it predicts the minimum structural requirement for a ligand to enter and anchor at the respective functional sites of LAAO that may facilitate the design of suicidal inhibitors.

Resistance of bromelain to SDS binding

Interaction of the plant cysteine protease bromelain with SDS has been studied using CD spectroscopy, intrinsic fluorescence emission, extrinsic fluorescence probe pyrene, isothermal calorimetric (ITC) investigations and inhibition of hydrolyzing activity. Results exhibit number of synchronous transitions when plotted against the total SDS concentration. SDS at submicellar level caused conformation change of bromelain leading to a stable entity. ITC and pyrene experiments suggest that the structural modifications below 5 mM, the cmc(app) of SDS solutions containing bromelain, are the result of alterations of solvent hydrophobicity or non-specific weak binding and/or adsorption of SDS monomers. Melting temperature (T(m)) and the free energy change for thermal unfolding (DeltaG(unf)) of the SDS induced conformers was decreased by 5 degrees C and 0.5 kcal/mol respectively, compared to native bromelain. Below 5 mM, SDS caused large decrease in V(max) without affecting K(m) for the substrate Z-Arg-Arg-NHMec. Analysis of kinetic data imply that SDS acts as a partial non-competitive inhibitor since even at 100 mM, the residual activity of bromelain was retained by 3%. Inhibition studies show an IC(50) of 0.55 mM and a high K(i) of 0.145 mM. These demonstrate that bromelain is resistant to SDS binding and denaturation, a property known for beta-sheet rich kinetically stable proteins.

Extended application of gel-permeation chromatography by spin column.

Separation of small volumes of proteins from unbound ligands or reequilibration with buffer by passing through a 1-ml Sephadex G-50 column under mild centrifugal force is a popular technique. Here it has been demonstrated that other Sephadex matrix could similarly be used for complete or partial separation of protein molecules. Proteins to be eluted at void volume are recovered near quantitatively, while others are partly or almost completely retained depending on molecular size. Calibration curves using standard proteins of Mw 12.5 to 440 kDa with Sephadex G-50-G-200 representing recovery versus molecular weight show profiles as expected from the fractionation ranges of the column matrix. The procedure may be applied to follow protein association-dissociation reactions if the molecular weights of the species concerned are known and a proper matrix exists for separating them. Equilibrium unfolding transitions constructed with model proteins in presence of 0-8M urea using recovery as an index correspond to profiles obtained from other physical measurements. This may be a convenient approach to follow change of protein hydrodynamic volume quickly when a parallel methodology is not readily available.

Enzymatic, antimicrobial and toxicity studies of the aqueous extract of Ananas comosus (pineapple) crown leaf ☆

ETHNOPHARMACOLOGICAL RELEVANCE Various parts of the plant pineapple (Ananas comosus) are used in traditional medicine worldwide for treatment of a number of diseases and disorders. In folk medicine, pineapple leaf extract was used as an antimicrobial, vermicide, purgative, emmenagoogue, abortifacient, anti-oedema and anti-inflammatory agent. Compared to the fruit and stem extracts of pineapple, information about its leaf extract is limited. The potential of pineapple crown leaf extract as an ethno-medicine has been evaluated in terms of its enzymatic activities related to wound healing, antimicrobial property and toxicity. MATERIALS AND METHODS Major protein components of the extract were revealed by 2-D gel electrophoresis followed by MS/MS analysis. Zymography, DQ-gelatin assay were performed to demonstrate proteolytic, fibrinolytic, gelatinase and collagenase activities. DNase and RNase activities were revealed from agarose gel electrophoresis. Antimicrobial activity was evaluated spectrophotometrically from growth inhibition. Sprague-Dawley rat model was used to measure acute and sub-acute toxicity of the extract by analyzing blood markers. RESULT The extract contains several proteins that were clustered under native condition. Proteomic studies indicated presence of fruit bromelain as major protein constituent of the extract. It showed nonspecific protease activity, gelatinolytic, collagenase, fibrinolytic, acid and alkaline phosphatase, peroxidase, DNase and RNase activities along with considerable anti-microbial property. The leaf extract did not induce any toxicity in rats after oral administration of acute and sub-acute doses. CONCLUSION Pineapple leaf extract is nontoxic, contains enzymes related to damage tissue repairing, wound healing and possibly prevents secondary infections from microbial organisms.

Induction of Cr(VI) reduction activity in an Anoxybacillus strain under heat stress: a biochemical and proteomic study

A bacterial strain, designated as TSB-6, was isolated from the sediments of a Tantloi (India) hot spring at 65 °C. The strain showed 98% 16S rRNA gene sequence similarity with Anoxybacillus kualawohkensis strain KW12 and was found to grow optimally at 37 °C. However, growing cells, cell suspensions, and cell-free extracts from 65 °C cultures showed higher Cr(VI) reduction activities when assayed at either 37 or 65 °C than those obtained from 37 °C cultures. On fractionation of extracts from cells grown at 65 °C, the chromate reductase activity assayed at 65 °C was found mostly in the soluble fraction. When log-phase cells growing at 37 °C were shifted to 65 °C, the stressed cells produced larger quantities of reactive oxygen species. Consequently, growth of the cells was retarded, but specific Cr(VI) reduction activity increased. 2D gel electrophoresis followed by MALDI-TOF MS/MS identified the proteins whose expression level changed as a result of heat stress. The upregulated set included proteins involved in cellular metabolism of sugar, nucleotide, amino acids, lipids and vitamins, oxidoreductase activity, and protein folding. The downregulated proteins are also involved in cellular metabolism, DNA binding, and environmental signal processing.

Analysis of free and bound NADPH in aqueous extract of human placenta used as wound healer.

NADPH is an important biomolecule involved in cellular regeneration. The distribution of free and bound NADPH in aqueous extract of human placenta used as a potent wound healer has been analyzed. Quantification from fluorescence and immuno-affinity chromatography indicates that 75.1+/-2.2% of NADPH present in the extract exists as free nucleotide or bound to very small peptides or amino acids whereas the rest remains bound to large peptides. Inability to dissociate the bound form of the nucleotide from the large peptides using urea or guanidium hydrochloride indicates that the binding is covalent. Identification of a fragmented mass of m/z 382.94 (nicotinamide+sugar+phosphate) from the NADPH-peptide conjugates supported the intactness of the nicotinamide moiety. Glutathione reductase assay indicated that 95.2+/-3.5% of the total NADPH pool of the extract can act as cosubstrate of the enzyme. This indicates that while a major fraction of free NADPH of the extract is easily available for cellular processes, the rest can also function locally where the conjugated peptides are deposited.

PI3K-Mediated Proliferation of Fibroblasts by Calendula officinalis Tincture: Implication in Wound Healing

Calendula officinalis, a member of the Asteraceae family, is a flowering plant and has been used for its antibacterial, antifungal, antiviral, antiinflammatory, anticancer and wound healing activity. The mode of action of C. officinalis tincture on wound healing is poorly understood. Here, we investigated the role of C. officinalis tincture (CDOT) on cell viability and wound closure. C. officinalis tincture stimulated both proliferation and migration of fibroblasts in a statistically significant manner in a PI3K‐dependent pathway. The increase in phosphorylation of FAK (Tyr 397) and Akt (Ser 473) was detected after treatment of CDOT. Inhibition of the PI3K pathway by wortmannin and LY294002 decreased both cell proliferation and cell migration. HPLC‐ESI MS revealed the presence of flavonol glycosides as the major compounds of CDOT. Altogether, our results showed that CDOT potentiated wound healing by stimulating proliferation and migration of fibroblast in a PI3K‐dependent pathway, and the identified compounds are likely to be responsible for wound healing activity. Copyright