Sesamol-mediated targeting of EPHA2 sensitises cervical cancer for cisplatin treatment by regulating mitochondrial dynamics, autophagy, and mitophagy.

BACKGROUND: Cervical cancer ranks as the fourth most prevalent cancer among women globally, presenting a significant therapeutic challenge due to its resistance to cisplatin. Ephrin type-A receptor 2 (EPHA2) is prominently overexpressed in cervical cancer and plays a vital role in cisplatin resistance, although the underlying mechanisms remain incompletely elucidated. Mitochondrial dynamics, autophagy, and mitophagy are critical in mediating cisplatin resistance. Sesamol, a phytochemical compound, has exhibited promising anticancer properties. This study aims to investigate the regulatory role of EPHA2 in these pathways underlying cisplatin resistance and to investigate the potential of sesamol in overcoming this resistance and inhibiting cervical cancer progression. METHODS AND RESULT: In this study, we knocked down EPHA2 in the SiHa cell line and evaluated the resulting changes in molecular markers associated with mitochondrial dynamics, mitophagy, and autophagy. Our results indicated that EPHA2 knockdown (EPHA2-KD) led to enhanced mitochondrial fusion and reduced mitochondrial fission, mitophagy, and autophagy. Furthermore, we investigated the effect of EPHA2-KD and sesamol treatment on sensitising cervical cancer to cisplatin treatment. Our data revealed that EPHA2-KD and sesamol treatment significantly increases cellular sensitivity to cisplatin-induced cytotoxicity. Additionally, we demonstrated that sesamol effectively targets EPHA2, as evidenced by decreased EPHA2 expression levels following sesamol treatment. CONCLUSION: In summary, targeting EPHA2 through knockdown or sesamol treatment enhances cisplatin sensitivity in cervical cancer by modulating mitochondrial dynamics, autophagy and mitophagy, suggesting promising therapeutic strategies to overcome chemoresistance.

Differential Interaction of Metal Ions with Gold Nanoclusters and Application in Detection of Cobalt and Cadmium.

Interest in biosensing platforms using protein fluorescent gold nanoclusters (FGNCs) has grown significantly in the past due to the unique optical properties they offer. This study investigates the interaction of metal ions with FGNCs, and the structural modifications brought about by the interaction resulting in fluorescence changes of the cluster and its successful application in the detection of two heavy metals, cobalt and cadmium. The binding of cobalt and cadmium to FGNCs synthesized from BSA significantly altered the secondary structure of the protein, causing a change in its hydrophobicity. It also resulted in a change in fluorescence properties of FGNCs by intersystem crossing (ICT) and fluorescence resonance energy transfer (FRET). Cobalt and cadmium could successfully be detected in the range of 5-165 ng/mL (R2 = 0.95) and 20-1000 ng/ mL (R2 = 0.91), respectively, with appreciable sensitivity. The principle was also applied for the detection of Vitamin B12 in commercially available ampoules, validating the proposed method. Graphical Abstract Proposed detection method of cadmium and cobalt using FGNCs.

Preparation of horsegram protein concentrate with improved protein quality, in vitro digestibility and available lysine.

Horsegram (Macrotyloma uniflorum or Dolichos biflorus), an inexpensive pulse crop with high lysine and iron contents, is underutilized due to the presence of antinutrients like lectin and trypsin inhibitors, which limit protein digestibility and availability. Horsegram protein concentrate (HGPC) was prepared, which had 80.4 ± 3.5% protein and 94.2 ± 1.4% in vitro protein digestibility compared to dehulled horsegram flour (22.8 ± 0.8% and 82.3 ± 1.2%, respectively). Available lysine content in concentrate was increased by 64% compared to dehulled horsegram flour. The trypsin inhibitory activity in the protein concentrate decreased by 51% from 36.6 ± 3.5 TIU/mg in horsegram flour to 17.6 ± 2.5 TIU/mg in HGPC. Nutritional indices, including PDCAAS values for different age groups, were calculated and found to be slightly lower due to the loss of methionine and tryptophan in the concentrate. However, branched amino acids and lysine contents were higher. Thus, a vegetarian source with high protein digestibility and available lysine content could be prepared as a protein ingredient for the food industry.

Production of highly active fungal milk-clotting enzyme by solid-state fermentation.

Cheese production is projected to reach 20 million metric tons by 2020, of which 33% is being produced using calf rennet (EC 3.4.23.4). There is shortage of calf rennet, and use of plant and microbial rennets, hydrolyze milk proteins non-specifically resulting in low curd yields. This study reports fungal enzymes obtained from cost effective medium, with minimal down streaming, whose activity is comparable with calf and Mucor rennet. Of the fifteen fungi that were screened, Mucor thermohyalospora (MTCC 1384) and Rhizopus azygosporus (MTCC 10195) exhibited the highest milk-clotting activity (MCA) of 18,383 ± 486 U/ml and 16,373 ± 558 U/ml, respectively. Optimization exhibited a 33% increase in enzyme production (30 g wheat bran containing 6% defatted soy meal at 30 °C, pH 7) for M. thermohyalospora. The enzyme was active from pH 5-10 and temperature 45-55 °C. Rhizopus azygosporus exhibited 31% increase in enzyme production (30 g wheat bran containing 4% defatted soy meal at 30 °C, pH 6) and the enzyme was active from pH 6-9 at 50 °C. Curd yields prepared from fungal enzyme extract decreased (5-9%), when compared with calf rennet and Mucor rennet. This study describes the potential of fungal enzymes, hitherto unreported, as a viable alternative to calf rennet.

Bioactive lignans from sesame (Sesamum indicum L.): evaluation of their antioxidant and antibacterial effects for food applications.

Antioxidants protect the body from various disease conditions through their ability to neutralize the effects of free radicals. Synthetic antioxidants are extensively used in processed foods for prevention of oxidation and retention of sensory quality. Consumer awareness and preference has led to a vigorous interest in the search for natural antioxidants. Sesamin and sesamolin, the major lignans present in sesame oil, are known for their antioxidative properties. Roasted sesame oil has a higher concentration of sesamol, the thermally degraded product of sesamolin, which is considered a more potent antioxidant compared to its parent molecule. The isolated lignans and sesamol were tested for their antioxidant, free radical scavenging and antibacterial properties. Sesamol is the best antioxidant and free radical scavenger amongst the molecules studied with IC50 value of 5.44 µg / mL (DPPH radical scavenging activity). Antibacterial assays against food borne pathogens revealed sesamol to be an antimicrobial agent with minimal inhibitory concentration (MIC) of 2 mg /mL in the culture. Its activity was synergistic with γ-tocopherol, also present in sesame seeds. Inhibition of browning (60-65 %) in fruit pulps (apple, banana and potato) was observed in presence of 20 µM sesamol.

Citrus nutraceutical eriocitrin and its metabolites are partial agonists of peroxisome proliferator-activated receptor gamma (PPARγ): a molecular docking and molecular dynamics study.

Peroxisome proliferator-activated receptor gamma (PPARγ) agonists are potent insulin sensitizers in treating type 2 diabetes. Despite being very effective in the fight against diabetes-mediated complications, PPARγ agonists are accompanied by severe side effects leading to complicated health problems, making the discovery of novel safe ligands highly pertinent. A significant intense research effort is in progress to explore the PPARγ activating potential of a wide range of natural compounds. Lemon (Citrus limon) contains various bioactive flavonoids, and eriocitrin is the major flavonoid. It possesses substantial antioxidant and anticancer, lipid-lowering activities and prevents obesity-associated metabolic diseases. Eriocitrin is metabolized to eriodictyol in the intestine, and the absorbed eriodictyol undergoes conversion to numerous metabolites in vivo. It is unclear if eriocitrin or its metabolites are responsible for their beneficial effects. We have used molecular docking, ADMET properties, drug-likeness score and molecular dynamics simulation study to find if eriocitrin and its metabolites are potent binders for PPARγ. Docking studies revealed that eriocitrin binds to PPARγ with the highest binding energy, but ADMET properties and in vivo studies show that the bioavailability of eriocitrin is very poor. Molecular dynamics studies were carried out to validate the docking results, and multiple parameters like RMSD, RMSF, Radius of gyration, SASA, hydrogen bond analysis, interaction energy, principal component analysis, Gibbs free energy and MM-PBSA were calculated. Based on our studies, eriodictyol, eriodictyol 7-O-glucuronide, eriodictyol 3'-O-glucuronide, homoeriodictyol and homoeriodictyol 7-O-glucuronide which are metabolites of eriocitrin appear to be potent partial agonists of PPARγ under physiological conditions.Communicated by Ramaswamy H. Sarma.

Aspartic protease-pepstatin A interactions: Structural insights on the thermal inactivation mechanism.

Aspartic proteases are the targets for structure-based drug design for their role in physiological processes and pharmaceutical applications. Structural insights into the thermal inactivation mechanism of an aspartic protease in presence and absence of bound pepstatin A have been obtained by kinetics of thermal inactivation, CD, fluorescence spectroscopy and molecular dynamic simulations. The irreversible thermal inactivation of the aspartic protease comprised of loss of tertiary and secondary structures succeeded by the loss of activity, autolysis and aggregation The enthalpy and entropy of thermal inactivation of the enzyme in presence of pepstatin A increased from 81.2 to 148.5 kcal mol-1, and from 179 to 359 kcal mol-1 K-1 respectively. Pepstatin A shifted the mid-point of thermal inactivation of the protease from 58 °C to 77 °C. The association constant (K) for pepstatin A with aspartic protease was 2.5 ± 0.3 × 10 5 M-1 and ΔGo value was -8.3 kcal mol-1. Molecular dynamic simulation studies were able to delineate the role of pepstatin A in stabilizing backbone conformation and side chain interactions. In the Cα-backbone, the short helical segments and the conserved glycines were part of the most unstable segments of the protein. Understanding the mechanism of thermal inactivation has the potential to develop re-engineered thermostable proteases.

Production and characterization of a milk-clotting enzyme from Aspergillus oryzae MTCC 5341.

Microbial milk-clotting enzymes are valued as calf rennet substitutes in the cheese industry. Aspergillus oryzae MTCC 5341 was identified to produce the highest milk-clotting activity during screening of 16 fungal strains. Solid state fermentation using wheat bran along with 4% defatted soy flour and 2% skim milk powder as substrate was optimal for growth of A. oryzae and production of the enzyme. Nearly 40,000 U/g bran of milk-clotting activity was present at the end of 120 h. The enzyme could be recovered by percolating the bran with 0.1 M sodium chloride for 60 min at 4 degrees Celsius. The decolorized enzyme preparation had high ratio of milk clotting to proteolytic activity. Affinity precipitation with alginate and subsequent elution with 0.5 M sodium chloride containing 0.2 M CaCl(2) resulted in an enzyme preparation with specific activity of 3,500 U/mg and 72% yield. Optimum pH and temperature for activity of the enzyme were characterized as 6.3 and 55 degrees Celsius, respectively. Milk-clotting enzyme showed differential degree of hydrolysis on casein components. High ratio of milk clotting to proteolytic activity coupled with low thermal stability strengthens the potential usefulness of milk-clotting enzyme of A. oryzae MTCC 5341 as a substitute for calf rennet in cheese manufacturing.

Acid protease production by solid-state fermentation using Aspergillus oryzae MTCC 5341: optimization of process parameters.

Aspergillus oryzae MTCC 5341, when grown on wheat bran as substrate, produces several extracellular acid proteases. Production of the major acid protease (constituting 34% of the total) by solid-state fermentation is optimized. Optimum operating conditions obtained are determined as pH 5, temperature of incubation of 30 degrees C, defatted soy flour addition of 4%, and fermentation time of 120 h, resulting in acid protease production of 8.64 x 10(5) U/g bran. Response-surface methodology is used to generate a predictive model of the combined effects of independent variables such as, pH, temperature, defatted soy flour addition, and fermentation time. The statistical design indicates that all four independent variables have significant effects on acid protease production. Optimum factor levels are pH 5.4, incubation temperature of 31 degrees C, 4.4% defatted soy flour addition, and fermentation time of 123 h to yield a maximum activity of 8.93 x 10(5) U/g bran. Evaluation experiments, carried out to verify the predictions, reveal that A. oryzae produces 8.47 x 10(5) U/g bran, which corresponds to 94.8% of the predicted value. This is the highest acid protease activity reported so far, wherein the fungus produces four times higher activity than previously reported [J Bacteriol 130(1): 48-56, 1977].

Anti-hyperglycemic activity of myricetin, through inhibition of DPP-4 and enhanced GLP-1 levels, is attenuated by co-ingestion with lectin-rich protein.

Dipeptidyl peptidase-4 (DPP-4) is a proteolytic enzyme responsible for the rapid degradation of Glucagon-like peptide 1 (GLP-1) that is required for the secretion of insulin. DPP-4 also influences activation of node like receptor family, pyrin domain containing 3 (NLRP3) inflammasome under diabetic conditions. Although several polyphenols are reported for various bioactivities, they are consumed as part of the food matrix and not in isolation. Horsegram (Macrotyloma uniflorum) is a rich source of myricetin (Myr) (35 µg/g flour), reported for its anti-hyperglycemic effect. In this investigation, we aimed to study the effect of Myr, singly, and in the presence of co-nutrient horsegram protein (HP) on DPP-4 activity and its consequential impact on GLP-1, insulin, and NLRP3 inflammasome in high-fat diet and single low dose streptozotocin (STZ)-induced diabetic male Wistar rats. In diabetic control (DC), the activity of DPP-4 and its expression were higher compared to treated groups. The consequential decrease in the circulating GLP-1 levels in the DC group, but not treated groups, further indicated the effectiveness of our test molecules under diabetic conditions. Specifically, Myr decreased DPP-4 activity and its expression levels with enhanced circulating GLP-1 and insulin levels. Myr administration also resulted in a lessening of diabetes-induced NLRP3 inflammasome activation and enhanced antioxidant enzyme activities. HP also proved to be efficient in reducing elevated blood glucose levels and enhancing antioxidant enzyme activities. However, Myr, in the presence of HP as a co-nutrient, had diminished capacity to inhibit DPP-4 and, consequently, reduced potential in ameliorating diabetic conditions. Myr proved to be a potent inhibitor of DPP-4 in vitro and in vivo, resulting in enhanced circulating GLP-1 and insulin levels, thereby improving diabetic conditions. Though Myr and HP, individually ameliorate diabetic conditions, their dietary combination had reduced efficiency.

Purification, characterization and specificity of a new GH family 35 galactosidase from Aspergillus awamori.

Galactosidases, ubiquitous in nature, are complex carbohydrate-active enzymes and find extensive applications in food, pharma, and biotechnology industries. The present study deals with the production of galactosidases from fungi by solid-state fermentation. Fifteen fungi were screened and Aspergillus awamori (MTCC 548), exhibited the highest α and ß-galactosidase activities of 75.11±0.29 U/g and 155.34±1.26 U/g, respectively. 30 g of wheat bran substituted with 6% defatted soy flour, at 28°C, pH 5.0 for 120 h, was established as the optimum production conditions by one-factor approach. The enzyme was purified to homogeneity with an apparent mass of 118 ± 2 kDa by ammonium sulfate precipitation (50-80%), ion exchange and hydrophobic interaction chromatography. Specific activities for α and ß-galactosidase were 22 and 74 U/mg, respectively. Optimum temperature and pH ranges for enzyme activities were 55-60 °C, 5.0-5.5, respectively. The thermal inactivation mid-point was 65 °C. The purified enzyme not only exhibited α and ß-galactosidase activities, but also exhibited ß-xylosidase and ß-glucosidase activities, indicating the enzyme has broad substrate specificity. Sequence analysis by in-gel digestion and tandem mass spectrometry (MS/MS) revealed that the enzyme was a probable ß-galactosidase A, belonging to glycoside hydrolase 35 family, and is being reported for the first time.

Aspartic protease from Aspergillus niger: Molecular characterization and interaction with pepstatin A.

In the pursuit of industrial aspartic proteases, aspergillopepsin A-like endopeptidase from the fungi Aspergillus niger, was identified and cultured by solid state fermentation. Conventional chromatographic techniques were employed to purify the extracellular aspartic protease to apparent homogeneity. The enzyme was found to have single polypeptide chain with a molecular mass of 50 ±â€¯0.5 kDa. The optimum pH and temperature for the purified aspartic protease was found to be 3.5 and 60 °C respectively. The enzyme was stable for 60 min at 50 °C. The purified enzyme had specific activity of 40,000 ±â€¯1800 U/mg. The enzyme had 85% homology with the reported aspergillopepsin A-like aspartic endopeptidase from Aspergillus niger CBS 513.88, based on tryptic digestion and peptide analysis. Pepstatin A reversibly inhibited the enzyme with a Ki value of 0.045 µM. Based on homology modeling and predicted secondary structure, it was inferred that the aspartic protease is rich in ß-structures, which was also confirmed by CD measurements. Interaction of pepstatin A with the enzyme did not affect the conformation of the enzyme as evidenced by CD and fluorescence measurements. Degree of hydrolysis of commercial substrates indicated the order of cleaving ability of the enzyme to be hemoglobin > defatted soya flour > gluten > gelatin > skim milk powder. The enzyme also improved the functional characteristics of defatted soya flour. This aspartic protease was found to be an excellent candidate for genetic manipulation for biotechnological application in food and feed industries, due to its high catalytic turn over number and thermostability.

Dual-function protein in plant defence: seed lectin from Dolichos biflorus (horse gram) exhibits lipoxygenase activity.

Plant-pathogen interactions play a vital role in developing resistance to pests. Dolichos biflorus (horse gram), a leguminous pulse crop of the subtropics, exhibits amazing defence against attack by pests/pathogens. Investigations to locate the possible source of the indomitable pest resistance of D. biflorus, which is the richest source of LOX (lipoxygenase) activity, have led to a molecule that exhibits LOX-like functions. The LOX-like activity associated with the molecule, identified by its structure and stability to be a tetrameric lectin, was found to be unusual. The evidence for the lectin protein with LOX activity has come from (i) MALDI-TOF (matrix-assisted laser-desorption ionization-time-of-flight) MS, (ii) N-terminal sequencing, (iii) partial sequencing of the tryptic fragments of the protein, (iv) amino acid composition, and (v) the presence of an Mn2+ ion. A hydrophobic binding site of the tetrameric lectin, along with the presence of an Mn2+ ion, accounts for the observed LOX like activity. This is the first ever report of a protein exhibiting both haemagglutination and LOX-like activity. The two activities are associated with separate loci on the same protein. LOX activity associated with this molecule adds a new dimension to our understanding of lectin functions. This observation has wide implications for the understanding of plant defence mechanisms against pests and the cellular complexity in plant-pathogen interactions that may lead to the design of transgenics with potential to impart pest resistance to other crops.

Effect of arginine:lysine and glycine:methionine intake ratios on dyslipidemia and selected biomarkers implicated in cardiovascular disease: A study with hypercholesterolemic rats.

The effect of intake ratios of arginine (Arg): lysine (Lys) and glycine (Gly): methionine (Met) on lipid profile and selected cardiovascular disease markers, was studied, in rats maintained on a hypercholesterolemic diet. The rise in blood cholesterol was countered by 32%, 24%, and 49%, respectively, through increased oral supplementation of Arg, Gly, and Arg+Gly; a corresponding increase in plasma phospholipids at the end of the 8-week study was observed. The elevated plasma cholesterol to phospholipids ratio was countered by 27, 40, and 57%, respectively, through oral supplementation of Arg, Gly, and Arg+Gly. The elevation in hepatic cholesterol was lowered by 18, 29, and 51%, respectively, while phospholipids concentration was concomitantly increased by these amino acids. The elevated cholesterol to phospholipids ratio was, thus, significantly countered in the hypercholesterolemic situation by orally supplemented Arg, Gly, and Arg+Gly. Increased plasma asymmetric dimethylarginine (ADMA) levels, under hypercholesterolemic conditions, were lowered by 12, 15 and 34%, respectively, while plasma symmetric dimethylarginine (SDMA) levels were lowered by 14, 10 and 17%, respectively, with orally supplemented Arg, Gly and Arg+Gly. Only Gly and Arg+Gly decreased plasma homocysteine levels. Total nitric oxide (NO) concentration was considerably increased by Gly supplementation in hypercholesterolemic rats. Thus, altered ratios of Arg:Lys or Gly:Met offered beneficial influence on the lipid profile and plasma levels of ADMA, SDMA and homocysteine in hypercholesterolemic rats. Optimal beneficial effects, among ratios tested, was observed when Arg:Lys and Gly:Met ratios were maintained in ratios of 1:1 and 2:1, respectively.

In vivo modulation of LPS induced leukotrienes generation and oxidative stress by sesame lignans.

The role of inflammation and oxidative stress is critical during onset of metabolic disorders and this has been sufficiently established in literature. In the present study, we evaluated the effects of sesamol and sesamin, two important bioactive molecules present in sesame oil, on the generation of inflammatory and oxidative stress factors in LPS injected rats. Sesamol and sesamin lowered LPS induced expression of cPLA2 (61 and 56%), 5-LOX (44 and 51%), BLT-1(32 and 35%) and LTC4 synthase (49 and 50%), respectively, in liver homogenate. The diminished serum LTB4 (53 and 64%) and LTC4 (67 and 44%) levels in sesamol and sesamin administered groups, respectively, were found to be concurrent with the observed decrease in the expression of cPLA2 and 5-LOX. The serum levels of TNF-α (29 and 19%), MCP-1 (44 and 57%) and IL-1ß (43 and 42%) were found to be reduced in sesamol and sesamin group, respectively, as given in parentheses, compared to LPS group. Sesamol and sesamin offered protection against LPS induced lipid peroxidation in both serum and liver. Sesamol, but not sesamin, significantly restored the loss of catalase and glutathione reductase activity due to LPS (P<.05). However, both sesamol and sesamin reverted SOD activities by 92 and 98%, respectively. Thus, oral supplementation of sesamol and sesamin beneficially modulated the inflammatory and oxidative stress markers, as observed in the present study, in LPS injected rats. Our report further advocates the potential use of sesamol and sesamin as an adjunct therapy wherein, inflammatory and oxidative stress is of major concern.

Escherichia coli Braun Lipoprotein (BLP) exhibits endotoxemia - like pathology in Swiss albino mice.

The endotoxin lipopolysaccharide (LPS) promotes sepsis, but bacterial peptides also promote inflammation leading to sepsis. We found, intraperitoneal administration of live or heat inactivated E. coli JE5505 lacking the abundant outer membrane protein, Braun lipoprotein (BLP), was less toxic than E. coli DH5α possessing BLP in Swiss albino mice. Injection of BLP free of LPS purified from E. coli DH5α induced massive infiltration of leukocytes in lungs and liver. BLP activated human polymorphonuclear cells (PMNs) ex vivo to adhere to denatured collagen in serum and polymyxin B independent fashion, a property distinct from LPS. Both LPS and BLP stimulated the synthesis of platelet activating factor (PAF), a potent lipid mediator, in human PMNs. In mouse macrophage cell line, RAW264.7, while both BLP and LPS similarly upregulated TNF-α and IL-1ß mRNA; BLP was more potent in inducing cyclooxygenase-2 (COX-2) mRNA and protein expression. Peritoneal macrophages from TLR2-/- mice significantly reduced the production of TNF-α in response to BLP in contrast to macrophages from wild type mice. We conclude, BLP acting through TLR2, is a potent inducer of inflammation with a response profile both common and distinct from LPS. Hence, BLP mediated pathway may also be considered as an effective target against sepsis.

Thermal stability of alpha-amylase from malted jowar (Sorghum bicolor).

Malted cereals are rich sources of alpha-amylase, which catalyzes the random hydrolysis of internal alpha-(1-4)-glycosidic bonds of starch, leading to liquefaction. Amylases play a role in the predigestion of starch, leading to a reduction in the water absorption capacity of the cereal. Among the three cereal amylases (barley, ragi, and jowar), jowar amylase is found to be the most thermostable. The major amylase from malted jowar, a 47 kDa alpha-amylase, purified to homogeneity, is rich in beta structure ( approximately 60%) like other cereal amylases. T(m), the midpoint of thermal inactivation, is found to be 69.6 +/- 0.3 degrees C. Thermal inactivation is found to follow first-order kinetics at pH 4.8, the pH optimum of the enzyme. Activation energy, E(a), is found to be 45.3 +/- 0.2 kcal mol(-)(1). The activation enthalpy (DeltaH), entropy (DeltaS*), and free energy change (DeltaG) are calculated to be 44.6 +/- 0.2 kcal mol(-)(1), 57.1 +/- 0.3 cal mol(-)(1) K(-)(1), and 25.2 +/- 0.2 kcal mol(-)(1), respectively. The thermal stability of the enzyme in the presence of the commonly used food additives NaCl and sucrose has been studied. T(m) is found to decrease to 66.3 +/- 0.3, 58.1 +/- 0.2, and 48.1 +/- 0.5 degrees C, corresponding to the presence of 0.1, 0.5, and 1 M NaCl, respectively. Sucrose acts as a stabilizer; the T(m) value is found to be 77.3 +/- 0.3 degrees C compared to 69.6 +/- 0.3 degrees C in the control.

The contribution of ionic interactions to the conformational stability and function of polygalacturonase from A. niger.

Aspergillus niger produces multiple forms of polygalacturonases with molecular masses ranging from 30 to 60 kDa. The high molecular weight polygalacturonase (61+/-2 kDa) from A. niger possesses a pH optimum of 4.3 and a pI of 3.9. The enzyme exhibited high sensitivity, both in terms of activity and structure, in the pH range of 4.3-7.0. The enzyme was irreversibly inactivated at pH 7.0. The enzyme is predominantly rich in parallel beta structure. There is unfolding of the enzyme molecule between 4.3 and 7.0 resulting in irreversible loss of secondary and tertiary structure with the exposure of hydrophobic surfaces. ANS binding measurements, intrinsic fluorescence and acrylamide quenching measurements have confirmed the unfolding and exposure of hydrophobic surfaces. The midpoint of pH transition for both activity and secondary structure is 6.2+/-0.1. The pH-induced changes of polygalacturonase confirm the role of histidine residues in structure and activity of the enzyme. The irreversible nature of inactivation is due to the unfolding induced exposure of hydrophobic surfaces leading to association/aggregation of the molecule. Size exclusion chromatography measurements have established the association of enzyme at higher pH. Urea induced unfolding measurements at pH 4.3 and 7.0 have confirmed the loss in stability as we approach neutral pH.

Interaction of curcumin with ß-lactoglobulin-stability, spectroscopic analysis, and molecular modeling of the complex.

Curcumin (diferuloyl methane) is the physiologically and pharmacologically active component of turmeric (Curcuma longa L.). Solubility and stability of curcumin are the limiting factors for realizing its therapeutic potential. ß-Lactoglobulin (ßLG), the major whey protein, can solubilize and bind many small hydrophobic molecules. The stability of curcumin bound to ßLG in solution is enhanced 6.7 times, in comparison to curcumin alone (in aqueous solution). The complex formation of curcumin with ßLG has been investigated employing spectroscopic techniques. ßLG interacts with curcumin at pH 7.0 with an association constant of 1.04 ± 0.1 × 10(5) M(-1) to form a 1:1 complex at 25 °C. Entropy and free energy changes for the interaction derived from the van't Hoff plot are 18.7 cal mol(-1) K(-1) and -6.8 kcal mol(-1) at 25 °C, respectively; the interaction is hydrophobic in nature. The interaction of ßLG with curcumin does not affect either the conformation or the state of association of ßLG. Competitive ligand binding measurements, binding studies with denatured ßLG, effect of pH on the curcumin-ßLG interaction, Förster energy transfer measurements, and molecular docking studies suggest that curcumin binds to the central calyx of ßLG. These binding studies have prompted the preparation and encapsulation of curcumin in ßLG nanoparticles. Nanoparticles of ßLG prepared by desolvation are found to encapsulate curcumin with >96% efficiency. The solubility of curcumin in ßLG nanoparticle is significantly enhanced to ∼625 µM in comparison with its aqueous solubility (30 nM). Nanoparticles of ßLG, by virtue of their ability to enhance solubility and stability of curcumin, may fit the choice as a carrier molecule.

Interaction of alphaS1-casein with curcumin and its biological implications.

alpha(S1)-Casein is one of the major protein components of the casein fraction of milk. Curcumin (diferuloyl methane), the major curcuminoid, constituting about 2-5% of turmeric (Curcuma longa ) is the active ingredient with many physiological, biochemical, and pharmacological properties. On the basis of spectroscopic measurements, it is inferred that curcumin binds to alpha(S1)-casein at pH 7.4 and 27 degrees C with two binding sites, one with high affinity [(2.01 +/- 0.6) x 10(6) M(-1)] and the other with low affinity [(6.3 +/- 0.4) x 10(4) M(-1)]. Binding of curcumin to alpha(S1)-casein is predominantly hydrophobic in nature. The anisotropy of curcumin or conformation of alpha(S1)-casein did not change on interaction. The stability of curcumin in solution at pH 7.2 was enhanced on binding with alpha(S1)-casein. The chaperone-like activity of alpha(S1)-casein gets slightly enhanced on its binding to curcumin. The ability of curcumin to protect erythrocytes against hemolysis was not affected due to curcumin- alpha(S1)-casein interaction. The two binding sites of alpha(S1)-casein for curcumin, along with enhanced solution stability on interaction, may offer an alternative approach in physiological and nutritional applications.