ABSTRACT
The treatment of infected wounds in patients with highly sensitive skin is challenging. Some of the available wound dressings cause further skin tear and bleeding upon removal thereby hindering the healing process. In this study, dissolvable antibacterial wound dressing patches loaded with cephalexin monohydrate were prepared from different amounts of sodium alginate (SA) and carboxymethyl cellulose (CMC) by the solvent casting evaporation technique. The patches displayed good tensile strength (3.83-13.83 MPa), appropriate thickness (0.09 to 0.31 mm) and good flexibility (74-98 %) suitable for the skin. The patches displayed good biodegradability and low moisture uptake suitable to prevent microbial invasion on the wound dressings upon storage. The release profile of the drug from the patches was sustained in the range of 47-80 % for 48 h, revealing their capability to inhibit bacterial infection. The biological assay showed that the patches did not induce cytotoxic effects on HaCaT cells, revealing good biocompatibility. The antimicrobial effect of the patches on the different strains of bacteria used in the study was significant. The cell migration (66.7-74.3 %) to the scratched gap was promising revealing the patches' capability to promote wound closure. The results obtained show that the wound dressings are potential materials for the treatment of infected wounds.
Subject(s)
Alginates , Anti-Bacterial Agents , Humans , Alginates/pharmacology , Anti-Bacterial Agents/pharmacology , Wound Healing , Bandages/microbiology , SkinABSTRACT
This paper presents a comparison on the effects of blending chitin and/or starch with poly(lactic acid) (PLA). Three sets of composites (PLA-chitin, PLA-starch and PLA-chitin-starch) with 92%, 94%, 96% and 98% PLA by weight were prepared. The percentage weight (wt.%) amount of the chitin and starch incorporated ranges from 2% to 8%. The mechanical, dynamic mechanical, thermal and microstructural properties were analyzed. The results from the tensile strength, yield strength, Young's modulus, and impact showed that the PLA-chitin-starch blend has the best mechanical properties compared to PLA-chitin and PLA-starch blends. The dynamic mechanical analysis result shows a better damping property for PLA-chitin than PLA-chitin-starch and PLA-starch. On the other hand, the thermal property analysis from thermogravimetry analysis (TGA) shows no significant improvement in a specific order, but the glass transition temperature of the composite increased compared to that of neat PLA. However, the degradation process was found to start with PLA-chitin for all composites, which suggests an improvement in PLA degradation. Significantly, the morphological analysis revealed a uniform mix with an obvious blend network in the three composites. Interestingly, the network was more significant in the PLA-chitin-starch blend, which may be responsible for its significantly enhanced mechanical properties compared with PLA-chitin and PLA-starch samples.
ABSTRACT
In the present study, the effect of irradiance on growth performance of Scenedesmus obliquus was investigated, and various non-linear growth models were evaluated to predict its temporal evolution. This microalga was cultured in a LED-illuminated flat-panel gas-lift photobioreactor operated in batch mode at varying irradiance ranging from 50 to 200 µmol/m2/s keeping all the other physico-chemical parameters constant. When growth data in terms of optical density were fitted in sigmoidal growth models, three non-linear models, namely, Richards model, Gompertz model, and logistic model, were found to be the best fit. Comparing these models based on statistical information, the logistic model could more appropriately and precisely describe algal growth under varying light intensity. Finally, the parameters of the logistic model were determined using regression analysis and were incorporated in the logistic equation to investigate the kinetic characteristics of S. obliquus. The optimum light intensity (Iopt) for growth was found to be 150 µmol/m2/s, at which a maximum specific growth rate (µopt) of 0.35/day was obtained. The model developed was validated experimentally and could successfully explain the photo-inhibition phenomenon occurring at light intensity above 150 µmol/m2/s.
Subject(s)
Biofuels , Biomass , Microalgae/growth & development , Photobioreactors , Scenedesmus/growth & development , Industrial Microbiology/methods , Kinetics , Light , Nonlinear Dynamics , Regression Analysis , Reproducibility of ResultsABSTRACT
The gas-detecting ability of nanostructured ZnO has led to significant attention being paid to the development of a unique and effective approach to its synthesis. However, its poor sensitivity, cross-sensitivity to humidity, long response/recovery times and poor selectivity hinder its practical use in environmental and health monitoring. In this context, the addition of noble metals, as dopants or catalysts to modify the ZnO surface has been examined to enhance its sensing performance. Herein, we report preparation of Pd-loaded ZnO nanoparticles via a chemical precipitation approach. Various Pd loadings were employed to produce surface-modified ZnO nanostructure sensors, and their resulting NH3 sensing capabilities both in dry and humid environments were investigated. Through a comparative gas sensing study between the pure and Pd-loaded ZnO sensors upon exposure to NH3 at an optimal operating temperature of 350 °C, the Pd-loaded ZnO sensors were found to exhibit enhanced sensor responses and fast response/recovery times. The influence of Pd loading and its successful incorporation into ZnO nanostructure was examined by X-ray diffraction, high resolution-transmission electron microscopy, and X-ray photoelectron spectroscopy. XPS studies demonstrated that in all samples, Pd existed in two chemical states, namely Pd° and Pd2+. The possible sensing mechanism related to NH3 gas is also discussed in detail.
ABSTRACT
The effects of stearic acid addition followed by hydrothermal treatment on the functional properties of maize starch were studied. Addition of stearic acid followed by hydrothermal treatment resulted in non-gelling starch. Starch with stearic acid had significantly (Pâ¯<â¯0.05) higher viscosity as compared to heat-moisture treated starch. There was no significant difference on the pasting properties of starch with stearic acid alone and in combination with annealing. Stearic acid addition followed by heat-moisture treatment significantly reduced starch susceptibility to acid hydrolysis as compared to stearic acid alone and heat-moisture treatment alone. These changes related well with the increased amylose lipid complexes and relative crystallinity observed by the DSC and XRD, suggesting that heat-moisture treatment promoted amylopectin side chain crosslinking and amylose-stearic acid complex formation. Stearic acid addition followed by hydrothermal treatment produced a 'clean label' starch that can potentially substitute chemically cross-linked and non-gelling starch in the food industry.
Subject(s)
Chemical Phenomena , Food Handling/methods , Starch/chemistry , Stearic Acids/administration & dosage , Zea mays/chemistry , Amylopectin/chemistry , Amylose/chemistry , Hot Temperature , Hydrolysis , Viscosity , Water , X-Ray DiffractionABSTRACT
Acidic crude oil transportation and processing in petroleum refining and petrochemical operations cause corrosion in the pipelines and associated components. Corrosion monitoring is invariably required to test and prove operational reliability. Thin Layer Activation (TLA) technique is a nuclear technique used for measurement of corrosion and erosion of materials. The technique involves irradiation of material with high energy ion beam from an accelerator and measurement of loss of radioactivity after the material is subjected to corrosive environment. In the present study, TLA technique has been used to monitor corrosion of carbon steel (CS) in crude oil environment at high temperature. Different CS coupons were irradiated with a 13â¯MeV proton beam to produce Cobalt-56 radioisotope on the surface of the coupons. The corrosion studies were carried out by subjecting the irradiated coupons to a corrosive environment, i.e, uninhibited straight run gas oil (SRGO) containing known amount of naphthenic acid (NA) at high temperature. The effects of different parameters, such as, concentration of NA, temperature and fluid velocity (rpm) on corrosion behaviour of CS were studied.
ABSTRACT
The life cycle assessment of several zinc oxide (ZnO) nanostructures, fabricated by a facile microwave technique, is presented. Key synthesis parameters such as annealing temperature, varied from 90°C to 220°C, and microwave power, varied from 110W to 710W, are assessed. The effect of these parameters on both the structural characteristics and the environmental sustainability of the nanostructures is examined. The nanostructures were characterized by means of X-ray diffraction (XRD), focused ion beam scanning electron microscopy (FIB-SEM), ultraviolet-visible spectroscopy (UV-Vis), Photoluminescence (PL) and Brunauer-Emmett-Teller (BET) analysis. Crystalline size was found to be 22.40nm at 110W microwave power, 24.83nm at 310W, and 24.01nm at 710W. Microwave power and synthesis temperature were both directly proportional to the surface area. At 110W the surface area was 10.44m2/g, at 310W 12.88m2/g, and at 710W 14.60m2/g; while it was found to be 11.64m2/g at 150°C and 18.09m2/g at 220°C. Based on these, a life cycle analysis (LCA) of the produced ZnO nanoparticles was carried out, using the ZnO surface area (1m2/g) as the functional unit. It was found that the main environmental weaknesses identified during the production process were; (a) the use of ethanol for purifying the produced nanomaterials and (b) the electricity consumption for the ZnO calcination, provided by South Africa's fossil-fuel dependent electricity source. When the effect of the key synthesis parameters on environmental sustainability was examined it was found that an increase of either microwave power (from 110 to 710W) or synthesis temperatures (from 90 to 220°C), results in higher sustainability, with the environmental footprint reduced by 27% and 41%, respectively. Through a sensitivity analysis, it was observed that an electricity mix based on renewable energy could improve the environmental sustainability of the nanoparticles by 25%.
ABSTRACT
Pueraria tuberosa is known for its therapeutic potentials in cardiovascular disorders, but its effect in angiogenesis has not been studied so far. In this study, a computational approach has been applied to elucidate the role of the phytochemicals in inhibition of angiogenesis through modulation of vascular endothelial growth factor receptors: Vascular endothelial growth factor receptor-1 and vascular endothelial growth factor receptor-2, major factors responsible for angiogenesis. Metabolite structures retrieved from PubChem and KNApSAcK - 3D databases, were docked using AutoDock4.2 tool. Hydrogen bond and molecular docking, absorption, distribution, metabolism and excretion and toxicity predictions were carried out using UCSF Chimera, LigPlot(+) and PreADMET server, respectively. From the docking analysis, it was observed that puerarone and tuberostan had significant binding affinity for the intracellular kinase domain of vascular endothelial growth factor receptors-1 and vascular endothelial growth factor receptor-2 respectively. It is important to mention that both the phytochemicals shared similar interaction profile as that of standard inhibitors of vascular endothelial growth factor receptors. Also, both puerarone and tuberostan interacted with Lys861/Lys868 (adenosine 5'-triphosphate binding site of vascular endothelial growth factor receptors-1/vascular endothelial growth factor receptors-2), thus providing a clue that they may enforce their inhibitory effect by blocking the adenosine 5'-triphosphate binding domain of vascular endothelial growth factor receptors. Moreover, these molecules exhibited good drug-likeness, absorption, distribution, metabolism and excretion properties without any carcinogenic and toxic effects. The interaction pattern of the puerarone and tuberostan may provide a hint for a novel drug design for vascular endothelial growth factor tyrosine kinase receptors with better specificity to treat angiogenic disorders.
ABSTRACT
Polyamidoamine conjugates containing curcumin and bisphosphonate were synthesized via a one-pot aqueous phase Michael addition reaction. In the design of the conjugate, bisphosphonate formed an integral part of the polymer carrier backbone. Curcumin was incorporated onto the polyamidoamine backbone via piperazine linker. The conjugates were characterized by Fourier transform spectroscopy, energy-dispersive X-ray analysis, atomic force spectroscopy and nuclear magnetic resonance spectroscopy and it confirmed the successful incorporation of the antiproliferative agents onto the carriers. The weight percentage incorporation of bisphosphonate to the carriers was found to be between 2.56% and 3.34%. The in vitro release studies of curcumin from the polyamidoamine conjugate were performed in dialysis bag at selected pH values. The release of curcumin was significantly slower at pH 7.4 when compared to pH 5.8. The release profiles indicate that the conjugates are more stable at pH 7.4 and are potential sustained drug-delivery systems for combination therapy.
Subject(s)
Diphosphonates , Polyamines , Delayed-Action Preparations/chemical synthesis , Delayed-Action Preparations/chemistry , Delayed-Action Preparations/pharmacokinetics , Diphosphonates/chemical synthesis , Diphosphonates/chemistry , Diphosphonates/pharmacokinetics , Hydrogen-Ion Concentration , Polyamines/chemical synthesis , Polyamines/chemistry , Polyamines/pharmacokineticsABSTRACT
Natural polymer hydrogels are useful for controlling release of drugs. In this study, hydrogels containing gum acacia were synthesized by free-radical polymerization of acrylamide with gum acacia. The effect of gum acacia in the hydrogels on the release mechanism of nitrogen-containing bisphosphonate (BP) was studied at pH 1.2 and 7.4. The hydrogels exhibited high swelling ratios at pH 7.4 and low swelling ratios at pH 1.2. The release study was performed using UV-Visible spectroscopy via complex formation with Fe(III) ions. At pH 1.2, the release profile was found to be anomalous while at pH 7.4, the release kinetic of BP was a perfect zero-order release mechanism. The hydrogels were found to be pH-sensitive and the release profiles of the BP were found to be influenced by the degree of crosslinking of the hydrogel network with gum acacia. The preliminary results suggest that these hydrogels are promising devices for controlled delivery of bisphosphonate to the gastrointestinal region.
Subject(s)
Diphosphonates/chemistry , Gum Arabic/chemistry , Hydrogels/chemistry , Nitrogen/chemistry , Diphosphonates/administration & dosage , Drug Carriers/chemistry , Drug Delivery Systems , Drug Liberation , Kinetics , Molecular Structure , Polymers/chemistry , Spectrum AnalysisABSTRACT
This study reports the microwave-assisted synthesis of gum-ghatti (Gg)-grafted poly(acrylamide-co-methacrylic acid) (AAm-co-MAA) hydrogels for the development of biodegradable flocculants and adsorbents. The synthesized hydrogels were characterized using TGA, FTIR and SEM. TGA studies revealed that the synthesized hydrogels were thermally more stable than pristine Gg and exhibited maximum swelling capacity of 1959% at 60°C in neutral pH. The optimal Gg-cl-P(AAm-co-MAA) hydrogel was successfully employed for the removal of saline water from various petroleum fraction-saline emulsions. The maximum flocculation efficiency was achieved in an acidic clay suspension with a 15 mg polymer dose at 40°C. Moreover, the synthesized hydrogel adsorbed 94% and 75% of Pb(2+) and Cu(2+), respectively, from aqueous solutions. Finally, the Gg-cl-P(AAm-co-MAA) hydrogel could be degraded completely within 50 days. In summary, the Gg-cl-P(AAm-co-MAA) hydrogel was demonstrated to have potential for use as flocculants and heavy metal absorbents for industrial waste water treatment.
Subject(s)
Hydrogels/chemistry , Plant Gums/chemistry , Polymethacrylic Acids/chemistry , Adsorption , Bacteria/metabolism , Copper/chemistry , Flocculation , Hydrogels/metabolism , Hydrogen-Ion Concentration , Kaolin/chemistry , Lead/chemistry , Microscopy, Electron, Scanning , Petroleum , Plant Gums/metabolism , Polymethacrylic Acids/metabolism , Soil Microbiology , Spectroscopy, Fourier Transform Infrared , TemperatureABSTRACT
We report on the room temperature ferromagnetism of various highly crystalline zinc oxide (ZnO) nanostructures, such as hexagonally shaped nanorods, nanocups, nanosamoosas, nanoplatelets, and hierarchical nano "flower-like" structures. These materials were synthesized in a shape-selective manner using simple microwave assisted hydrothermal synthesis. Thermogravimetric analyses demonstrated the as-synthesized ZnO nanostructures to be stable and of high purity. Structural analyses showed that the ZnO nanostructures are polycrystalline and wurtzite in structure, without any secondary phases. Combination of electron paramagnetic resonance, photoluminescence, and X-ray photoelectron spectroscopy studies revealed that the zinc vacancies (VZn) and singly ionized oxygen vacancies (VO(+)) located mainly on the ZnO surface are the primary defects in ZnO structures. A direct link between ferromagnetism and the relative occupancy of the VZn and VO(+) was established, suggesting that both VZn and VO(+) on the ZnO surface plays a vital role in modulating ferromagnetic behavior. An intense structure- and shape-dependent ferromagnetic signal with an effective g-value of >2.0 and a sextet hyperfine structure was shown. Moreover, a novel low field microwave absorption signal was observed and found to increase with an increase in microwave power and temperature.
ABSTRACT
A total of 50 women having uterine enlargement of > 12 weeks' size due to severe adenomyosis with raised CA125, were carefully studied and compared with other cases. The greater the enlargement of the uterus over 240 cm(3) volume or 12 weeks' uterine size due to severe adenomyosis, the greater was the rise of CA125 levels and these were disturbingly high in some cases. However, similarly enlarged uteri due to fibroids did not show a rise in CA125 levels above normal. Raised CA125 thus need not instil anxiety, when the clinical findings are benign.
Subject(s)
Adenomyosis/blood , CA-125 Antigen/blood , Leiomyoma/blood , Membrane Proteins/blood , Uterine Neoplasms/blood , Adenomyosis/pathology , Female , Humans , Leiomyoma/pathology , Uterine Neoplasms/pathology , Uterus/pathologyABSTRACT
The outcome of different studies on the role of Zn & Mo in esophageal cancer (EC) is conflicting. Here, the levels of those elements in hair as well as food grain of two different ethnic populations across two continents have been studied to explore their role in EC. Two different ethnic populations are taken from (i) Eastern Cape, South Africa (RSA), an area of very high incidence of EC and (ii) West Bengal, India, an area of low incidence of that disease. Each ethnic population is divided into two groups: case and control (n=30 for all groups). Hair samples from all groups and food grain from RSA and India are analyzed for Zn & Mo content. This study shows a strong correlation between reduced levels of those elements in hair and the development of EC in RSA (both Zn &Mo: p < 0.0001), though it is only suggestive in Indian context (both Zn & Mo p ? 0.05). Interestingly, control group of RSA shows significantly reduced level of those elements in hair even with respect to Indian case group (Zn: p<0.001 & Mo: p< 0.00001). Food grain from RSA has significantly reduced level of those elements with respect to India (both Zn & Mo: p <0.0001). This deficiency of Zn & Mo in food grains can be correlated to the deficiency of those elements in hair of RSA population. The deficiency of Zn & Mo can be correlated to the development of EC.
Subject(s)
Edible Grain/chemistry , Esophageal Neoplasms/epidemiology , Hair/chemistry , Molybdenum/isolation & purification , Trace Elements/isolation & purification , Zinc/isolation & purification , Female , Humans , India/epidemiology , Male , South Africa/epidemiology , Young AdultABSTRACT
STUDY DESIGN: To determine whether kinematic measures of reach excursion are valid measures of postural control in individuals with motor-incomplete spinal cord injury (MISCI), seated reach test (SRT; obtained from a wrist marker) values and associated trunk excursion values (obtained from a C7 marker) were compared with center of pressure excursion (COPE). Data were obtained from individuals with MISCI and from non-disabled individuals for each of four directions. To assess the reliability of these measures in subjects with MISCI, these values were collected on two separate days. OBJECTIVE: Dynamic seated postural control is essential for individuals who perform their daily activities from a wheelchair. Although seated reach distance is used as a proxy measure for postural control, it is unknown whether this is an accurate and reliable measure of the limits of stability in individuals with MISCI. RESULTS: In subjects with MISCI, the SRT was significantly correlated with the COPE in three directions of reaching (r ≥ 0.71), with the exception being leftward reaching. Trunk excursion was correlated with COPE in all directions (r ≥ 0.93). In non-disabled individuals, both SRT (r ≥ 0.56) and trunk excursion (r ≥ 0.91) were correlated with COPE for all directions. In individuals with MISCI, there was significant intersession agreement for both reach distance (intraclass correlation coefficient (ICC)≥ 0.78) and trunk excursion (ICC ≥ 0.77) measured in all directions. CONCLUSIONS: Although both SRT and trunk excursion are reasonable reflections of COPE, measurement of postural control based on trunk excursion has advantages for individuals who may have difficulty maintaining arm position during reaching. Reach distance is highly reliable in individuals with MISCI in all directions of reaching.
Subject(s)
Motor Activity/physiology , Postural Balance/physiology , Posture/physiology , Spinal Cord Injuries/physiopathology , Activities of Daily Living , Adult , Biomechanical Phenomena , Female , Humans , Male , Middle Aged , Nonlinear Dynamics , Reproducibility of Results , Rotation , Upper Extremity/innervation , Wheelchairs , Wrist/innervationABSTRACT
The binding of 1-anilino-8-naphthalene-sulfonic acid (ANS) to various globular proteins at acidic pH has been investigated by electrospray ionization mass spectrometry (ESI-MS). Maximal ANS binding is observed in the pH range 3-5. As many as seven species of dye-bound complexes are detected for myoglobin. Similar studies were carried out with cytochrome c, carbonic anhydrase, triosephosphate isomerase, lysozyme, alpha-lactalbumin, and bovine pancreatic trypsin inhibitor (BPTI). Strong ANS binding was observed wherever molten globule states were postulated in solution. ANS binding is not observed for lysozyme and BPTI, which have tightly folded structures in the native form. Alpha-lactalbumin, which is structurally related to lysozyme but forms a molten globule at acidic pH, exhibited ANS binding. Reduction of disulfide bonds in these proteins leads to the detection of ANS binding even at neutral pH. Binding was suppressed at very low pH (<2.5), presumably due to neutralization of the charge on the sulfonate moiety. The distribution of the relative intensities of the protein bound ANS species varies with the charge state, suggesting heterogeneity of gas phase conformations. The binding strength of these complexes was qualitatively estimated by dissociating them using enhanced nozzle skimmer potentials. The skimmer voltages also affected the lower and higher charge states of these complexes in a different manner.
Subject(s)
Anilino Naphthalenesulfonates/chemistry , Proteins/chemistry , Aprotinin/chemistry , Carbonic Anhydrases/chemistry , Cytochrome c Group/chemistry , Disulfides/chemistry , Lactalbumin/chemistry , Muramidase/chemistry , Myoglobin/chemistry , Oxidation-Reduction , Protein Binding , Protein Folding , Spectrometry, Mass, Electrospray Ionization , Triose-Phosphate Isomerase/chemistryABSTRACT
BACKGROUND: The limited proteolytic cleavage of proteins can result in distinct polypeptides that remain noncovalently associated so that the structural and biochemical properties of the 'nicked' protein are virtually indistinguishable from those of the native protein. The remarkable observation that rabbit muscle triosephosphate isomerase (TIM) can be multiply nicked by subtilisin and efficiently religated in the presence of an organic solvent formed the stimulus for our study on a homologous system, Plasmodium falciparum triosephosphate isomerase (PfTIM). RESULTS: The subtilisin nicked form of PfTIM was prepared by limited proteolysis using subtilisin and the major fragments identified using electrospray ionization mass spectrometry. The order of susceptibility of the peptide bonds in the protein was also determined. The structure of the nicked form of TIM was investigated using circular dichroism, fluorescence and gel filtration. The nicked enzyme exhibited remarkable stability to denaturants, although significant differences were observed with the wild-type enzyme. Efficient religation could be achieved by addition of an organic cosolvent, such as acetonitrile, in the presence of subtilisin. Religation was also demonstrated after dissociation of the proteolytic fragments in guanidinium chloride, followed by reassembly after removal of the denaturant. CONCLUSIONS: The eight-stranded beta8/alpha8 barrel is a robust, widely used protein structural motif. This study demonstrates that the TIM barrel can withstand several nicks in the polypeptide backbone with a limited effect on its structure and stability.
Subject(s)
Peptide Fragments/chemistry , Plasmodium falciparum/enzymology , Triose-Phosphate Isomerase/chemistry , Amino Acid Sequence , Animals , Chromatography, Gel , Chromatography, High Pressure Liquid , Circular Dichroism , Electrophoresis, Polyacrylamide Gel , Guanidine , Hydrolysis , Mass Spectrometry , Molecular Sequence Data , Muscle, Skeletal/enzymology , Peptides/chemistry , Protein Conformation , Protein Denaturation , Rabbits , Spectrometry, Fluorescence , Subtilisins/chemistryABSTRACT
The crystal structure of a covalently cross-linked Lactobacillus casei thymidylate synthase has been determined at 2.8 A resolution. The sites for mutation to achieve the bis-disulfide linked dimer were identified using the disulfide modeling program MODIP. The mutant so obtained was found to be remarkably thermostable. This increase in stability has been reasoned to be entirely a consequence of the covalent gluing between the two subunits.
Subject(s)
Crystallography, X-Ray , Lacticaseibacillus casei/enzymology , Thymidylate Synthase/chemistry , Models, Molecular , Mutagenesis, Site-Directed , Protein ConformationABSTRACT
The conformational stability of Plasmodium falciparum triosephosphate isomerase (TIMWT) enzyme has been investigated in urea and guanidinium chloride (GdmCl) solutions using circular dichroism, fluorescence, and size-exclusion chromatography. The dimeric enzyme is remarkably stable in urea solutions. It retains considerable secondary, tertiary, and quaternary structure even in 8 M urea. In contrast, the unfolding transition is complete by 2.4 M GdmCl. Although the secondary as well as the tertiary interactions melt before the perturbation of the quaternary structure, these studies imply that the dissociation of the dimer into monomers ultimately leads to the collapse of the structure, suggesting that the interfacial interactions play a major role in determining multimeric protein stability. The Cm(urea)/Cm(GdmCl) ratio (where Cm is the concentration of the denaturant required at the transition midpoint) is unusually high for triosephosphate isomerase as compared to other monomeric and dimeric proteins. A disulfide cross-linked mutant protein (Y74C) engineered to form two disulfide cross-links across the interface (13-74') and (13'-74) is dramatically destablized in urea. The unfolding transition is complete by 6 M urea and involves a novel mechanism of dimer dissociation through intramolecular thiol-disulfide exchange.
Subject(s)
Cross-Linking Reagents/chemistry , Disulfides/chemistry , Guanidine , Plasmodium falciparum/enzymology , Triose-Phosphate Isomerase/chemistry , Triose-Phosphate Isomerase/genetics , Urea , Animals , Chromatography, Gel , Circular Dichroism , Cysteine/genetics , Models, Molecular , Mutagenesis, Site-Directed , Plasmodium falciparum/genetics , Protein Conformation , Protein Denaturation , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification , Spectrometry, Fluorescence , Triose-Phosphate Isomerase/isolation & purification , Tryptophan , Tyrosine/geneticsABSTRACT
Disulfide engineering across subunit interfaces provides a means of inhibiting dissociation during unfolding of multimeric enzymes. Two symmetry-related intersubunit disulfide bridges were introduced across the interface of the dimeric enzyme triosephosphate isomerase from Plasmodium falciparum. This was achieved by mutating a tyrosine residue at position 74 at the subunit interface to a cysteine, thereby enabling it to form a covalent cross-link with a pre-existing cysteine at position 13 of the other subunit. The wild-type enzyme (TIMWT) and the oxidized (Y74Cox) and reduced (Y74Cred) forms of the mutant have similar enzymatic activity, absorption, and fluorescence spectra. All three proteins have similar far-UV CD spectra. The Y74Cred shows a distinct loss of near-UV CD. Thermal precipitation studies demonstrate that TIMWT and Y74Cox have very similar Tm values (Tm approximately 60 degreesC) whereas Y74Cred is surprisingly labile (Tm approximately 38 degreesC). The Y74C mutant results in the creation of a large cavity (approximately 100 A3) at the dimer interface. The crystal structure for the oxidized form of Y74C mutant, crystallized in the presence of low concentrations of dithiothreitol, reveals an asymmetric dimer containing a disulfide bridge at one site and a reduced dithiol cysteine at the other. The crystal structure of the mutant offers insights into the destabilization effects of the interfacial cavities and the role of disulfide tethering in restoring protein stability.