Nanostructures of bismuth sulphide: synthesis and electrical properties.

Bismuth ammonium citrate complex (C24H20Bi4O28 x 6NH3 x 10H2O) interacted with sodium sulphide (Na2S) in presence of beta-cyclodextrin (beta-CD) yielding Bi2S3 nanospheres. Solvothermal treatment of the bismuth complex and dimethyl sulphoxide (DMSO) produced Bi2S3 nanorods. Reaction conditions were optimized to investigate the morphology evolution of the product. Electrical properties of the nanorods were monitored in details.

Morphological modifications of selenium by recrystallization of its aqueous complex solutions.

Recrystallization of elemental selenium (Se) from aqueous solution in presence of sodium sulphite (Na2SO3) and sodium sulphide (Na2S) acting as complexing agents has resulted in the formation of nano and microstructures of Se having five different morphological modifications. (1) An aqueous solution of sodium selenosulphate (Na2SO3Se) obtained by dissolving Se in Na2SO3 under refluxing condition yields hemispherical microcrystals. (2) The filtrate of the above reaction mixture on aging produces hexagonal prismatic microrods of Se. Addition of Na2SO3Se solution to formalin (HCHO) at room temperature and refluxing conditions generates (3) Se nanorods, and (4) spherical microcrystals, respectively. (5) Recrystallization of Se from aqueous solution of Na2S develops flower shaped microcrystals.

Nanorods of tellurium: synthesis and self-assembly.

A simple solution phase approach is described to prepare tellurium nanorods which undergo morphological modifications to yield different microstructures under varied experimental conditions. The morphology of the prepared products is drastically altered in presence of a few oxidizing agents such as sodium oxychloride (NaOCl), hydrogen peroxide (H2O2) etc. The effects of poly (sodium 4-styrene sulphonate) (PSS) and Isooctyl phenoxy poly oxyethanol (TritonX-100) on the size and shape of the products in presence of air/NaOCl have also been monitored.

Kinetics and inhibition of carbonic anhydrase-catalyzed hydrolysis of 2-hydroxy-5-nitro-alpha-toluenesulfonic acid sultone. Comparison with reactions of other substrates.

The catalytic activities of human red cell carbonic anhydrase (EC 4.2.1.1) isozymes B and C for the hydrolysis of 2-hydroxy-5-nitro-alpha-toluenesulfonic acid sultone have been compared with their activities towards three other substrates. The substrate specificity (measured as kcat/Km) for either isozyme decreases in this order: CO2 greater than 2-hydroxy-5-nitro-alpha-toluenesulfonic acid sultone greater than acetaldehyde greater than p-nitrophenyl acetate. Unlike CO2 hydration, enzyme B is slightly more active towards sultone hydrolysis than C. Despite these widely differing activities of both isozymes with regard to different substrates, the inhibition constants for anion and sulfonamide inhibitors are nearly independent of the substrate used. This suggests that the binding sites of these substrates in the enzyme are the same or nearly the same. Earlier studies on 2-hydroxy-5-nitro-alpha-toluenesulfonic acid sultone from this and other laboratories had underestimated both the intrinsic activity and the susceptibility to anion inhibition of human carbonic anhydrase B. We now find that this was due to the use of acetonitrile as the substrate solvent, which is often contaminated with cyanide, a powerful inhibitor of carbonic anhydrase. The inhibition of human carbonic anhydrase B by several industrial batches of acetonitrile agrees completely with the spectrophotometrically determined cyanide content of these batches.

Kinetics and inhibition of membrane-bound carbonic anhydrase from canine renal cortex.

The membrane-bound carbonic anhydrase (carbonate hydro-lyase, EC 4.2.1.1) in the canine renal cortex has been characterized in terms of its CO2 hydration kinetics and inhibition by sulfonamides and inorganic anions. Comparing these properties with those of the renal cytoplasmic and the human red cell B and C isozymes, it appears that the membrane enzyme is quite different from the soluble carbonic anhydrases. The turnover number of the particulate enzyme is about 3-times lower than that of the cytoplasmic enzyme. The membrane-bound enzyme is also different from its cytoplasmic counterpart in being more resistant against inhibition, particularly against Cl-. Microsomes from the renal cortex were purified to yield luminal and anti-luminal fractions. Carbonic anhydrase activity was found in both. The luminal and anti-luminal carbonic anhydrases appeared similar in terms of their kinetic properties and susceptibility to inhibition.

Spectroscopic characterization of tick anticoagulant peptide.

Tick anticoagulant peptide (TAP) is a disulfide rich potent inhibitor of factor Xa. Although this peptide is of potential clinical utility, very little is known about its higher order structure. Therefore, the secondary structure of recombinant TAP (rTAP) has been examined by circular dichroism (CD) and Fourier transform infrared (FTIR) spectroscopy. Both techniques suggest that rTAP is rich in beta-sheet structure. Disulfide bonds play a significant role in the folding and structural stability of rTAP. This is apparent from the resistance of rTAP to fluorescence-detected unfolding by guanidinium chloride (Gdn-HCl), unless disulfides are first reduced. The protein's tryptophan and tyrosine residues exhibit greater solvent exposure upon reduction of the cystines as indicated by fluorescence spectra and second derivative UV spectroscopy. A considerable amount of beta-structure appears to be retained after reduction of disulfides, although the CD spectrum manifests an increased amount of disordered structure in the reduced peptide. While rTAP does not bind the hydrophobic fluorescence probe 2-p-toludinylnaphthalene-6-sulfonate (TNS) at neutral or acidic pH, the reduced peptide binds TNS at pH 2.0 but not at pH 7.0. The secondary structure of the reduced peptide at pH 2 is, however, similar to that at pH 7 as judged by CD spectroscopy. The reduced form of rTAP at acidic pH thus resembles a molten globule-like state.

Direct biophysical characterization of human apolipoprotein A-1 in ISCOMs.

Human apolipoprotein A-1 was formulated in "Immune Stimulating Complexes" (ISCOMs). The structure of the protein in ISCOMs was examined directly using several biophysical techniques including Fourier transform infrared (FTIR) spectroscopy, near UV circular dichroism (CD), and fluorescence spectroscopy. Amide I FTIR data indicate that human apolipoprotein A-1 displays a slightly increased alpha-helical content after its incorporation into ISCOMs. Near UV CD and tryptophan fluorescence data suggest that association with ISCOMs results in the tryptophan residues of the protein experiencing a relatively hydrophobic environment, motional restriction, and local electrostatic interactions. These observations are consistent with an increased order in the protein structure upon incorporation in ISCOMs. In addition, biomolecular interaction analysis (BIA), based on surface plasmon resonance (SPR) measurements, suggests that the binding affinity of human apolipoprotein A-1 to a monoclonal anti-human apolipoprotein A-1 antibody is moderately decreased (by 20%) after its incorporation into ISCOMs. This study demonstrates that these biophysical techniques can be used to noninvasively monitor integrity of or changes in secondary and tertiary structure of proteins within the ISCOM particles without the need for protein extraction.

The carbon dioxide hydration activity of the sulfonamide-resistant carbonic anhydrase from the liver of male rat: pH independence of the steady-state kinetics.

The steady-state kinetic constants for the catalysis of CO2 hydration by the sulfonamide-resistant and testosterone-induced carbonic anhydrase from the liver of the male rat has been determined by stopped-flow spectrophotometry. The turnover number was 2.6 +/- 0.6 X 10(3) s-1 at 25 degrees C, and was invariant with pH ranging from 6.2 to 8.2 within experimental error. The Km at 25 degrees C was 5 +/- 1 mM, and was also pH independent. These data are in quantitative agreement with earlier findings of pH-independent CO2 hydration activity for the mammalian skeletal muscle carbonic anhydrase isozyme III. The turnover numbers for higher-activity isozymes I and II are strongly pH dependent in this pH range. Thus, the kinetic status of the male rat liver enzyme is that of carbonic anhydrase III. This finding is consistent with preliminary structural and immunologic data from other laboratories.

Thermodynamics of carbonic anhydrase catalysis. A comparison between human isoenzymes B and C.

The CO2 hydration and HCO3- dehydration activities of human red cell carbonic anhydrase isozymes B and C (HCAB and HCAC) have been studied as a function of temperature from 0 degrees to 37 degrees C. The Arrhenius plots of ln kcat versus 1/T are linear for both isozymes in both hydration and dehydration reactions, indicating that the rate-determining steps remain unchanged over this temperature range. The 37 degrees C hydration kcat, at pH 7.5, is 13 X 10(5) s-1 for isozyme C and 0.71 X 10(5) s-1 for isozyme B. Km, for hydration, is 10 mM for C and 5 mM for B, and invariant with temperature. The uncatalyzed reactions are significantly affected by temperature, 30- to 40-fold rate enhancements being observed from 0 degrees to 37 degrees C. The enzyme-catalyzed processes are much less sensitive to temperature, the rate enhancements being 2- to 3-fold for HCAB and 5- to 6-fold for HCAC in this temperature range. These observations are consistent with a significant lowering of the free energy of activation by both isozymes. This effect is greater for C accounting for its higher catalytic power. The enthalpy of activation, at pH 7.5 and 8.2, in the rate-limiting step is considerably less for the B enzyme compared to C. This is, however, more than offset by a large negative entropy of activation in the case of HCAB. This observation indicates either a mechanistic difference in the rate-limiting events or a difference in the structural organizations of the active sites of the two isozymes, or both.

Quantitative determination of saccharide surfactants in protein samples by liquid chromatography coupled to electrospray ionization mass spectrometry.

A direct and highly selective method, combining liquid chromatography (LC) with electrospray ionization mass spectrometry (ESI-MS), has been developed for quantifying saccharide surfactants. Saccharide surfactants, such as n-octyl-beta-d-glucopyranoside (NOG), are widely used to solubilize or refold membrane-bound or lipophilic proteins. In the present study, we have developed an LC-MS method to quantify NOG in protein samples. Protein-bound NOG was completely dissociated from proteins by reversed-phase LC, allowing the total amount of saccharide surfactant in protein samples to be quantified by MS. A chemical analog of NOG was used as an internal standard for improving the reproducibility of the method. Linearity was found in the range of 10 microg/mL-1.0 mg/mL NOG concentrations. Seven major surfactant oligomeric ions were detected under the ionization conditions applied and their relative abundance was essentially unchanged over the range of 0.05-1.0 mg/mL NOG concentrations. Consequently, ions with characteristic mass-to-charge ratios could be used for quantification of NOG. Analytical accuracy of the method was examined by determining the amounts of NOG recovered from apolipoprotein A-I and myoglobin samples spiked with NOG.

Effect of polyanions on the refolding of human acidic fibroblast growth factor.

Acidic fibroblast growth factor (aFGF) is unstable at physiological temperatures in the absence of polyanions such as heparin. Therefore, the effect of temperature on the kinetics of refolding of aFGF has been examined in the presence and absence of several polyanions. The protein folds into its native state at temperatures up to 30 degrees C without polyanions with an activation energy of approximately 14 kcal/mol, but does not acquire native structure above this temperature. When heparin, inositol hexasulfate, or sulfate ion are present, aFGF refolds below 30 degrees C with a slightly reduced activation energy (10-11 kcal/mol). In addition, the protein now also renatures between 30 and 50 degrees C with activation energies of 1-2 (heparin), 16 (inositol hexasulfate), and 7 (sulfate) kcal/mol. Trace heavy metals appear to inhibit the refolding process, but a molecular chaperone (bovine 70-kDa heat shock cognate protein) and a peptidylprolyl isomerase (the FK506-binding protein) have no effect. It is concluded that the rate of refolding of aFGF at physiological temperatures is probably controlled by the interaction of a native-like state of the protein with an unknown polyanionic species.

Probable role of amphiphilicity in the binding of mastoparan to calmodulin.

Two-dimensional helical wheel diagrams and calculations of mean hydrophobic moments show mastoparan, mastoparan X, and Polistes mastoparan to have all the properties expected for amphiphilic helices. Circular dichroic properties are consistent with a random form for these peptides in dilute aqueous solution, but greater than 50% helix is apparent when the peptides are dissolved in 70% trifluoroethanol/water mixtures (v/v) or when the peptides are bound to calmodulin. Changes in the fluorescence spectra, anisotropy, and accessibility of tryptophan whose indole side chain is on the apolar surface of the amphiphilic helix imply a significant role for the apolar surface in the binding of the mastoparans and another amphiphilic peptide, melittin, to calmodulin. These data provide a useful model for designing high-affinity synthetic peptide inhibitors of calmodulin.

In vivo evidence that theophylline is metabolized principally by CYP1A in rats.

The role of various subfamilies of rat hepatic cytochrome P-450 in the oxidation of theophylline was evaluated by comparing theophylline clearance in control rats and those pretreated with relatively selective inducers and inhibitors of the cytochromes P-450. Pretreatment with the CYP1A inducer, beta-naphthoflavone (BNF), increased theophylline clearance 4.5-fold (p < 0.001), and the CYP1A inhibitor, alpha-naphthoflavone, significantly attenuated the BNF effect. Pretreatment with phenobarbital, an inducer of CYP2B/C in rats, had a far more modest effect, increasing theophylline clearance only 1.6-fold (p < 0.005). The phenobarbital-mediated increase in theophylline clearance was attenuated by orphenadrine, a CYP2B/C inhibitor. The CYP2E inducer, isoniazid and the CYP2E inhibitor, diallyl sulfide were virtually without effect, as was the CYP4A inducer, clofibrate, and the CYP4A inhibitor, 10-undecynoic acid. Ajmaline, and inhibitor of CYP2D, was also without any effect on theophylline clearance. While the powerful CYP3A inducer clotrimazole did not increase theophylline clearance, troleandomycin, an inhibitor of CYP3A, did slow theophylline clearance by about 25% (p < 0.002). Together, these findings suggest that CYP1A is principally responsible for the overall oxidation of theophylline in rats, and that CYP2B/C probably also mediates some theophylline oxidation. The involvement of CYP2D, CYP2E, CYP4A, and CYP3A is relatively trivial.

A transforming growth factor-alpha-Pseudomonas exotoxin hybrid protein undergoes pH-dependent conformational changes conducive to membrane interaction.

TP40 is a chimeric protein containing transforming growth factor alpha (TGF-alpha) at the N-terminus and a derivative of a 40,000-Da segment (PE40 delta cys) of Pseudomonas exotoxin (PE). PE40 delta cys contains domains Ib, II, and III of PE in which the cysteines are mutated to alanines. The rationale for inclusion of TGF-alpha is to provide TP40 with selective targeting toward cells expressing the epidermal growth factor receptor (EGFr) on their surface [Pastan, I., & FitzGerald, D. (1989) J. Biol. Chem. 264, 15157-15160]. Translocation across endosomal membranes is thought to be a required step for cytotoxic activity of PE. This step is presumably facilitated by the low pH in endosomes which induces exposure of a hydrophobic surface of the protein, which in turn becomes available to interact with and translocate across the membrane. We have employed the hydrophobic fluorescence probe 2-p-toludinylnaphthalene-6-sulfonate (TNS) and the intrinsic tryptophan fluorophores of TP40 to investigate pH-induced changes in the tertiary structure of this protein. The pH dependence of TP40 interaction with liposomes also provided a model for studying protein-membrane interactions. TNS fluorescence was markedly enhanced in the presence of TP40 below pH 4 and to a lesser degree between pH 7 and 5. A progressive red shift of tryptophan fluorescence with decreasing pH was also seen with the approximate midpoint for this transition occurring around pH 3. Both observations suggest that acidic pH induces exposure of hydrophobic regions of TP40, making them accessible to solvent and TNS. No major alteration of the secondary structure was manifested in the far-UV CD spectrum of TP40 upon a reduction in pH from 7 to 2. Thus, the low-pH-induced structural change of TP40 appears to involve a subtle exposure of one or more hydrophobic surfaces without an extensive unfolding of the protein's secondary structure. In the presence of anionic liposomes, a low-pH-induced blue shift of the TP40 tryptophan fluorescence was observed, suggesting that interaction with liposomes also required the low-pH conformation of the protein. However, the midpoint of this fluorescence blue shift occurred at approximately pH 5, which is presumably closer to the physiological pH within endosomes. Neutral liposomes failed to induce these spectral changes in TP40, implying a lack of interaction with these lipids. At acidic pH values between 2 and 4, self-association of TP40 in solution was detected by equilibrium sedimentation and quasielastic light scattering measurements. This probably results from intermolecular interaction between exposed hydrophobic surfaces.(ABSTRACT TRUNCATED AT 400 WORDS)

Evidence for an equilibrium intermediate in the folding-unfolding pathway of a transforming growth factor-alpha-Pseudomonas exotoxin hybrid protein.

TP40 is a chimeric protein containing transforming growth factor-alpha (TGF-alpha) at the N-terminus and a Cys-->Ala mutant (PE40 delta Cys) of a 40,000-dalton segment (PE40) of Pseudomonas exotoxin (PE). The guanidine hydrochloride (Gdn-HCl)-induced unfolding of TP40 and PE40 delta Cys has been studied by tryptophan fluorescence, circular dichroism (CD), and high-performance size exclusion chromatography (HPSEC). The equilibrium unfolding of both proteins involves at least one intermediate (I). In the I state(s), which may be induced by 1.3-2.0 M Gdn-HCl, the tertiary structure is fully or partially collapsed as detected by tryptophan fluorescence and near-UV CD, but the protein largely retains the native secondary structure and a semicompact shape as judged by far-UV CD and HPSEC, respectively. Soluble aggregates of TP40 and PE40 delta Cys are observed in addition to monomers at these intermediate (but not at higher) Gdn-HCl concentrations, suggesting that self-association is possibly mediated by thermodynamically stable, partially unfolded I states. The kinetics of refolding of TP40 upon dilution of Gdn-HCl involve two or more phases. Re-formation of secondary structure occurs rapidly (t 1/2 < 10 s) as determined by CD and is followed by a biphasic refolding of the native tertiary structure as detected by changes in tryptophan fluorescence. The midpoint (Tm) of the thermal unfolding transition occurs at a lower temperature when measured by tryptophan fluorescence than when detected by DSC and CD. These data suggest that Gdn-HCl and temperature can induce conformation(s) of TP40 that are distinct from native (N) and unfolded (U) states.(ABSTRACT TRUNCATED AT 250 WORDS)

Analysis of vaccine stability.

The complexity of vaccines creates a unique formulation challenge. Vaccines may consist of one or more types of antigenic component including live attenuated or killed viral or bacterial particles, polysaccharides, proteins, polynucleotides and particle conjugates. In addition, other excipients such as adjuvants may be present. Not only must the chemical and structural integrity of the various components be maintained, but immunogenicity must be ensured. The inherent lability of vaccines can critically limit their distribution, administration, and efficacy in parts of world where it is difficult to maintain a cold chain. Combination with other vaccines and oral administration may also compromise vaccine stability. Successful vaccine stabilization strategies include both empirical efforts to screen and identify appropriate stabilizers and environmental conditions and more rational approaches toward developing an understanding of the causes and mechanisms of vaccine inactivation. In principle, by elucidating the conformational and chemical pathways of macromolecular inactivation, more rational strategies to minimize their occurrence can be adopted. This presentation will review the application of classical techniques such as viral plaque assays to identify vaccine stabilizers by empirical testing. The potential of using various biophysical techniques (both hydrodynamic and spectroscopic methods) to characterize the physicochemical stability of purified vaccine preparations (Hepatitis A and B) is also explored.

The pH dependence of the hydration of CO2 catalyzed by carbonic anhydrase III from skeletal muscle of the cat. Steady state and equilibrium studies.

We have measured the pH dependence of the kinetics of CO2 hydration catalyzed by carbonic anhydrase III from the skeletal muscle of the cat. Two methods were used: an initial velocity study in which the change in absorbance of a pH indicator was measured in a stopped flow spectrophotometer, and an equilibrium study in which the rate of exchange of 18O between CO2 and H2O was measured with a mass spectrometer. We have found that the steady state constants kCO2 cat and KCO2 m are independent of pH within experimental error in the range of pH 5.0 to 8.5; the rate of release from the enzyme of the oxygen abstracted from substrate HCO-3 in the dehydration is also independent of pH in this range. This behavior is very different from that observed for carbonic anhydrase II for which kCO2 cat and the rate of release of substrate oxygen are very pH-dependent. The rate of interconversion of CO2 and HCO-3 at equilibrium catalyzed by carbonic anhydrase III is not altered when the solvent is changed from H2O to 98% D2O and 2% H2O. Thus, the interconversion probably proceeds without proton transfer in its rate-limiting steps, similar to isozymes I and II.