The molecular disposition of p-nitrophenol and sodium p-nitrophenolate in the cyclohexaamylose cavity: A 13C probe

The direction in which both sodium p-nitrophenolate and p-nitrophenol penetrate the cyclohexaamylose cavity in aqueous solution has been examined by ¹³C-nmr. Both sodium p-nitrophenolate and p-nitrophenol penetrate the cavity asymetrically and quite specifically nitro group first with the phenolic oxanion or hydroxyl group pointing out into solution as evidenced by the nature of the changes in the meta-carbon-¹³ shifts. The stoichiometries of the complexes can be defined for various host-to-guest molar ratios. Finally, the potential of these cycloamylose complexes as models for studying the effects of intermolecular interaction of the enzyme substrate type on the ¹³C-nmr of both host and guest molecules is discussed.     

The molecular disposition of sodium p-nitrophenolate in the cavities of cycloheptaamylose and cyclohexaamylose in solution

The extent to which sodium p-nitrophenolate penetrates the cycloheptaamylose and cyclohexaamylose cavities has been defined by nmr studies of the complexes in aqueous solution. Measurements of changes in the ¹H nmr spectra of both the sodium p-nitrophenolate guest and the cyclohexaamylose host, along with an intermolecular nuclear Overhauser effect, reveal that this guest only partially penetrates the cyclohexaamylose cavity and does so nitro end first. With cyclohepaamylose, sodium p-nitrophenolate penetrates more deeply, but the orientation may be less specific. These findings are in accord with the notion that both London dispersion forces and removal of high energy cavity water contribute to substrate binding.     

Calorimetric studies of p-nitrophenol binding to α- and β-cyclodextrin

The thermodynamics of the binding of p-nitrophenol to both α- and β-cyclodextrin has been investigated as a function of pH and solvent composition using the technique of flow microcalorimetry. A preferential binding of the anionic form of the ligand is found for both cyclodextrins. It is postulated that the additional affinity for p-nitrophenolate is due to a dispersion interaction between the cyclodextrin cavity and the delocalized charge of the ligand. Studies of the binding of p-nitrophenol analogs and of the binding of p-nitrophenol as a function of ionic strength and DMSO cosolvent composition are consistent with this theory.     

C nuclear magnetic resonance observations on the interaction between p-amidinophenylpyruvic acid and trypsin

The formation of an enzyme-inhibitor adduct between bovine trypsin and [2-¹³C]p-amidinophenylpyruvic acid has been investigated by ¹³C NMR spectroscopy. The observation of a resonance at 100.8 ppm demonstrates that the hemiketal formed between the hydroxyl of serine-195 and the 2-¹³C carbon of p-amidinophenylpyruvic acid is sp³ hybridized with no significant deviation from tetrahedral geometry. It is shown that stabilization of the hemiketal oxyanion if it occurs is less effective than in chloromethylketone inhibitor complexes. The tetrahedral adduct is stable from pH 3 to 8. The mechanisms of breakdown of the tetrahedral adduct at pH extremes are discussed.     

Effect of the sugar moiety on complex formation of cycloamyloses with p-nitrophenyl glycosides

Circular diochroism (CD) spectra of four p-nitrophenyl glycosides and their cycloamylose complexes were investigated at various concentrations of cycloamylose and at temperatures ranging from 20 to 60°C. The CD spectra of p-nitrophenyl glycosides changed remarkably in the presence of cycloamyloses, and significant differences in spectral shape and intensity were observed between the cyclohexaamylose complex and the cycloheptaamylose complex. The difference CD spectra between the free guest and its complex indicates that the nitrophenyl group is included in the cycloamylose cavity but its disposition is different between the complexes with cyclohexaamylose and cycloheptaamylose. Values of enthalpy and entropy of the cyclohexaamylose complex are considerably larger than those of the corresponding cycloheptaamylose complex, although the free energy differs only slightly. It is suggested that the nitrophenyl group is more loosely bound to the cycloheptaamylose cavity than to the cyclohexaamylose cavity, and has much more flexibility in its disposition.     

Biodegradation of p-nitrophenol by methyl parathion-degrading Ochrobactrum sp. B2

Ochrobactrum sp. B2, a methyl parathion-degrading bacterium, was proved to be capable of using p-nitrophenol (PNP) as carbon and energy source. The effect of factors, such as temperature, pH value, and nutrition, on the growth of Ochrobactrum sp. B2 and its ability to degrade p-nitrophenol (PNP) at a higher concentration (100 mg l⁻¹) was investigated in this study.The greatest growth of B2 was observed at a temperature of 30 °C and alkaline pH (pH 9–10). pH condition was proved to be a crucial factor affecting PNP degradation. Enhanced growth of B2 or PNP degradation was consistent with the increase of pH in the minimal medium, and acidic pH (6.0) did not support PNP degradation. Addition of glucose (0.05%, 0.1%) decreased the rate of PNP degradation even if increased cell growth occurred. Addition of supplemental inorganic nitrogen (ammonium chloride or ammonium sulphate) inhibited PNP degradation, whereas organic nitrogen (peptone, yeast extract, urea) accelerated degradation.     

C.P.-M.A.S. 13C-N.M.R. study of some solid-state inclusion complexes of cyclomalto-oligosaccharides with para-substituted benzenes

Cross polarisation—magic-angle sample spinning ¹³C-n.m.r. spectral have been measured in the solid state for p-nitrophenol, p-iodophenol, and their inclusion complexes with cyclomaltohexaose, cyclomaltoheptaose, and methylated cyclomaltohexaose. Analysis of the line-shapes of the resonances and the dipolar-dephasing experiments indicate that the guest molecules undergo motion in the host cavities, whereas the host molecules are almost static. The mode and rate of guest motion depend on the size of the cavity.     

Crystal structures of cyclomaltohexaose (α-cyclodextrin) complexes with p-chlorophenol and p-cresol

Crystal structures of cyclomaltohexaose (α-cyclodextrin) complexes with p-chlorophenol and p-cresol have been determined by single-crystal X-ray diffraction studies. The space group of the α-cyclodextrin–p-chlorophenol complex is P2₁2₁2₁ with unit cell dimensions of a=15.299(3), b=24.795(5), c=13.447(5) Å, and that of the α-cyclodextrin–p-cresol complex is P2₁ with unit cell dimensions of a=7.927(7), b=13.568(7), c=24.54(1) Å, β=90.41(8)°. In spite of the similar structures of guest molecules, both complexes have different inclusion modes and packing structures.     

1H and13C NMR studies of the spatial arrangement ofp-nitrophenol and acetate at the catalytic sites of Mn(II)-and Co(II)-substituted human carbonic anhydrase C

Nuclear relaxation studies were performed on Mn²⁺ and Co²⁺-substituted human carbonic anhydrase C (HCAC) to determine the active site configurations of the products ofp-nitrophenylacetate hydrolysis. Metal-ligand distances calculated from the magnitudes of the paramagnetic effect on longitudinal nuclear relaxation rates indicate direct coordination of the phenolic oxygen ofp-nitrophenol to the enzyme-bound Mn²⁺ in the ternary HCAC-Mn²⁺-p-nitrophenol complex. The Mn²⁺ top-nitrophenol distances in the quaternary HCAC-Mn²⁺-acetate-p-nitrophenol and HCAC-Co²⁺-p-nitrophenol-acetate complexes are also consistent with coordination ofp-nitrophenol to the enzyme-bound metal via the phenolic oxygen. However, the orientation of the aromatic ring ofp-nitrophenol appears to be different for the Co²⁺ and Mn²⁺-enzymes. The carboxyl carbon of acetate is in the range of innersphere distances expected for direct coordination of a carboxyl oxygen to the enzyme-bound Mn²⁺, and the acetate molecule is folded around the metal. Although direct coordination of acetate to the metal in the HCAC-Co²⁺-p-nitrophenol-acetate complex cannot be inferred from our data, weak innersphere complexation with a somewhat longer Co²⁺-carboxyl oxygen bond distance is not excluded. We found small but significant differences in the arrangement of the products at the active sites of the Co²⁺ and Mn²⁺-enzymes which may be responsible for the differences in their reactivities in the hydrolysis reaction.     

Secondary structure of peptides. 18. 15N-nmr and 13C-nmr CP/MAS investigation of the primary and secondary structure of (Ala/Val) copolypeptides

Various copolypeptides were prepared by benzylamine or tertiary amine-initiated copolymerizations of alanine–N-carboxyanhydride (Ala-NCA) and valine–N-carboxyanhydride (Val-NCA). The number-average molecular weights of these copolypeptides were detemined by ¹H-nmr spectroscopic end-group analyses and viscosity measurements. The sequences were characterized by ¹⁵N-nmr spectra in solution, and the average lengths of the homogeneous blocks were determined from the signal intensities. The 50.3-and 75.4-MHz ¹³C-nmr CP/MAS spectra of the solid copolypeptides are not sensitive to sequence effects, but allow qualitative and quantitative analyses of the secondary structures. In contrast to other methods, the ¹³C-nmr spectra allow determination of the extent to which individual amino acids are incorporated into β-sheet or α-helix phases. Depending on primary structure and molecular weight, the secondary structure of (Ala/Val) copolypeptides may vary significantly. Both monomer units may be predominantly helical or predominantly β-sheet structure, or the Val units may prefer the β-sheet structure with most Ala-units forming β-helices. However, these secondary structures are more or less thermodynamically unstable and revert to the stable conformations on reprecipitation from trifluoroacetic acid/water.     

Interactions of D-cellobiose with p-toluenesulfonic acid in aqueous solution: a C NMR study

The effects of adding D₂SO₄, and p-toluenesulfonic acid-d to D-cellobiose dissolved in D₂O were investigated at 23 °C by plotting ¹³C NMR chemical shift changes (Δδ) against the acid to D-cellobiose molar ratio. ¹³C Chemical shifts of all 18 carbon signals from α and β anomers of D-cellobiose showed gradual decreases due to increasing acidity in aqueous D₂SO₄ medium. The C-1 of the α anomer showed a slightly higher response to increasing D⁺ concentration in the surrounding. In the aqueous p-toluenesulfonic acid-d medium, C-6′ and C-4′ carbons of both α, and β anomeric forms of D-cellobiose are significantly affected by increasing the sulfonic acid concentrations, and this may be due to a 1:1 interaction of p-toluenesulfonic acid-d with the C-6′, C-4′ region of the cellobiose molecule.     

An easy colorimetric assay for glycosyltransferases

Glycosyltransferases are involved in biosynthesis of both protein-bound and non-bound glycans that have multiple and important biological functions in all species. A variety of methods for assaying glycosyltransferase activity have been developed driven by the specific interests and type of information required by researchers. In this work, a novel colorimetric assay for the glycosyltransferase-catalyzed reaction was established. Compared with measuring the newly formed product, which might not exhibit visible absorption, the unreacted acceptor could be readily detected by measuring the visible absorption of the hydrolysis product. In the assay, 4-nitrophenyl-β-D-glycoside (glycosyl-β-pNP) is used as the glycosyl acceptor, which can be hydrolyzed by a special exoglycosidase to release the p-nitrophenol before glycosylation reactions. Absorbance change of the p-nitrophenolate corresponds to unreacted glycosyl acceptor that accompanied the glycosyl transfer. The assay is demonstrated to be useful in the initial characterization of recombinant glycosyltransferases for their kinetic parameters, optimal metal cofactor, and pH value. It provides a simple, sensitive, and quantitative method for assessing glycosyltransferase activity and is thus expected to have broad applications including automated high-throughput screening.     

13C-NMR chemical shifts and the conformations of rigid polypeptides

¹³C-nmr chemical shifts of backbone carbonyl and side-chain β-carbons in polypeptides provide structural information. Recent utilization of substituent effects on ¹³C-nmr chemical shifts (principally γ-effects) has permitted the rationalization of their sequence and conformation dependence when observed in linear, flexible polypeptides. In this report, we apply the γ-effect method to study the ¹³C-nmr chemical shifts observed in solution and in the solid state for the backbone carbonyl and side-chain β-carbons in conformationally rigid polypeptides, which are usually cyclic. As found previously for flexible, linear polypetides, the relative ¹³C-nmr chemical shifts observed for the backbone carbonyl and side-chain β-carbons in conformationally rigid polypeptides are predictable from knowledge of their peptide residue sequence (primary structure) and conformation (secondary structure) via the γ-effect method.     

Interactions between bovine plasma albumin and sodium dodecyl sulfate studied by means of 13C-NMR spectra.

The interactions between the protein, bovine plasma albumin, and surfactant, sodium dodecyl sulfate, have been studied by ¹³C-nmr spectroscopy at pH 5.4–6.8 in D₂O solution. The ¹³C chemical shifts and the ¹³C spin-lattice relaxation time of the individual carbons of the surfactant were measured as a function of the molar ratio of the surfactant to albumin in order to analyze the surfactant-protein interaction and the molecular motion of the surfactant. It was found that in the region of initial binding of the surfactant to the high-affinity sites on the protein, both the surfactant head group and alkyl chain interact with the protein. With an excess of high-affinity sites at the beginning of the reaction, surfactant molecules are in a micellelike environment in which the surfactant's alkyl chains are associated with nonpolar groups of the protein. Even after the denaturation by many surfactant bindings, much of the secondary and higher structure seems to remain intact.     

Degradation of p-nitrophenol by the phototrophic bacterium Rhodobacter capsulatus

The phototrophic bacterium Rhodobacter capsulatus detoxified p-nitrophenol and 4-nitrocatechol. The bacterium tolerated moderate concentrations of p-nitrophenol (up to 0.5 mM) and degraded it under light at an optimal O₂ pressure of 20 kPa. The bacterium did not metabolize the xenobiotic in the dark or under strictly anoxic conditions or high O₂ pressure. Bacterial growth with acetate in the presence of p-nitrophenol took place with the simultaneous release of nonstoichiometric amounts of 4-nitrocatechol, which can also be degraded by the bacterium. Crude extracts from R. capsulatus produced 4-nitrocatechol from p-nitrophenol upon the addition of NAD(P)H, although at a very low rate. A constitutive catechol 1,2-dioxygenase activity yielding cis,cis-muconate was also detected in crude extracts of R. capsulatus. Further degradation of 4-nitrocatechol included both nitrite- and CO₂-releasing steps since: (1) a strain of R. capsulatus (B10) unable to assimilate nitrate and nitrite released nitrite into the medium when grown with p-nitrophenol or 4-nitrocatechol, and the nitrite concentration was stoichiometric with the 4-nitrocatechol degraded, and (2) cultures of R. capsulatus growing microaerobically produced low amounts of ¹⁴CO₂ from radiolabeled p-nitrophenol. The radioactivity was also incorporated into cellular compounds from cells grown with uniformly labeled ¹⁴C-p-nitrophenol. From these results we concluded that the xenobiotic is used as a carbon source by R. capsulatus, but that only the strain able to assimilate nitrite (E1F1) can use p-nitrophenol as a nitrogen source. Received: 30 December 1996 / Accepted: 3 September 1997     

13C-nmr spectroscopy of red algal galactans

¹³C-nmr spectra of red seaweed galactans, belonging to the agar and carrageenan groups or having the “intermediate” type of structure, were interpreted on the basis of ¹³C-nmr spectra of model compounds. Signal assignments have been made for most of the known extreme structures of such galactans. ¹³C-nmr spectroscopy was shown to be a rapid and convenient method of structural analysis, which permits one to determine the type of galactan structure, the absolute configurations of its constituents (galactose and 3,6-anhydrogalactose), and the positions of the sulfate and O-methyl groups in a polysaccharide molecule.     

Metabolism of nitrophenols by bacteria isolated from parathion-amended flooded soil

Two bacterial isolates from parathion-amended flooded soil, Pseudomonas sp. and Bacillus sp., were examined for their ability to decompose nitrophenols. Uniformly labelled ¹⁴C-p-nitrophenol was metabolized by both bacteria, ¹⁴CO₂ and nitrite being end products. A substantial portion (23% for Pseudomonas sp. and 80% for Bacillus sp.) of radioactivity applied as p-nitrophenol was accounted for as ¹⁴CO₂ at the end of a 72-h period; 8 to 16% remained in the water phase after solvent extraction. Pseudomonas sp. produced nitrite also from 2,4-dinitrophenol, but only after a lag, and not from o- and m-nitrophenols. Interestingly, m-nitrophenol, known for its resistance to biodegradation because of meta substitution, was decomposed by Bacillus sp., resulting in the formation of nitrite and phenol; o-nitrophenol and 2,4-dinitrophenol resisted degradation by this bacterium.     

Optimization of activated carbon fiber preparation from Kenaf using K2HPO4 as chemical activator for adsorption of phenolic compounds

The present work reports the preparation of activated carbon fiber (ACF) from Kenaf natural fibers. Taguchi experimental design method was used to optimize the preparation of ACF using K₂HPO₄. Optimized conditions were: carbonization at 300 °C, impregnation with 30% w/v K₂HPO₄ solution and activation at 700 °C for 2 h with the rate of achieving the activation temperature equal to 2 °C min⁻¹. The surface characteristics of the ACF prepared at optimized conditions were also studied using pore structure analysis, scanning electron microscopy (SEM) and Fourier transform infrared (FT-IR) spectroscopy. Pore structure analysis shows that micropores constitute the most of the porosity of the prepared ACF. The ability of the ACF prepared at optimized conditions to adsorb phenol and p-nitrophenol from aqueous solution was also investigated. The equilibrium data of phenol and p-nitrophenol adsorption on the prepared ACF were well fitted to the Langmuir isotherm. The maximum adsorption capacities of phenol and p-nitrophenol on the prepared ACF are 140.84 and 136.99 mg g⁻¹, respectively. The adsorption process follows the pseudo-first-order kinetic model.     

13C-Nmr studies of ACTH: Assignment of resonances and conformational features

The biologically active ACTH(1–32) and ACTH(1–24) and other shorter peptide segments of the native hormone ACTH(1–39) were studied in aqueous solution by ¹³C-nmr. In order to identify the ¹³C resonances—except those of the carbonyls—both high-field nmr spectroscopy measurements and substitution of residues with amino acids enriched to 85% in ¹³C were carried out. The main results are (1) the direct characterization of the cis–trans isomerism of proline 24 and its effects on the directly connected and sequentially neighboring residues and (2) findings that the conformational features agree with an α-helix type organization in the N-terminal part of the ACTH molecule which is responsible for the biological activity.     

Effects of molecular association on structure and dynamics of a collagenous peptide

Peptide GVKGDKGNPGWPGAPY from the triple-helix domain of type IV collagen aggregates in solution at a critical aggregation concentration of 18 mM. This molecular self association process is investigated by ¹H- and ¹³C-nmr spectroscopy. As a function of increasing peptide concentration, selective ¹H resonances are cooperatively chemically shifted by up to 0.04 ppm to apparently saturable values at high concentration. Pulsed field gradient nmr was used to derive translation diffusion constants that, as the peptide concentration is increased, also cooperatively and monotonically decrease to an apparent limiting value. An average number of 6 monomer units per aggregate have been estimated from diffusion constant and ¹³C relaxation data. Comparative ¹H nuclear Overhauser effect spectroscopy (NOESY) spectra accumulated at high and low peptide concentrations suggest that average internuclear distances are decreased as a result of peptide association. ¹³C-nmr multiplet spin-lattice relaxation and ¹³C- {¹H} NOE effects on ¹³C-enriched glycine methylene positions in the peptide demonstrate that overall molecular tumbling and backbone internal motions are attenuated in the aggregate state. Lowering the solution pD from pD 6 to pD 2 disrupts the aggregate state, suggesting a role for electrostatic interactions in the association process. Based on thermodynamic considerations, hydrophobic interactions also probably act to stabilize the aggregate state. These data are discussed in terms of an nmr/NOE constrained computer-modeled structure of the peptide. © 1993 John Wiley & Sons, Inc.