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

N.m.r. studies of cycloamylose inclusion-complexes with p-substituted phenols

Aqueous solutions of inclusion complexes of host cyclomaltohexa- and cyclomaltohepta-ose (cyclohexa- and cyclohepta-amylose) with guest phenols p-substituted with Br, Cl, CN, NO₂, CH₃, CHO, and COOH have been studied by ¹H- and ¹³C-n.m.r. spectroscopy. The stability of the complexes depends on the guests, the cycloamyloses, and the ionisation states of the host hydroxyl groups. The ¹H data for the host protons in the complexes show that the guests are more deeply inserted into the cavity of cyclomaltoheptaose than into that of cyclomaltohexaose.     

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.     

13C 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.     

Ovalbumin labeling with p-hydroxymercurybenzoate: The effect of different denaturing agents and the kinetics of reaction

The aim of our study was to investigate how denaturing agents commonly used in protein analysis influence the labeling between a reactive molecule and proteins. For this reason, we investigated the labeling of ovalbumin (OVA) as a globular model protein with p-hydroxymercurybenzoate (pHMB) in its native state (phosphate buffer solution) and in different denaturing conditions (8 mol L⁻¹ urea, 3 mol L⁻¹ guanidinium thiocyanate, 6 mol L⁻¹ guanidinium chloride, 0.2% sodium dodecyl sulfate, and 20% methanol). In addition to chemical denaturation, thermal denaturation was also tested. The protein was pre-column simultaneously denatured and derivatized, and the pHMB-labeled denatured OVA complexes were analyzed by size exclusion chromatography (SEC) coupled online with chemical vapor generation–atomic fluorescence spectrometry (CVG–AFS). The number of –SH groups titrated greatly depends on the protein structure in solution. Indeed, we found that, depending on the adopted denaturing conditions, OVA gave different aggregate species that influence the complexation process. The results were compared with those obtained by a common alternative procedure for the titration of –SH groups that employs monobromobimane (mBBr) as tagging molecule and molecular fluorescence spectroscopy as detection technique.     

Chitosan-graft poly(p-dioxanone) copolymers: preparation, characterization, and properties

A new biodegradable copolymer of chitosan and poly(p-dioxanone) (PPDO) was prepared through a protection-graft-deprotection procedure using N-phthaloyl-chitosan as an intermediate. PPDO terminated with the isocyanate group was allowed to react with hydroxyl groups of the N-phthaloyl-protected chitosan, and then the phthaloyl group was cleaved to give the free amino groups. The length of PPDO graft chains can be controlled easily by using the prepolymers of PPDO with different molecular weights. The resulting products were thoroughly characterized with FT-IR, ¹H NMR, TG, DSC, SEM, and WAXD. The copolymers were used as drug carriers for sinomenine (7,8-didehydro-4-hydroxy-3,7-dimethoxy-17-methyl-9α,13α,14α-morphinan-6-one) and these exhibited a significant controlled drug-releasing behavior whether in artificial gastric juice or in neutral phosphate buffer solution.     

Biosynthesis of p-hydroxybenzoic acid in poplar lignin

Biosynthetic pathways to p-hydroxybenzoic acid in polar lignin were examined by tracer experiments. High incorporation of radioactivity to the acid was observed when shikimic acid-[1-¹⁴C], phenylalanine-[3-¹⁴C], trans-cinnamic acid-[3-¹⁴C], p-coumaric acid-[3-¹⁴C] and p-hydroxybenzoic acid-[COOH-¹⁴C] were administered, while incorporation was low from shikimic acid-[COOH-¹⁴C], phenylalanine-[1-¹⁴C], phenylalanine-[2-¹⁴C], tyrosine-[3-¹⁴C], benzoic acid-[COOH-¹⁴C], sodium acetate-[1-¹⁴C] and d-glucose-[U-¹⁴C]. Thus p-hydroxybenzoic acid in poplar lignin is formed mainly via the pathway: shikimic acid → phenylalanine → trans-cinnamic acid → p-coumaric acid → p-hydroxybenzoic acid.     

Conformational changes resulting from the carbazoylation of bovine α-chymotrypsin by p-nitrophenyl N2-acetyl-N1-arylmethylcarbazates

Several (N²-acetyl-N¹-arylmethylcarbazoyl)-α-chymotrypsins with p-substituents in the N¹-arylmethyl group have been prepared. Measurements of (a) accessibility of tryptophyl residues to modification by 2-hydroxy-5-nitrobenzyl bromide, (b) intrinsic fluorescence spectra in the absence and presence of sodium dodecyl sulphate, (c) thermal perturbation spectra indicate that, in general, tryptophyl residues are less accessible to solvent than in the free enzyme and the modified enzymes are more stable than α-chymotrypsin to denaturation by heat or sodium dodecyl sulphate. N²-Acetyl-N¹-p-dimethylaminobenzylcarbazoyl-α-chymotrypsin, however, contains more accessible tryptophyl residues than the other derivatives and is thermally less stable although it is more stable than the free enzyme.     

Stereochemical structure determination of p-cymene Ru(II) complexes containing the PPh2Py ligand with 2-D NOESY and HMQC NMR experiments

Complexes [Ru(p-cymene)Cl₂(PPh₂Py)] (1) and [Ru(p-cymene)Cl(PPh₂Py)]BF₄ (2) were studied by means of ¹H, ¹³C{¹H} 2-D NOESY and HMQC NMR spectral methods. NMR data agree with C₁ and C_s symmetries for complexes 1 and 2, respectively. NOESY cross-peaks allowed the assignment of signals to CH arene protons and in the case of complex 2 the determination of the molecular stereochemistry. These results are in agreement with the X-ray molecular structures of both complexes.     

The effect of sodium on inorganic phosphate- and p-nitrophenyl phosphate-facilitated ouabain binding to (Na+ + K+)-activated ATPase

The effect of the hydrolysis product P_i and the artificial substrate p-nitrophenyl phosphate (p-nitrophenyl-P) on ouabain binding to (Na⁺ + K⁺)-activated ATPase was investigated.The hypothesis that (Mg²⁺ + p-nitrophenyl-P)-supported ouabain binding might be due to P_i release and thus (Mg²⁺ + P_i)-supported could not be confirmed.The enzyme · ouabain complexes obtained with different substrates were characterized according to their dissociation rates after removal of the ligands facilitating binding. The character of the enzyme · ouabain complex is determined primarily by the monovalent ion present during ouabain binding, but, qualitatively at least, it is immaterial whether binding was obtained with p-nitrophenyl phosphate or P_i.The presence or absence of Na⁺ during binding has a special influence upon the character of the enzyme · ouabain complex. Without Na⁺ and in the presence of Tris ions the complex obtained with (Mg²⁺ + P_i) and that obtained with (Mg²⁺ + p-nitrophenyl-P) behaved in a nearly identical manner, both exhibiting a slow decay. High Na⁺ concentration diminished the level of P_i-supported ouabain binding, having almost no effect on p-nitrophenyl phosphate-supported binding. Both enzyme · ouabain complexes, however, now resembled the form obtained with (Na⁺ + ATP), as judged from their dissociation rates and the K⁺ sensitivity of their decay. The complexes obtained at a high Na⁺ concentration underwent a very fast decay which could be slowed considerably after adding a low concentration of K⁺ to the resuspension medium. The most stable enzyme · ouabain complex was obtained in the presence of Tris ions only, irrespective of whether p-nitrophenyl phosphate or P_i facilitated complex formation. The presence of K⁺ gave rise to a complex whose dissociation rate was intermediate between those of the complexes obtained in the presence of Tris and a high Na⁺ concentration.It is proposed that the different ouabain dissociation rates reflect different reactive state of the enzyme. The resemblance between the observations obtained in phosphorylation and ouabain binding experiments is pointed out.     

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.     

Acid dissociation of cyclooctaamylose

Acid dissociation constants of aqueous cyclooctaamylose (8-Cy) have been determined at 15–45°C by pH potentiometry. Standard enthalpies and entropies of dissociation are derived from the temperature dependences of these pK_a's. These results are compared to corresponding measurements of aqueous cyclohexaamylose and cycloheptaamylose, and the observed trends are interpreted in terms of complexation of cycloamylose with hydroxide ion. ¹³C-nmr spectral measurements are reported for 8-Cy in 99.8% D₂O solution, and assignments of the observed lines are made with the help of deuterium-induced differential isotopic shift experiments.     

Ditopic tris(2-mercaptoimidazol-1-yl)borate ligands and their coordination behavior toward [Ru(p-cymene)]

The ditopic tris(2-mercaptoimidazol-1-yl)borate ligand K₂[(mt^Et)₃B-B(mt^Et)₃] cannot be prepared from B₂(NMe₂)₄/4 Hmt^Et/2 Kmt^Et, because the stable intramolecular diadduct (mt^Et)B(μ-mt^Et)₂B(mt^Et) is generated instead (Hmt^Et = 2-mercapto-1-ethylimidazole). Introduction of a meta- or para-phenylene spacer between the two boron atoms precludes the 2-mercaptoimidazol-1-yl groups from adopting a bridging position so that the potassium salts K₂[(mt^Et)₃B-(m-C₆H₄)-B(mt^Et)₃] and K₂[(mt^Et)₃B-(p-C₆H₄)-B(mt^Et)₃] become readily accessible. These ligands react with [(p-cym)RuCl₂]₂ to give the dinuclear Ru^II complexes [(p-cym)Ru(mt^Et)₃B-(m-C₆H₄)-B(mt^Et)₃Ru(p-cym)]Cl₂ and [(p-cym)Ru(mt^Et)₃B-(p-C₆H₄)-B(mt^Et)₃Ru(p-cym)]Cl₂ (p-cym = p-cymene). After the exchange of the Cl⁻ counterions for [PF₆]⁻, both complexes have been crystallized and structurally characterized by X-ray diffraction.     

Carbon-13 nmr spectra of vitamin B6 compounds: Aqueous solution equilibria and determination of microscopic acid dissociation constants

The aqueous solution equilibria and solute structure of vitamin B₆ compounds and several model compounds have been investigated using ¹³C-nmr spectroscopy. The unsubstituted α-carbon of these compounds is a very good probe for data which permits assignment of the ionization steps to indicidual groups. While the ionizations of the pyridinium and phenolic groups take place simultaneously in 3-hydroxypyridine, they take place in well-separated steps in pyridoxamine (PM), pyridoxamine phosphate (PMP), and pyridoxal phosphate. It has been established that the ionization with a pK_a value of 3.7 is predominantly phenolic in origin in PM and PMP. A zwitterionic structure consistent with the earlier spectroscopic investigations is proposed for the vitamin B₆ compounds in neutral aqueous solution.     

Giardia lamblia: Characterization of ecto-phosphatase activities

Ecto-phosphatase activities of Giardia lamblia were characterized in intact cells, which are able to hydrolyze the artificial substrate p-nitrophenylphosphate (p-NPP) to p-nitrophenol (p-NP) at a rate of 8.4 ± 0.8 nmol p-NP/h/10⁷ cells. The ecto-phosphatase activities were inhibited at high pH as well as by classical inhibitors of acid phosphatases, such as sodium fluoride and sodium molybdate and by inorganic phosphate, the final product of the reaction. Experiments using a classical inhibitor of phosphotyrosine phosphatase, sodium orthovanadate, also showed that the ecto-phosphatase activity was inhibited in a dose-dependent manner. Different phosphorylated amino acids were used as substrates for the G. lamblia ecto-phosphatase activities the highest rate of phosphate release was achieved using phosphotyrosine. Not only p-NPP hydrolysis but also phosphotyrosine hydrolysis was inhibited by sodium orthovanadate. Phosphotyrosine but not phospho-serine or phospho-threonine inhibited the p-nitrophenylphosphatase activity. We also observed a positive correlation between the ecto-phosphatase activity and the capacity to encystation of G. lamblia trophozoites.     

Antimicrobial agents

Among 15 antimycotic agents tested, maximum activity was displayed by 5,7-dichloro-8-oxyquinoline, both as regards the microorganism spectrum and the minimum inhibitory concentration. The antimycotic effect of 2-aminotridecane-undecylenate and 2-aminotridecane-2-chloro-4-nitrophenolate was greater than that of 2-aminotridecane, undecylenic acid and 2-chloro-4-nitrophenol alone. Biochemical resistance develops relatively rapidly in aTrichophyton mentagrophytes strain on contact with fungicidin, 2-chloro-4-nitrophenol and 5,7-dichloro-8-oxyquinoline. ATrichophyton mentagrophytes strain resistant to one of these three agents is also several times less sensitive to all the other test substances except septonex.     

Structure-activity relationship study of the cell-penetrating peptide pVEC

The peptide pVEC is a recently described cell-penetrating peptide, derived from the murine vascular endothelial-cadherin protein. In order to define which part of this 18-amino acid long peptide is important for the cellular translocation, we performed a structure-activity relationship study of pVEC. Together with the l-alanine substituted peptides, the retro-pVEC, D-pVEC and the scramble pVEC are studied for comparison. The peptide analogues are labeled with carboxyfluorescein at the N-terminus for monitoring the cellular uptake into human Bowes melanoma cells with different efficacy. We show that all the Fl-pVEC analogues internalize in live Bowes melanoma cells. l-Alanine substitution of the five respective N-terminal hydrophobic amino acids significantly decreases the translocation property, while replacing of Arg⁶, Arg⁸ or Ser¹⁷ by alanine enhances the uptake. The uptake of pVEC is significantly reduced by treatment with an endocytosis inhibitor wortmannin. Treatment with heparinase III, nystatin and EIPA had no effect on the peptide uptake. The data presented here show that the N-terminal hydrophobic part of pVEC is crucial for efficient cellular translocation.