Light-scattering studies of pectic substances in aqueous solution

The behaviour of pectic substances in aqueous solution has been studied by the light-scattering technique. Pectic substances were found to be present as high molecular aggregates having strong, intermolecular interactions, and their behaviour depends upon the nature of the carbohydrate chain. Additives can cause dissociation of the polysaccharide aggregates, resulting, in some cases, in particles having similar weight-average ( _w) and number-average ( _n) molecular weights.     

Conformation of poly-L-tyrosine in aqueous solution

The conformational transition of poly-L -tyrosine in 0.1M KCl was investigated by ORD and infrared spectroscopy, potentiometric titration, and sedimentation velocity experiments. It is shown that the fully ordered conformer is obtained by slow titration of the random coil with 0.1N HCl at 25°C. The charge-induced transition, at variance with other poly-α-amino acids, is completed in a narrow range of α. An aggregation process was detected both by potentiometric titration and sedimentation velocity. The polyamino acid aggregates around α = 0.7 at 25°C when the conformational transition is almost complete. Infrared spectra, in the region of the amide I band (1650 cm^?1) showed that the transition is a random coil → antiparallel β one. Evidence exists that the form is of the intramolecular type. The foregoing interpretations of ORD and CD spectra in terms of the α-helix conformation are discussed.     

Circular dichroic spectrum of poly-L-tyrosine in aqueous solution

The UV and CD spectra of poly-L-tyrosine were investigated at pH 10.6 and pH 11.2. At pH 10.6 (μ=0.1), the CD spectrum exhibits a medium positive band at 230mμ, an extremely small negative band at 217mμ, and a large positive band at 200mμ. At pH 11.2 (μ=0.1), a new positive CD band appears at 277mμ while the bands at 230mμ and 217mμ are shifted to longer wavelengths by 15 and 10mμ respectively. These results, together with UV spectral data and a specific rotation- pH profile, suggest that at pH 10.6, poly-L-tyrosine exists in the helical conformation with only a small fraction of its side chains ionized; at pH 11.2, the polypeptide retains its helical structure but with a considerable increase in ionization.     

Conformation of poly-L-tyrosine in trimethyl phosphate solution

Absorption, circular dichroism (CD), and optical rotatory dispersion (ORD) measurements were carried out on poly-L -tyrosine in trimethyl phosphate solution over the spectral range 185–600 mμ. There is evidence in the CD spectrum for side chain-side chain interactions in poly-L -tyrosine. ORD and CD data in dimethylformamide and pyridine closely parallel those in trimethyl phosphate, indicating a similarity in conformation of the polymer in all three solvents. In the polarized infrared spectrum both position and polarization of amide A, I, and II bands are characteristic of α-helical polypeptides. Bands corresponding to side chain also exhibit dichroism, suggesting that the side chains are not randomly oriented. Viscosity and light-scattering studies are consistent with α-helical structure for the polymer that, remains rigid over a temperature range of 15–50°C and becomes somewhat flexible at higher temperatures. Optical rotatory properties were found to vary gradually and continuously with temperature over the range of ?30 to +100°C. This suggested that all three electronic transitions of tyrosyl side chain are optically active, and that the side chains have some freedom of motion that decreases with decreasing temperature, disappearing only at about ?30°C.     

Hydrodynamic studies on chitosans in aqueous solution

Three commercial chitosans with a degree of acetylation of 25–30% were studied by light scattering (static and dynamic), analytical ultracentrifugation (sedimentation velocity and sedimentation equilibrium), and capillary viscometry in 0.02 M acetate buffer/0.1 M NaCl, pH 4.5. The molecular masses obtained by sedimentation equilibrium measurements or sedimentation and diffusion coefficients according to the Svedberg equation agreed well or fairly well with those from static light scattering whereas the molecular masses calculated via the Scheraga–Mandelkern equation were found too low by almost 50%. The various Mark–Houwink type relationships suggested a nearly free-draining flexible worm-like chain. A prolate ellipsoid of revolution with an axial ratio a/b25 was shown to be a hydrodynamically equivalent body of the flexible worm-like chain that had been derived from static light scattering. The findings illustrate the fact that a hydrodynamically strongly asymmetric shape need not mean a strongly elongated shape of the molecules in reality.     

Radiation chemical studies of Gly-Met-Gly in aqueous solution

Abstract

Important biological consequences are related to the reaction of HO^? radicals with methionine (Met). Several fundamental aspects remain to be defined when Met is an amino acid residue incorporated in the interior of peptides and proteins. The present study focuses on Gly-Met-Gly, the simplest peptide where Met is not a terminal residue. The reactions of HO^? with Gly-Met-Gly and its N-acetyl derivative were studied by pulse radiolysis technique. The transient absorption spectra were resolved into contributions from specific components of radical intermediates. Moreover, a detailed product analysis is provided for the first time for Met-containing peptides in radiolytic studies to support the mechanistic proposal. By parallel radiolytical and electrochemical reactions and consequent product identification, the formation of sulfoxide attributed to the direct HO^? radical attack on the sulfide functionality of the Met residue could be excluded, with the in situ generated hydrogen peroxide responsible for this oxidation. LC–MS and high resolution MS/MS were powerful analytical tools to envisage the structures of five products, thus allowing to complete the mechanistic picture of the overall Met-containing peptide reactivity.     

Conformational studies of nikkomycin X in aqueous solution

Nikkomycin X is a peptidyl-nucleoside antibiotic that inhibits chitin synthesis in fungi. Information on its conformation in aqueous solution was obtained from pH titration studies in which the nmr shifts of exchangeable and nonexchangeable protons were monitored. These studies and nuclear Overhauser effects support an unfolded or conformationally flexible structure for the antibiotic, and the syn and anti conformations of the nucleoside moiety were found to coexist. The conformation of the ribose ring was determined using a two-state model; a slight shift from type N to type S conformers occurred as the pH was raised from 1 to 6.     

Light-scattering studies on supercoil unwinding

It has been shown previously that supercoiled [unk]X174 bacteriophage intracellular DNA (mol.wt. 3.2x10(6)) with superhelix density, sigma=-0.025 (-12 superhelical turns) at 25 degrees C is best represented as a Y shape. In this work two techniques have been used to unwind the supercoil and study the changes in tertiary structure which result from changes in the secondary structure. The molecular weights from all experiments were in the range 3.2x10(6)+/-0.12x10(6). In experiments involving temperature change little change in the Y shape was observed between sigma=-0.027 (-13 superhelical turns, 14.9 degrees C) and sigma=-0.021 (-10 superhelical turns, 53.4 degrees C) as evidenced by the root-mean-square radius and the particle-scattering factor P(theta). However, at sigma=-0.0176 (-8 superhelical turns, 74.5 degrees C) the root-mean-square radius fell to between 60 and 70nm from 90nm indicating a large structural change, as did alterations in the P(theta) function. In experiments with the intercalating dye proflavine from values of bound proflavine of 0-0.06mol of dye/mol equiv. of nucleotide which correspond to values of sigma from -0.025 to -0.0004 (-12 to 0 superhelical turns) a similar transition was found when the superhelix density was changed by the same amount, and the molecule was shown to go through a further structural change as the unwinding of the duplex proceeded. At sigma=-0.018 (-9 superhelical turns) the structure was compatible with a toroid, and at sigma=-0.0004 it was compatible with a circle but at no point in the sequence of structure transitions was the structure compatible with the conventional straight interwound model normally visualized as the shape of supercoiled DNA.     

Stability studies of hydrazide and hydroxylamine-based glycoconjugates in aqueous solution

Glycoconjugates can be readily formed by the condensation of a free-reducing terminus and a strong α-effect nucleophile, such as a hydrazide or a hydroxylamine. Further characterization of a series of glycoconjugates formed from xylose, glucose and N-acetylglucosamine, and either p-toluenesulfonyl hydrazide or an N-methylhydroxylamine, was carried out to gain insight into the optimal conditions for the formation of these useful conjugates, and their stability. Their apparent association constants (9-74 M⁻¹) at pH 4.5; as well, as rate constants for hydrolysis, at pH 4.0, 5.0 and 6.0 (37 °C), were determined. The half-lives of the conjugates varied between 3 h and 300 days. All the compounds were increasingly stable as the pH approached neutrality. Conjugate hydrolysis rates mirrored those found for O-glycoside hydrolysis where conjugates formed from electron-rich monosaccharides hydrolyzed more rapidly.     

Dielectric studies on self-associating nucleosides and bases in aqueous solution

Measurements have been made of the dielectric properties of aqueous solutions in which aggregates are formed by stacking. The nucleosides cytidine, uridine and thymidine, and the bases purine, pyrimidine and 6-methylamino-9-methyl-purine (N⁶N⁹-dimethyladenine) were investigated at around 1 MHz, where the static increments can be determined, and for cytidine, dimethyladenine, uridine and pyrimidine measurements were also made in the 100–2000 MHz range where the main relaxation of the solute dipoles is found. Whereas cytidine and purine show a positive static dielectric increment increasing linearly with concentration, dimethyladenine, uridine, thymidine and pyrimidine show a similar negative effect. Also, within the experimental accuracy, single retaxation times are found for the solute dispersions investigated.It is suggested that these relaxations correspond to the effects of free rotation of individual polar molecules in the plane of stacking. This phenomenon would also account for the linear variation of the dielectric increments with concentration. These increments are thought to be positive or negative due to the varying balance in the solutions between the loss of polarization due to displaced and “bound” water and the corresponding gain due to the polarity of the solute molecules.     

Two-dimensional NMR studies and secondary structure of cobrotoxin in aqueous solution

The 1H-NMR spectra of cobrotoxin, a neurotoxic protein isolated from Formosan cobra Naja naja atra, have been studied by two-dimensional NMR techniques. Of 62 amino acid residues in cobrotoxin, the complete assignments of 58 residues have been made. The resonances from several of the remaining residues have been identified but not yet specifically assigned. The secondary structure of an antiparallel triple- and double-stranded beta-sheet has also been determined by observing the NOE.     

Light-scattering and infrared-spectrophotometric studies of chitin and chitin derivatives

A sample of chitin isolated from the shell of the crab Scylla serrata had, when in lithium thiocyanate solution, an average, weight-average molecular weight (1) of 1.036 x 10⁶ daltons, an intrinsic dissymmetry (2) of 1.93, and a Z-average radius of gyration (3) of 64.14 nm. Carboxymethylchitin and glycol chitin, in 0.5M sodium chloride, had, respectively, (1) 1.896 and 1.819 x 10⁶ daltons, (2) 3.25 and 4.31. and (3) 143.49 and 251.57 nm. They had similar degrees of polymerization, they underwent dissociation as the concentration of sodium chloride was increased to 2.5M, and the molecular packing of the chains was essentially side-by-side. Chitin in 5.55M lithium thiocyanate and carboxymethylchitin in 2.5M sodium chloride had similar degrees of polymerization. It is concluded that a small but significient number of the amino groups in the chitin molecule are not acetylated.     

Chromatographic studies on the radiolysis of DNA in aqueous solution

Radiation-induced degradation of double-stranded DNA from calf thymus in aqueous solution with sodium phosphate was studied by conventional gel chromatography and by high-performance liquid-gel permeation chromatography. Comparison of the data after radiolysis of aqueous solutions of DNA under anaerobic and aerobic conditions indicates that double-strand breakage is not enhanced by oxygen. An increase of ionic strength impedes the break-down of the DNA molecules, so that loss of DNA can only be observed at doses above 100 Gy. Only reactions of OH-radicals contribute to the fragmentation of DNA, while the presence of hydrated electrons, H.-or formate radicals does not lead to a loss of highly polymerized DNA up to doses of 1500 Gy. High-performance liquid-chromatography proved to be an excellent method of studying the degradation of macromolecules as a function of dose.     

Proton NMR studies of angiotensin II and its analogs in aqueous solution

The¹H nuclear magnetic resonance (NMR) spectra of angiotensin II (Asp-Arg-Val-Tyr-Ile-His-Pro-Phe) and five of its octapeptide analogs as well as angiotensin I (Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu) and angiotensin III (Arg-Val-Tyr-Ile-His-Pro-Phe) in aqueous solutions (90% H₂O/10% D₂O) were completely assigned by two-dimensional COSY and ROESY experiments. All of the peptides give rise to two distinct sets of signals. The minor set accounts for about 5% of the total population belowpH 5.5 and increases to 12–20% aroundpH 7.0. The two sets of signals result from acis-trans isomerization of the His-Pro peptide bond with the major resonances arising from thetrans isomer. One analog in which the Pro is replaced with a D-Pro displays a very different isomerization behavior. The measured coupling constants J_NH-CH, the temperature dependence of the amide proton shifts and the relative intensities of the intraresidue and sequential NH-CH ROEs, are all indicative of an extended backbone conformation for ANGII. However, some evidence for the existence of conformers with local structure involving preferred sidechain positions for the Tyr, His, Phe, and the carboxyl group of the Phe was found, particularly in the ROESY andpH-titration experiments. Moreover,pH effects and the unusual amide exchange behavior of the Arg NH suggests the presence of interactions between the Asp and Arg sidechains of ANGII. At low temperatures the Arg guanidinium NH₂ protons were detected as two broad peaks which are related by sizeable exchange peaks in ROESY experiments. This behavior could be useful as a general probe for the study of Arg sidechain mobility and accessibility in other peptides and proteinsPreliminary results of this work have been presented at the XIIth International Conference on Magnetic Resonance in Biological Systems in abstract form (1988).