Conformation of amylose in aqueous solution: Optical rotatory dispersion and circular dichroism of amylose–iodine complexes and dependence on chain length of retrogradation of amylose

The dependence on chain length of two characteristic properties of amylose, i.e., retrogradation and complex formation with iodine, have been studied by using enzymatically synthesized, homodisperse amyloses. The association rates of amyloses in water containing 5% dimethyl sulfoxide have a sharp maximum at a degree of polymerization P?_n of 80; shorter and longer molecules are much more soluble. The iodine complexes of amylose exhibit a strong Cotton effect in the range of the long-wave absorption maximum (position depending on chain length) and two weaker Cotton effects at 480 and 350 nm. The long-wave Cotton effect is most intense at about P?_n 50 and decreases rapidly for shorter and longer chains. This behavior is unexpected and is not in accordance with the further increase of λ_max and λ_max. The experiments can best be interpreted by assuming well ordered, stiff chains in the low molecular weight range (P?_n 50–80). For longer chains, the findings are discussed in the light of current concepts of amylose conformation in aqueous solution, namely the model of the broken helical chain (alternating stiff helical segments and unordered regions) and the model of a flexible coil without a significant helical content. However, according to the results given in this paper, a wormlike helical chain seems to be the most adequate model for amylose conformation in neutral solution.     

Ozonolytic depolymerization of polysaccharides in aqueous solution.

The selective oxidation of beta-D-glycosidic linkages of polysaccharides by ozone has great utility as a general method for depolymerization of polysaccharides. Here we describe a 'one-step' method whereby polysaccharides dissolved in water or basic solutions are depolymerized by ozonolysis. The oxidation of glycosidic linkages of unprotected carbohydrates by ozone is complicated by several side reactions. We describe here optimized conditions for carrying out ozonolysis degradation. We also characterize the major pathways for unwanted degradation by various side reactions. In the preferred oxidation pathway, the aldosidic linkage is oxidized to an aldonic ester function that hydrolyzes under the basic conditions employed to give a free aldonate, with cleavage of the polysaccharide chain. Nonselective degradation pathways include oxidative degradation by radical species that oxidize glycosyl residues to formic, acetic, and oxalic acids. The nonselective degradation caused by acids is minimized by basic buffers. The products of polysaccharide depolymerization form a size distribution around a nominal molecular weight, and the average molecular weight of the products can be controlled by the rate or amount of ozone passed through the reaction mixture. The ozonolysis method described herein provides a convenient, inexpensive, and controllable means for generating small polysaccharides or large oligosaccharide fragments.     

Helical conformation of amylose in aqueous solution. I. Viscosity measurements

Two amylose samples, amylose V (DP_w = 2300) and amylose HE 15 (a low-substituted hydroxyethylamylose, DP_W = 1600) were studied. The intrinsic viscosity of the polymers in aqueous solution was measured with regard to its dependence on the alkalinity (0 to 5MNaOH), the ionic strength (0 to 5M), and the temperature (0 to 75°C). Additionally, the temperature dependence of the viscosity of the amylose-iodine complex was measured. It was found that the two amylose samples show the same dependence on the studied parameters. Therefore, it was concluded that the conformation is unchanged by the hydroxyethylation in the present case. In a discussion, steric, hydrodynamic, and thermodynamic data on amylose in solution are compared with the corresponding data on cellulose and dextran. The comparison leads to the conclusion that amylose in a neutral solution must have a helical conformation, corresponding to the well-accepted rod conformation of cellulose. The helical conformation also explains several viritual anomalies in the behavior of amylose. The results of the experiments support the helix model for amylose. The conclusion of the whole work, therefore, is that the amylose molecule in neutral aqueous solution can be regarded as a random coil, built up by helical segments. The average number of monomers per segment exceeds 100. This value decreases with increasing alkalinity.     

Helical conformation of amylose in aqueous solution. II. Electron spin resonance,fluorescence depolarization,and sedimentation measurements

In the previous study [Elmgren, H. (1984) Biopolymers 23, 2525–2541] concerning the conformation of amylose in aqueous solution, it was stated that amylose in a neutral aqueous solution is a random coil consisting of helical segments. In terms of Kuhn statistics, each segment contains more than 100 monomers. The number of monomers per segment decreases by alkali addition. In an attempt to verify these statements, a combined electron spin resonance (esr) and ultracentrifugation (uc) study of a weakly hydroxyethylated amylose sample in water and alkaline solvents was performed. This combination of measuring techniques makes it possible to estimate the Brownian motion, and thus the mass of the polymer segments. As a control for the obtained esr data, fluorescence depolarization (fdp) measurements were performed on the polymer sample in a bicarbonate buffer at pH 10. The result of the study confirms that the amylose segments are very heavy in water. In strong alkaline solvents, the segment mass corresponds to that of a few monomers. Our findings thus support the statements made in the preceding article, and the data obtained by others. [Kitamura S., Yunokawa H., & Kuge T., (1982) Polym. J. 14, 85–91; Kitamura S., Yunokawa H., Mitsu'ie S., & Kuge T., (1982) Polym J. 14, 93–99].     

Protein rotational motion in solution measured by polarized fluorescence depletion.

A microscope-based system is described for directly measuring protein rotational motion in viscous environments such as cell membranes by polarized fluorescence depletion (PFD). Proteins labeled with fluorophores having a high quantum yield for triplet formation, such as eosin isothiocyanate (EITC), are examined anaerobically in a fluorescence microscope. An acousto-optic modulator generates a several-microsecond pulse of linearly polarized light which produces an orientationally-asymmetric depletion of ground state fluorescence in the sample. When the sample is then probed with light polarized parallel to the excitation pulse, fluorescence recovers over 0-1,000 microseconds as the sum of two exponentials. One exponential corresponds to triplet decay and the other to the rotational relaxation. An exciting pulse perpendicular to the probe beam is then applied. Fluorescence recovery following this pulse is the difference of the same two exponentials. Equations for fluorescence recovery kinetics to be expected in various experimentally significant cases are derived. Least-squares analysis using these equations then permits the triplet lifetime and rotational correlation time to be determined directly from PFD data. Instrumentation for PFD measurements is discussed that permits photobleaching recovery measurements of lateral diffusion coefficients using the same microscope system. With this apparatus, both rotational and translational diffusion coefficients (Dr, Dt) were measured for EITC-labeled bovine serum albumin in glycerol solutions. Values obtained for Dr and Dt are discussed in light of both the PFD models and the experimental system.     

Investigation of the effect of amylose/iodine complexation on the conformation of amylose in aqueous solution

Using a potato amylose fraction of 8 × 10⁵, molecular-weight viscosity studies were carried out at 25°C on solutions containing 0.176–0.042% polymer, 8.67 mM KI, 1% ethanol, and different concentrations of iodine. By a novel extrapolation method, the intrinsic viscosities of the amylose/iodine complex were determined under various conditions of iodine binding (0–0.133 g I₂/g amylose). Contrary to the view long held in this research area, it was found that the intrinsic viscosity of amylose solutions decreases significantly upon complex formation with iodine. Taking into account the results of our previous kinetic studies, the present findings are interpreted in terms of an amylose model characterized by loose, extended helical regions which are interrupted by short disordered regions. It is proposed that the intrinsic viscosity decrease observed is due to a shortening of the linear dimension of the polymer chain. This conformation change is apparently caused by the contraction of loose helical regions of the amylose macromolecule due to the entrapment of iodine (and perhaps other) atoms inside the helical cavities.     

Rate enhancement by guided diffusion. Chain length dependence of repressor-operator association rates

Association rates are calculated for cases where one reaction partner belongs to a chain that has an unspecific affinity to the other. Provided that the unspecific attachment does not completely suppress diffusion along the chain, this channeling may considerably speed up the association. Explicit formulae are derived to show how this effect depends on the chain length and other parameters. The influence of electrostatic forces and reaction barriers is discussed. Time dependent solutions of the diffusion equations are analyzed in order to test the usual steady state assumptions. Experiments on the repressor-operator system seem to be in good agreement with our theory.     

Changes of fluorescence spectra of thymine in aqueous solution by radiation

Aqueous solution of thymine (5 X 10(-4) M, buffered at pH 7.0) was irradiated with 60Co gamma-rays under four different atmospheric conditions. In the presence of t-BuOH-N2, there was little increase in the fluorescence intensity as was previously reported in the radiolysis of cytosine. Under O2 there was also no significant increase differing from the case of cytosine. The fluorescence intensity was found to increase appreciably under N2O but it was less under N2 indicating that OH radical is mainly responsible for the formation of the highly fluorescent products. However, the fluorescence yields under these conditions were much lower in thymine radiolysis than cytosine radiolysis.     

Chain length dependence of phosphatidylcholine hydrolysis catalyzed by lipoprotein lipase. Effect of apolipoprotein C-II

The effect of apolipoprotein C-II (apoC-II) on the bovine milk lipoprotein lipase (LpL)-catalyzed hydrolysis of a homologous series of saturated phosphatidylcholines was examined with respect to the fatty acyl chain length of the substrates. Dilauryl-, dimyristoyl-, dipalmitoyl-, and distearoylphosphatidylcholine solubilized by Triton X-100 and sonicated vesicles of dimyristoylphosphatidylcholine were used as substrates. The maximal rate of the LpL-catalyzed hydrolysis of each of these lipids was determined in the absence and presence of apoC-II. The activation factor (the ratio of enzyme activity with apoC-II to that without the activator protein) increased with increasing mol ratios of apoC-II to LpL and was maximal at a ratio of approximately 50. At all apoC-II/LpL mole ratios tested, the activation factor increased as a function of fatty acyl chain length. A quantitative relationship between fatty acyl chain length and the extent of maximal activation of LpL by apoC-II was observed: the logarithm of the activation factor is a linear function of the number of carbon atoms of a single fatty acyl chain of the substrates.     

Acrylamide quenching of Yt-base fluorescence in aqueous solution

Acrylamide was found to be an effective quencher of Yt-base (Y-4,9-dihydro-4,6-dimethyl-9-oxo-1H-imidazo-1,2a-purine) in water. In the absence of collisional quenching the decay of Yt-base in water is predominantly a single exponential. The intensity decays become increasingly heterogeneous when quenched by acrylamide. The frequency-domain data were analyzed using the radiation model, which provides estimates of molecular parameters characteristic of the system. The mutual diffusion coefficient at 20 degrees C was found to be 0.5 x 10(5) cm2/s, the Yt-base acrylamide interaction radius was 8 A, and the rate constant for quenching was 100 cm/s. These values indicate that quenching is diffusion-limited, i.e., the encounter complex is deactivated at least 2-fold faster than the rate of diffusive encounters.     

The chain length dependence of the conformation for oligomers of L-lysine in aqueous solution: Optical rotatory dispersion studies

The optical rotatory dispersion of L -lysine oligopeptides (Lys_n, n = 2–22) in solution was measured in water and in 50% methanol. A gradual change with increasing chain length in the ORD curves of the oligomers was observed at pH 4. 3. Not even a chain of 22 residues had ORD identical with that of high molecular weight poly-L -lysine. A plot of the average molar residue rotation at 233 nm versus 1/n (where n is the chain length) resulted in a straight line with an intercept of ?1900, representing the internal residue rotation of a lysine residue in the random conformation, and a slope of +6200 representing the large end effect. At pH 11.9 a stright line is obtained up to n = 12 after which it deviates from the initial slope indicating onset of helicity. Extrapolation of the initially straight line to tire higher n's provided the necessary zero-helicity values for calculation of helicity. The highest oligolysine (n = 22) showed at pH 11. 9 13% helicity, which on adding methanol to 50% increased to about 50% helicity. It is shown that helix-coil data which are usually obtained from the temperature dependence of helicity can be obtained from the dependence of helicity on chain length applying the statistical theory. For the methanol-water system the cooperativity parameter v was calculated to be in the range 0.024–0.060, with corresponding equilibrium constants w of 1.32–1.43. The helical structure was calculated to be less stable in water than in methanol-water by about 250 calories per residue.     

Complexation study of europium(III) and curium(III) with urea in aqueous solution investigated by time-resolved laser-induced fluorescence spectroscopy

The complex formation of europium(III) and curium(III) with urea in aqueous solution has been studied at I = 0.1 M (NaClO₄), room temperature and trace metal concentrations in the pH-range of 1-8 at various ligand concentrations using time-resolved laser-fluorescence spectroscopy. While for curium(III) the luminescence maximum is red shifted upon complexation, in case of europium(III) emission wavelengths remain unaltered but a significant change in peak splitting occurs. Both heavy metals form weak complexes of the formulae ML³⁺ and MLOH²⁺ with urea. Stability constants were determined to be log β₁₁₀ = −0.12 ± 0.05 and log β_11-1 = −6.86 ± 0.15 for europium(III) and log β₁₁₀ = −0.28 ± 0.12 and log β_11-1 = −7.01 ± 0.15 for curium(III).     

Changes of fluorescence spectra of 2'-deoxyguanosine in aqueous solution by radiation

Aqueous solution of 2'-deoxyguanosine (5 X 10(-4) M, pH 7.0) was irradiated with 60Co gamma-rays under O2, N2, N2O, and t-BuOH-N2, respectively. A marked increase in fluorescence emission intensity was observed under all atmospheric conditions as was observed in aqueous solutions of adenine and 2'-deoxyadenosine. However, the fluorescence yield from 2'-deoxyguanosine with radiation was lower under O2 and much higher under t-BuOH-N2 than that from 2'-deoxyadenosine though it was not so different both under N2 and N2O. Such high fluorescence yield from 2'-deoxyguanosine especially under t-BuOH-N2 suggests that guanine base has a specific reactivity with hydrated electron or t-butanol radical differing from the other nucleobases.     

Changes of fluorescence spectra of 2'-deoxyadenosine in aqueous solution by radiation

Aqueous solution of 2'-deoxyadenosine (5 X 10(-4) M, buffered at pH 7.0) was irradiated with 60Co gamma-rays under N2, O2, N2O and t-BuOH-N2 atmospheres in order to compare with adenine radiolysis previously reported. By exposure to radiation, the fluorescence was found to increase more markedly than that from adenine under all conditions of radiolysis. This result indicates that not only base moiety but also sugar moiety participate in the formation of highly fluorescent products. In this 2'-deoxyadenosine radiolysis, both OH and e-aq take part in the formation of such products, but OH predominates over over e-aq when both active species are present, as observed in adenine radiolysis.     

Chain conformation and rheological behavior of an extracellular heteropolysaccharide Erwinia gum in aqueous solution

The chain conformation of a heteropolysaccharide Erwinia gum (EG) consisting of Glc, Gal, Fuc, and GlcA in aqueous solution was investigated by using viscometry and static and dynamic light scattering. The Huggins constants k′ ranging from 0.31 to 0.35, and the larger second virial coefficient A₂ of the order of 10⁻⁴ and even 10⁻³ mol g⁻² cm³ for EG fractions having different molecular weights in 0.03 M NaCl aqueous solution at 25 °C, suggested that 0.03 M NaCl aqueous solution is a good solvent for EG polysaccharide. Smidsrød’s ‘B-value’ characterizing chain stiffness was estimated to be 0.028-0.045 for EG fractions indicating that the backbone of EG polysaccharide is semi-stiff having similar stiffness to the semi-stiff Alginate and CMC. The hydrodynamic factor ρ (1.69-1.89), Flory-Fox factor Φ, and the product of ρΦ/N_A (0.16-0.22) also confirmed the semi-stiffness of EG polysaccharide chains. Compared with general flexible polymers, the first remarkable shear-thinning and then Newtonian flowing behaviors in steady shear tests for EG polysaccharides were ascribed to the alignment of extended semi-stiff chains on shearing. The dynamic oscillatory shear experiments indicated that addition of certain amount of NaCl effectively prohibited its gelation in pure water even at high concentration and low temperature for long time, suggesting that 0.03 M NaCl aqueous solution of EG has good stability and ability of antigelation, and thus is a promising additive in food field.