The dynamics of the DNA hydration shell at gigahertz frequencies

We have used Brillouin scattering to measure the linewidths and frequencies of GHz acoustic phonons in Na- and Li-DNA films as a function of temperature between 300 and 140 K for samples that were dry, lightly, and heavily hydrated. The linewidths decrease with falling temperature and water contents, indicating that coupling to a water relaxation is the main source of phonon damping. The strength of the relaxation was determined using measurements of the phonon linewidth as a function of frequency, and confirmed by comparison of measured and calculated spectral profiles. The relaxation strength is anisotropic, being greater for phonons propagating perpendicular to the helix axis. The hydrated DNA exhibits both a rapid relaxation (≤ 10^?11 s per radian) giving rise to a classical f² damping, and a slower motion with a relaxation time that varies from ～ 4 × 10^?11 s per radian (primary hydration shell) to ～ 2 × 10^?12 s per radian (secondary hydration shell) at room temperature. In the frequency interval that bounds these relaxation times (～ 4 to 80 GHz) we expect degrees of freedom associated with the primary hydration shell to be important. The sample with primary hydration follows a simple Arrhenius behavior with ΔH ～ 5 kcal mole^?1. The effective activation energy for the sample with secondary hydration is somewhat higher (indicating a more cooperative water relaxation) and varies strongly with temperature. The elastic moduli change much more than can be accounted for by relaxation, indicating the importance of water motion in softening interatomic potentials. The extent of the softening caused by the “unfreezing” of water motion is similar to the degree of softening caused by hydrating the sample.     

The origin of the A to B transition in DNA fibers and films

We have studied the hydration of Na-DNA and Li-DNA fibers and films, measuring water contents, x-ray fiber diffraction patterns, low-frequency Raman spectra (below 100 cm^?1), high-frequency Raman spectra (600–1000 cm^?1), and swelling, as a function of relative humidity. Most samples gain weight equilibrium (though not conformational equilibrium) in one day. The volume occupied by a base pair as the DNA is hydrated (obtained from the x-ray and swelling data) shows anomalies for the case of Na-DNA in the region where the A-form occurs. Our Raman and x-ray data reproduce the well-known features of the established conformational transitions, but we find evidence in the Raman spectra and optical properties of a transition to what may be a disordered B-like conformation in Na-DNA below 40% relative humidity. We have studied the effects of crystallinity on the A to B transition. We find that the transition to the B-form is impeded in highly crystalline samples. In most samples, the transition occurs in three days (after putting the sample at 92% relative humidity) but in highly crystalline samples, the transition may take months. By comparing the high-frequency Raman spectra of highly ordered and disordered films, we show that the extent of crystallinity controls the amount of A-DNA formed when ethanol is used to dehydrate the films. We show that rapid dehydration (by laser heating) does not result in a B to A transition. A fiber that gives A-type x-ray reflections probably contains B-like material in noncrystalline regions. The low-frequency Raman spectrum is dominated by a band at about 25 cm^?1 in both Na- and Li-DNA. Another band is seen near 35 cm^?1 in Na-DNA at humidities where the sample is in the A-form. In contrast to earlier reports, we find that the Raman intensity does not depend on fiber orientation relative to the scattering vector. The “35-cm^?1” band is largely depolarized (i.e. vertical polarization incident and horizontal polarization scattered, VH, or vice versa, HV) while the “25-cm^?1” band appears in both VV, VH and HV polarizations. These bands are all weaker in HH polarization. The “25-cm^?1” band may be due to a shearing motion of the phosphates and their associated counterions, while the “35-cm^?1” band may be characteristic of A-DNA crystallites. We consider mass-loading, relaxational coupling to the hydration shell, and softening of interatomic potentials as possible explanations of the observed softening of the low-frequency Raman bands on hydration. Relaxation data suggest that the added water binds tightly (on these time scales) and a mass-loading model accounts for the observed softening rather well. We conclude that the A to B transition is not driven by softening of the “25-cm^?1” band. Rather, it is most probably a consequence of crystal-packing forces, with the more regular A-form favored in crystals when these forces are strong.     

A 2H-NMR study of the A-DNA conformation in films of oriented Na-DNA: evidence of a disordered B-DNA contribution

Solid-state ²H-nmr spectra have been obtained from folded films of oriented Li- and Na-DNA molecules with the purine bases selectively deuterium labeled at the 8 position. From line shape simulations, we find that the Na-DNA sample at 75% relative humidity (rh) contains both A-DNA and surprisingly large amounts of B-DNA

1 Here, B-DNA refers to “B-DNA family” (i.e. B- or C-DNA).

(57%). For the A-DNA component the average base tilt is 23°, and the total distribution width of tilt angles and helix axis orientations is ～ 4° (standard deviation). In the B-DNA component the base tilt is ～ 0° and the total distribution width is ～ 20°. In contrast, films of Li-DNA only exhibit the B-form line shape, consistent with a base tilt of ～ 0° and a total distribution width of base tilt angles and helix axis orientations of 9°. The nmr results that demonstrate the presence of large amounts of B-DNA in the Na-DNA sample contrast with the x-ray diffraction measurements that indicated mainly A-form. The nmr spectra are used to monitor the B-DNA content in the Na-films and to evaluate procedures for increasing the A-DNA fraction.     

Interaction of water with oriented DNA in the A- and B-form conformations.

High resolution 2H nuclear magnetic resonance (NMR) was used to investigate the interaction of D2O with solid samples of uniaxially oriented Li-DNA (B-form DNA) and Na-DNA (A- and B-form DNA). At low levels of hydration, 0 approximately 4 D2O/nucleotide, the 2H spectra shows a very weak (due to short T2) broad single resonance, suggestive of unrestricted rotational diffusion of the water. At approximately 5 or more D2O/nucleotide, the Li-DNA (B-form) spectra suddenly exhibit a large doublet splitting, characteristic of partially ordered water. With increasing hydration, the general trend is a decrease of this splitting. From our analysis we show that the DNA water structure reorganizes as the DNA is progressively hydrated. The D2O interaction with Na-DNA is rather different than with Li-DNA. Below 10 D2O/nucleotide Na-DNA is normally expected to be in the A-form, and a small, or negligible splitting is observed. In the range 9-19 D2O/nucleotide, the splitting increases with increasing hydration. Above approximately 20 D2O/nucleotide Na-DNA converts entirely to the B-form and the D2O splittings are then similar to those found in Li-DNA. We show that the complex Na-DNA results obtained in the range 0-20 D2O/nucleotide are caused by a mixture of A- and B-DNA in those samples.     

Thermal denaturation of Na- and Li-DNA in salt-free solutions

Thermal denaturation of Na- and Li-DNA from chicken erythrocytes was studied by means of scanning microcalorimetry in salt-free solutions at DNA concentrations (C_p) from 4.5 · 10^?2 to 1 · 10^?3 moles of nucleotides/liter (M). Linear dependencies of DNA melting temperature (T_m) vs lgC_p were obtained: ((1)) ((2)) for Na- and Li-DNA, respectively. Microcalorimetry data were compared with the results of spectrophotometric studies at 260 nm of DNA thermal denaturation in Me-DNA + MeCl solutions at C_p ? (6–8) · 10^?5 M and C_s = 0–40 mM (Me is Na or Li, C_s is salt concentration). It was found that Eqs. (1) and (2) are valid in DNA salt-free solutions over the C_p range 6 · 10^?5?4.5 · 10^?2M. Protonation of DNA bases due to the absorption of CO₂ from air in Na-DNA + NaCl solutions affects DNA melting parameters at C_s < 4 mM. Linear dependence of T_m on lga₊ is found in Na-DNA + NaCl at C_s > 0.4 mMin the absence of contact of solutions with CO₂ from air (a₊ is cation activity). A dependence of [dT_m/dlga₊] on Li⁺ activity was observed in Li-DNA + LiCl solutions at C_s < 10 mM: [dT_m/dlga₊] increases from 17°–18° at C_s > 10 mM to 28°–30° at C_s ? 0.2–0.4 mM. Spectrophotometric measurements at 282 nm show that this effect was caused by protonation of bases in fragments of denatured DNA in neutral solutions. The Poisson–Boltzmann (PB) equation was solved for salt-free DNA at the melting point. The linear dependence of T_m vs lgC_p was interpreted in terms of Manning's condensation theory. PB and Manning's theories fit the experimental data if charge density parameter (ξ) of denatured DNA is in the range 1.8–2.1 (assuming for native DNA ξ = 4.2). Specificity of Li ions in interactions with DNA is discussed. © 1994 John Wiley & Sons, Inc.     

Hydrodynamic properties of mucins secreted by primary cultures of Guinea-pig tracheal epithelial cells: determination of diffusion coefficients by analytical ultracentrifugation and kinetic analysis of mucus gel hydration and dissolution

We have used two different approaches to determine hydrodynamic parameters for mucins secreted by guinea-pig tracheal epithelial cells in primary culture. Cells were cultured under conditions that promote mucous cell differentiation. Secreted mucins were isolated as the excluded fraction from a Sepharose CL-4B gel filtration column run under strongly dissociating conditions. Biochemical analysis confirmed the identity of the high molecular weight material as mucins. Analytical ultracentrifugation was used to study the physical properties of the purified mucins. The weight average molecular mass (M _w ) for three different preparations ranged from 3.3×10⁶ to 4.7×10⁶ g/mol (corresponding to an average structure of 1 – 2 subunits), and the sedimentation coefficient from 25.5 to 35 S. Diffusion coefficients ranging from 4.5×10^–8 to 6.4×10^–8 cm²/s were calculated using the Svedberg equation. A polydispersity index (M _z /M _w ) of ∼1.4 was obtained. Diffusivity values were also determined by image analysis of mucin granule exocytosis captured by videomicroscopy. The time course of hydration and dissolution of mucin was measured and a relationship is presented which models both phases, each with first order kinetics, in terms of a maximum radius and rate constants for hydration and dissolution. A median diffusivity value of 8.05×10^–8 cm²/s (inter-quartile range = 1.11×10^–7 to 6.08×10^–8 cm²/sec) was determined for the hydration phase. For the dissolution phase, a median diffusivity value of 6.98×10^–9 cm²/s (inter-quartile range = 1.47×10^–8 to 3.25×10^–9 cm²/sec) was determined. These values were compared with the macromolecular diffusion coefficients (D _20,w ) obtained by analytical ultracentrifugation. When differences in temperature and viscosity were taken into account, the resulting D _37,g was within the range of diffusivity values for dissolution. Our findings show that the physicochemical properties of mucins secreted by cultured guinea-pig tracheal epithelial cells are similar to those of mucins of the single or double subunit type purified from respiratory mucus or sputum. These data also suggest that measurement of the diffusivity of dissolution may be a useful means to estimate the diffusion coefficient of mucins in mucus gel at the time of exocytosis from a secretory cell. Received: 10 March 1998 / Accepted: 27 March 1998     

Effect of pressure on the sol-gel transition of carrageenans

The effects of pressure on the sol-gel transition of κ- and ι-carrageenans were studied in KCl solutions under high pressures up to 3000 kg/cm². The carrageenan gels were destabilized by pressure: the pressure depression of melting temperature, (dT/dP)_m, was ?5.7 × 10^?3 and ?4.0 × 10^?3 K cm²/kg independent of KCl concentration for κ- and ι-carrageenans, respectively. The enthalpy, entropy and volume changes accompanying the gel formation were calculated from the Eldridge-Ferry's plots and the Clausius-Clapeyron equation. The volume change per unit cross-link (two disaccharide residues) was estimated to be (2.5 ～ 4.9) and (1.7 ～ 3.4) ml/mol for κ- and ι-carrageenans, respectively. The compressibility of both carrageenan molecules appeared to be larger by (1.6 ～ 2.6) × 10^?12 (κ-form) and by (0.8 ～ 1.3) × 10^?12cm²/dyn (i-form) in gel state as compared with in sol state These increases in volume and compressibility on gelation were attributed to a reduction of water of hydration from the carrageenan molecules, which is mainly due to a replacement of the polymer-water hydrogen bond by the polymer-polymer hydrogen bond. These results seemed not inconsistent with the idea that a double helix structure of carrageenan gels may persist in solution as well as in the solid state.     

Histone-induced conformational changes in DNA as probed by quasi-elastic light scattering.

Quasi-elastic light scattering as measured by intensity fluctuation (self-beat) spectroscopy in the time domain can be profitably used to follow both the translational diffusion D and the dominant internal flexing mode τ_int of DNA and its complexes with various histones in aqueous salt solutions. Without histones, DNA is found to have D = 1.6 × 10^?8 cm²/sec and τ_int ? 5 × 10^?4 sec in 0.8 M NaCl, 2 M urea at 20°C. Total histone as well as fraction F2A induce supercoiling (D = 2.6 × 10^?8 cm²/sec, τ_int ? 2.8 × 10^?4 sec) whereas fraction F1 induces uncoiling (D = 1.0 × 10^?8 cm²/sec, τ_int ? 9.4 × 10^?4 sec). Upon increasing the salt concentration to 1.5 M the DNA–histone complex dissociates (D = 1.8 × 10^?8 cm²/sec). Upon decreasing the salt concentration to far below 0.8 M, the DNA–histone complex eventually precipitates as a chromatin gel.     

An investigation into possible quantum chaos in the H<Subscript>2</Subscript> molecule under intense laser fields via Ehrenfest phase space (EPS) trajectories

By employing the Ehrenfest "phase space" trajectory method for studying quantum chaos, developed in our laboratory, the present study reveals that the H₂ molecule under intense laser fields of three different intensities, I?=?1?×?10¹⁴ W/cm², 5?×?10¹⁴ W/cm², and 1?×?10¹⁵ W/cm², does not show quantum chaos. A similar conclusion is also reached through the Loschmidt echo (also called quantum fidelity) calculations reported here for the first time for a real molecule under intense laser fields. Thus, a long-standing conjecture about the possible existence of quantum chaos in atoms and molecules under intense laser fields has finally been tested and not found to be valid in the present case.     

Inactivation of Cronobacter spp. (Enterobacter sakazakii) in infant formula using lactic acid,copper sulfate and monolaurin

Aims: To investigate the effect of lactic acid (LA), copper (II), and monolaurin as natural antimicrobials against Cronobacter in infant formula. Methods and Results: The effect of LA (0·1, 0·2 and 0·3% v/v), copper (II) (10, 50 and 100 μg ml^?1) and monolaurin (1000, 2000, and 3000 μg ml^?1) suspended into tween‐80? or dissolved in ethanol against Cronobacter in infant formula was investigated. Reconstituted infant formula and powdered infant formula were inoculated with five strains of Cronobacter spp. at the levels of c. 1 × 10⁶ CFU ml^?1 and 1 × 10³ CFU g^?1, respectively. LA at 0·2% v/v had a bacteriostatic effect on Cronobacter growth, whereas 0·3% v/v LA resulted in c. 3 log₁₀ reduction. Copper (II) at the levels of 50 μg ml^?1 and 100 μg ml^?1 elicited c. 1 and 2 log₁₀ reductions, respectively. The combination of 0·2% LA and 50 μg ml^?1 copper (II) resulted in a complete elimination of the organism. Monolaurin exhibited a slight inhibitory activity against Cronobacter (c. 1·5 log₁₀ difference) compared to the control when ethanol was used to deliver monolaurin. Conclusions: A complete elimination of Cronobacter was obtained when a combination of sublethal concentrations of LA (0·2%) and copper (II) (50 μg ml^?1) was used. Significance and Impact of the Study: The use of the synergistic interactive combination of LA and copper (II) could be beneficial to control Cronobacter in the infant formula industry.     

Helix promotion in polypeptides by polyols

The helix content of [L -Lys(Me₃)]_n · ClO₄, and [L -Lys(Me₃)⁵⁰, L -Ala⁵⁰]_n · ClO₄ in water is markedly increased by the presence of sucrose and glycerol. For [L -Lys(Me₃)]_n · ClO₄ the ellipticity at 222 nm changes from +2 × 10³ deg cm² dmole^?1 in water to ?44 × 10³ in 50% glycerol. Sucrose does not promote helix formation in melittin at pH 7.2, but glycerol does. At pH 5.5 sucrose and, more so, glycerol, induce helix in melittin. Glycerol induces some helix in methylated melittin, but less than in melittin. The results are discussed in relation to excluded volume effects, ΔG of transfer of peptide and hydrophobic groups from water to mixed solvents, electrostatic effects, and preferential hydration. © 1993 John Wiley & Sons, Inc.     

In situ measurements of bioluminescence response of Gonyaulax spinifera to various mechanical stimuli

A conspicuous bioluminescence during nighttime was reported in an aquaculture farm in the Cochin estuary due to Gonyaulax spinifera bloom on March 20, 2020. In situ measurements on bioluminescence was carried out during nighttime to quantify the response of G. spinifera to various mechanical stimuli. The bioluminescence intensity (BI) was measured using Glowtracka, an advanced single channel sensor, attached to a Conductivity–Temperature–Depth Profiler. In steady environment, without any external stimuli, the bioluminescence generated due to the movement of fishes and shrimps in the water column was not detected by the sensor. However, stimuli such as a hand splash, oar and swimming movements, and a mixer could generate measurable bioluminescence responses. An abundance of?~?2.7?×?10⁶ cells L^?1 of G. spinifera with exceptionally high chlorophyll a of 25 mg m^?3 was recorded. The BI in response to hand splash was recorded as high as 1.6?×?10¹¹ photons cm^?2 s^?1. Similarly, BI of?~?1–6?×?10¹⁰ photons cm^?2 s^?1 with a cumulative bioluminescence of?~?2.51?×?10¹² photons cm^?2 (for 35 s) was recorded when there is a mixer with a constant force of 494 N/800 rpm min^?1. The response of G. spinifera was spontaneous with no time lapse between application of stimuli and the bioluminescence response. Interestingly, in natural environment, application of stimulus for longer time periods (10 min) does not lower the bioluminescence intensity due to the replenishment of water thrusted in by the mixer from surrounding areas. We also demonstrated that the bioluminescence intensity decreases with increase in distance from the source of stimuli (mixer) (av. 1.84?×?10¹⁰ photons cm^?2 s^?1 at 0.2 m to av. 0.05?×?10¹⁰ photons cm^?2 s^?1 at 1 m). The BI was highest in the periphery of the turbulent wake generated by the stimuli (av. 3.1?×?10¹⁰ photons cm^?2 s^?1) compared to the center (av. 1.8?×?10¹⁰ photons cm^?2 s^?1). When the stimuli was applied vertically down, the BI decreased from 0.2 m (0.3?×?10¹⁰ photons cm^?2 s^?1) to 0.5 m (0.10?×?10¹⁰ photons cm^?2 s^?1). Our study demonstrates that the BI of G. spinifera increases with increase in mechanical stimuli and decreases with increase in distance from the stimuli.

     

Static and dynamic light scattering from dilute insulin solutions

Static and dynamic light-scattering measurements are reported on zinc-insulin at room temperature (21 ± l°C) and pH = 6.88 in 0.1M NaCl aqueous solution. The experiments were performed at very low concentration, in the range 0.12 × 10^?4 to 0.90 × 10^?4 g cm^?3. Within experimental error, we find no evidence for a critical micellar concentration in this system. The aggregation phenomenon starts immediately after preparation of the solutions, and takes several days to come to stable equilibrium. The concentration dependence of the diffusion coefficients, D _z, = D_o (1 — k_DC), is negative, and k_D was observed to decrease as a function of time, while the aggregate size was found to increase. The equivalent concentration coefficient, ?2BM _W, obtained from static light scattering, showed a similar behavior, and, within experimental error, was found to be numerically equal to k_D. From the relation found between the diffusion coefficient at infinite dilution and the molecular weight of the aggregates, log D₀ = ?0.240 log M _w ? 5.077, we deduce that the insulin aggregates are compact structures with a characteristic radius of 0.71 Å/(dalton)^1/3, surrounded by a hydration layer of a thickness of 8.0 Å. The equilibrium aggregation number is approximately 10.     

Weak Electron–Phonon Coupling and Enhanced Thermoelectric Performance in n-type PbTe–Cu2Se via Dynamic Phase Conversion

This study investigates Ga-doped n-type PbTe thermoelectric materials and the dynamic phase conversion process of the second phases via Cu₂Se alloying. Introducing Cu₂Se enhances its electrical transport properties while reducing its lattice thermal conductivity (κ_lat) via weak electron–phonon coupling. Cu₂Te and CuGa(Te/Se)₂ (tetragonal phase) nanocrystals precipitate during the alloying process, resulting in Te vacancies and interstitial Cu in the PbTe matrix. At room temperature, Te vacancies and interstitial Cu atoms serve as n-type dopants, increasing the carrier concentration and electrical conductivity from ≈1.18 × 10¹⁹ cm⁻³ and ≈1870 S cm⁻¹ to ≈2.26 × 10¹⁹ cm⁻³ and ≈3029 S cm⁻¹, respectively. With increasing temperature, the sample exhibits a dynamic change in Cu₂Te content and the generation of a new phase of CuGa(Te/Se)₂ (cubic phase), strengthening the phonon scattering and obtaining an ultralow κ_lat. Pb_0.975Ga_0.025Te-3%CuSe exhibits a maximum figure of merit of ≈1.63 at 823 K, making it promising for intermediate-temperature device applications.     

Salt effects on the denaturation of DNA. V. Preferential interactions of native and denatured calf thymus DNA in Na2SO4 solutions of varying ionic strength.

Precision density measurements were performed at 25°C on Na-DNA-Na₂SO₄ mixtures in the presence of either 0.005 m cacodylic acid buffer (pH 6.8) or in the presence of 0.1 m NaOH (pH 12.3). From measurements executed under equilibrium dialysis conditions, the so-called “density increments” (?ρ/?c₂)_μ⁰ for native (pH 6.8), heat-denatured (pH 6.8), and alkali-denatured (pH 12.6) Na-DNA were evaluated as a function of Na₂SO₄ concentration. (?ρ/?c₂)_μ⁰ for native DNA was found to decrease almost linearly with ionic strength I^1/2 of the supporting electrolyte. The density increment for Na-DNA at pH 12.6, on the other hand, increases in more or less linear fashion with I^1/2. (?ρ/?c₂)_μ⁰ for heat-denatured DNA at pH 6.8 is not affected very much by increasing salt strength. From density measurements performed on the Na-DNA–Na₂SO₄ mixtures at fixed concentrations of diffusible components, the partial specific volumes ν ₂° of native (pH 6.8), heat-denatured (pH 6.8), and alkali-denatured (pH 12.6) Na-DNA were determined as a function of Na₂SO₄ concentration. All ν ₂° values, irrespective of the secondary structure of the DNA, increase with increasing salt concentration although the increase for heat denatured DNA (pH 6.8) is barely noticeable. ν ₂° of both native and heat-denatured DNA (pH 6.8) extrapolates to a value of 0.50_o ml/g at vanishing salt concentration; ν ₂° of DNA in 0.1 m NaOH, on the other hand, assumes the value 0.2_o ml/g. Distribution coefficients of diffusible components, expressed in terms of preferential water and salt interaction, were evaluated from the (?ρ/?c₂)_μ⁰ data, solvent densities, and partial specific volumes of all solution components. All interaction parameters depend strongly on salt concentration and on the conformation of DNA. From data collected and from information available in the literature it is concluded that Na₂SO₄, for instance, displaces water of hydration from native DNA much more readily with increasing salt concentration than does NaCl. The solvation properties of the denatured forms of Na-DNA are rather complex but appear to be in harmony with whatever information can be gathered from the literature.     

Rotational relaxation of macromolecules determined by dynamic light scattering. I. Tobacco mosaic virus

The intensity autocorrelation function for the depolarized component of forward-scattered light from a solution of large polymeric molecules is derived in terms of the correlation function for the amplitudes of the Y₂,±₁(θ,?) fluctuations in the anglar distribution of segments in the solution without any assumptions regarding the statistical properties of the scatterad light field. Effects arising from the use of polychromatic incident light and from the mixing of the scattered and polychromatic incident light beams are examined in detail. Apparatus for observing the depolarized forward-scattered light, digitizing and storing the fluctuating phototube current at rates from 10 to 540,000 times per second, and computing the correlation functions directly in the time-domain is described herein. Correlation functions were obtained for 0.05 mg/ml solution of tobacco mosaic virus at pH 9.1 and also at pH 6. The degree of association of the virus appears to be independent of pH, and the monomer relaxation times (corrected to 25°C) extracted from the data by a least-squares procedure lie in the range 0.44–0.49 msec, also independent of pH. The absence of faster component in the correlation function between 6 μsec and 0.5 msec is used in conjunction with thermal fluctuation theory to infer a lower limit for the effective Young's modulaus of the rod, E ≤ 2.5 × 10⁷ dynes/cm².     

A one-step procedure for immobilising the thermostable carbonic anhydrase (SspCA) on the surface membrane of Escherichia coli

The carbonic anhydrase superfamily (CA, EC 4.2.1.1) of metalloenzymes is present in all three domains of life (Eubacteria, Archaea, and Eukarya), being an interesting example of convergent/divergent evolution, with its seven families (α-, β-, γ-, δ-, ζ-, η-, and θ-CAs) described so far. CAs catalyse the simple, but physiologically crucial reaction of carbon dioxide hydration to bicarbonate and protons. Recently, our groups characterised the α-CA from the thermophilic bacterium, Sulfurihydrogenibium yellowstonense finding a very high catalytic activity for the CO₂ hydration reaction (k_cat?=?9.35?×?10⁵?s^?1 and k_cat/K_m?=?1.1?×?10⁸?M^?1?s^?1) which was maintained after heating the enzyme at 80?°C for 3?h. This highly thermostable SspCA was covalently immobilised within polyurethane foam and onto the surface of magnetic Fe₃O₄ nanoparticles. Here, we describe a one-step procedure for immobilising the thermostable SspCA directly on the surface membrane of Escherichia coli, using the INPN domain of Pseudomonas syringae. This strategy has clear advantages with respect to other methods, which require as the first step the production and the purification of the biocatalyst, and as the second step the immobilisation of the enzyme onto a specific support. Our results demonstrate that thermostable SspCA fused to the INPN domain of P. syringae ice nucleation protein (INP) was correctly expressed on the outer membrane of engineered E. coli cells, affording for an easy approach to design biotechnological applications for this highly effective thermostable catalyst.     

Ultrasonic absorption in aqueous solution of lysozyme.

The titration curve of ultrasonic absorption at 2.82 MHz in aqueous solutions of lysozyme measured by Zana and Lang [J. Phys. Chem., 74 , 2734 (1970)] is theoretically analyzed. The maxima at pH 3 and pH 11 are describable with proton-transfer reactions of dissociable carboxyl and amino groups by assuming that volume changes due to the reactions are 2.3 and 5.2 cm³/mole, respectively, which are appreciably smaller than those of simple amino acids. The remaining, pH-independent excess absorption over solvent is measured at frequencies ranging from 3 to 150 MHz. The absorption is ascribed to the internal loss of protein. The complex compressibility β′_p ? iβ″_p of lysozyme molecule is evaluated as β′_p = 7.2 × 10^?12 cm²/dyne and β″_P = 4.3 × 10^?14 cm²/dyne from the increments over solvent in absorption as well as in sound velocity.     

Carbonic anhydrase inhibitors: purification and inhibition studies of pigeon (Columba livia var. domestica) red blood cell carbonic anhydrase with sulfonamides

A carbonic anhydrase (CA, EC 4.2.1.1) from red blood cells of pigeons (Columba livia var. domestica), clCA, was purified to homogeneity. Its kinetic parameters for the CO₂ hydration reaction were measured. With a k_cat/K_m of 1.1?×?10⁸ M^?1 s^?1, and a k_cat of 1.3?×?10⁶ s^?1, clCA has a high activity, similar to that of the human isoform hCA II. A group of 25 aromatic/heterocyclic sulfonamides incorporating the sulfanilamide, homosulfanilamide, benzene-1,3-disulfonamide, and acetazolamide scaffolds showed variable inhibitory activity against the pigeon enzyme, with K_Is in the range of 1.9–3460?nM. Red blood cells of pigeons, like those of ostriches, contain thus just one CA isoform, unlike the blood of mammals, which normally contain two isoforms, one of low (CA I-like) and one of very high activity (CA II-like). However, from the sulfonamide inhibition viewpoint, the pigeon enzyme was more similar to hCA II than to the ostrich enzyme.     

Cloning,expression and biochemical characterization of a β-carbonic anhydrase from the soil bacterium Enterobacter sp. B13

A recombinant carbonic anhydrase (CA, EC 4.2.1.1) from the soil-dwelling bacterium Enterobacter sp. B13 was cloned and purified by Co²⁺ affinity chromatography. Bioinformatic analysis showed that the new enzyme (denominated here B13-CA) belongs to the β-class CAs and to possess 95% homology with the ortholog enzyme from Escherichia coli encoded by the can gene, whereas its sequence homology with the other such enzyme from E. coli (encoded by the cynT gene) was of 33%. B13-CA was characterized kinetically as a catalyst for carbon dioxide hydration to bicarbonate and protons. The enzyme shows a significant catalytic activity, with the following kinetic parameters at 20?°C and pH of 8.3: k_cat of 4.8?×?10⁵?s^?1 and k_cat/K_m of 5.6?×?10⁷ M^?1?×?s^?1. This activity was potently inhibited by acetazolamide which showed a K_I of 78.9?nM. Although only this compound was investigated for the moment as B13-CA inhibitor, further studies may reveal new classes of inhibitors/activators of this enzyme which may show biomedical or environmental applications, considering the posssible role of this enzyme in CaCO₃ biomineralization processes.