Vitaly Buckin

Hexamminecobalt(III)-induced condensation of calf thymus DNA: circular dichroism and hydration measurements

The interaction of hexamminecobalt(III), Co(NH(3))(6)(3+), with 160 and 3000-8000 bp length calf thymus DNA has been investigated by circular dichroism, acoustic and densimetric techniques. The acoustic titration curves of 160 bp DNA revealed three stages of interaction: (i) Co(NH(3))(6)(3+) binding up to the molar ratio [Co(NH(3))(6)(3+)]/[P] = 0.25, prior to DNA condensation; (ii) a condensation process between [Co(NH(3))(6)(3+)]/[P] = 0.25 and 0.30; and (iii) precipitation after [Co(NH(3))(6)(3+)]/[P] = 0.3. In the case of 3000-8000 bp DNA only two processes were observed: (i) binding up to [Co(NH(3))(6)(3+)]/[P] = 0.3; and (ii) precipitation after this point. In agreement with earlier observations, long DNA aggregates without changes in its B-form circular dichroism spectrum, while short DNA demonstrates a positive B-->Psi transition after [Co(NH(3))(6)(3+)]/[P] = 0.25. From ultrasonic and densimetric measurements the effects of Co(NH(3))(6)(3+) binding on volume and compressibility have been obtained. The binding of Co(NH(3))(6)(3+) to both short and long DNA is characterized by similar changes in volume and compressibility calculated per mole Co(NH(3))(6)(3+): DeltaV = 9 cm(3) mol(-1) and Deltakappa = 33 x 10(-4) cm(3) mol(-1) bar(-1). The positive sign of the parameters indicates dehydration, i.e. water release from Co(NH(3))(6)(3+) and the atomic groups of DNA. This extent of water displacement would be consistent with the formation of two direct, hydrogen bonded contacts between the cation and the phosphates of DNA.

Ultrasonic high-resolution longitudinal and shear wave measurements in food colloids: monitoring of gelation processes and detection of pathogens

In the present paper we describe the applications of the ultrasonic high-resolution longitudinal and shear wave measurements for food and bio-colloids. In the first example, both ultrasonic methods were used for the monitoring of the acidified milk gelation induced by glucono-δ-lactone (formation of gel network in yoghurt). Ultrasonic measurements demonstrated a high sensitivity to pre-gelation and gelation processes during the formation of acid milk gels. The hydration of colloidal calcium phosphate released into serum and the swelling of casein in micelles at pH 5.6–5.0 are suggested as the main contributors to the ultrasonic velocity and attenuation changes during the pre-gelation. The increase in shear loss modulus of acidified milk at pH 5.0-4.85 can be explained by the aggregation of the casein micelles into clusters. Subsequent reformation of these clusters into a gel network at pH 4.85–4.6 is observed as a sharp rise in the storage moduli of acid milk gels and an increase in the ultrasonic velocity. The second example is the application of the ultrasonic shear wave measurements for the detection of Salmonella in liquids. The antigen-antibody binding monitored by impedance measurements of a quartz crystal at 5, 15 and 25 MHz results in both the decrease in resonant frequency and an increase in the imaginary part of the quartz impedance. The analysis of the data indicates that the bacteria cells on the sensor surface do not exhibit pure mass-load behaviour, and the viscoelastic properties of the interfacial layer must be taken into account for quantitative analysis. Overall, our ultrasonic measurements demonstrate their high potential as non-destructive methods of analysis of complex foods and bio-colloids.

High-frequency shear and volume viscoelastic moduli of casein particle gel

Abstract Protein particle gels, which consist of a continuous three-dimensional network of flocculated particles, such as casein micelles, are the main constituents of many biological and food colloids. These gels have an extremely short region of linear elastic behaviour and small fracture strain, thus creating complications for their rheological analysis. In the present paper we describe the application of low amplitude ultrasonic measurements for the monitoring of casein particle gel formation in a suspension of casein particles (a process similar to the formation of gel network in yoghurt). Combination of high-resolution longitudinal and shear wave measurements allowed us to make a complete analysis of the shear and volume viscoelasticity of casein particle gels in the frequency range 5–25 MHz. Both shear moduli, the storage ( G ′ Gel ∼30 kPa, at 7 MHz) and the loss moduli ( G ″ Gel ∼100 kPa, at 7 MHz) of the gel in the megahertz frequency range are several orders higher than those determined previously by dynamic rheology at low frequency (0.1 Hz), indicating different contributions of the gel network to the changes in the viscoelastic parameters at low and high frequencies. The volume storage ( K ′ Gel ∼75 kPa, at 7 MHz) and loss ( K ″ Gel ∼15 kPa, at 7 MHz) moduli of the casein particle gel are of the same order as the shear modili of the gel. The contribution of the gel to the volume storage modulus of the whole suspension of casein particles is very small, about 0.001%. This indicates the importance of high-resolution ultrasonic measurements for analysis of these systems.

Volume and compressibility effects in the formation of metal-EDTA complexes

We used precise measurements of ultrasonic velocity and density to study the complexation of ethylendiaminetetraacetic acid (EDTA) with Mg2+, Ca2+, Sr2+, and Ba2+ at 25‡C and pH 12. From these measurements we obtained the changes in the molar concentration increment of the ultrasonic velocity δA, the apparent molar adiabatic compressibility δKsΦ, and the apparent molar volume δVΦ of complex formation. The hydration contributions δ(AVh) to the volume effect of binding range from 39.6 to 46.6 cm3-mol-1 while the hydration contribution to the adiabatic compressibility change in the binding, δ(δKh), ranges from 103.9X 10-4 to 131.1 X 10-4 cm3-mol-1-bar-1. These data are interpreted in terms of dehydration of interacting molecules,i.e., transfer of water molecules from the hydration shells of cations and EDTA into the bulk water. The ratio δ(δVh)/ δ(δVh) is in the range 0.35 to 0.38 bar, indicating a dominant contribution from the dehydration of charged atomic groups in the volume and the compressibility effects of complex formation.

Ultrasonic analysis of kinetic mechanism of hydrolysis of cellobiose by β-glucosidase.

High-resolution ultrasonic spectroscopy (HR-US) was applied for real-time analysis of enzymatic hydrolysis of cellobiose by a β-glucosidase from Aspergillus niger (Novozyme 188) at 50°C and pH 4.9. This technique is noninvasive, it does not require optical transparency and is suitable to continuously monitor the time dependence of the reaction progress in a broad range of experimental conditions. The time profiles of the amount of glucose released and the reaction rate were obtained from the time profile of ultrasonic velocity. The results are in good agreement with a discontinuous glucose assay (hexokinase method). The kinetic parameters of the reaction were estimated by fitting the ultrasonic time profiles of the reaction rates to several inhibition models. In addition, the equilibrium constant for the reaction of hydrolysis of cellobiose and the molar Gibbs free energy of hydrolysis were determined from the ultrasonic time profiles of concentration of glucose in the reverse reaction (glucose condensation). The results suggest the existence of more complex mechanisms regulating the activity of cellobiase than the combination of simple inhibitions. An extended kinetic model based on two sites for the competitive inhibitor (glucose) is proposed.

Ultrasonic analysis of heat-induced coagulation in calcium fortified milk

The key components determining the physical behaviour of milk are casein micelles. Heat-induced coagulation of casein micelles is a major problem in the design of new milk-based products in the food industry. Many of food additives reduce the stability of casein micelles in milk, leading to coagulation during heat treatment. Up to now there was a lack of experimental methods allowing fast, non-expensive and technically non-complicated analysis of the heat stability of milk colloids. In the present work we used a new high-resolution ultrasonic resonator technique to monitor the coagulation of calcium fortified low fat milks. The technique provides a relatively cheap, rapid and non-destructive analysis and requires small volumes of material. Two independent parameters, ultrasonic velocity and ultrasonic attenuation in milk, were measured as a function of temperature to analyse the effects of the addition of calcium and selected stabilisers on the coagulation temperature. Both parameters give the same value of coagulation temperature with a resolution better than 0.1 K. The results allowed us to evaluate the concentrations of stabilisers required to prevent the coagulation of calcium fortified milk at temperatures below 373.15 K.

Lipid membrane domain formation and alamethicin aggregation studied by calorimetry, sound velocity measurements, and atomic force microscopy

An experimental study of phosphocholine membranes made from one lipid, from mixtures of DPPC and DLPC, and also from lipids and small amounts of alamethicin is presented. We used atomic force microscopy to investigate the spatial organization and structure of lipid domains and also of the defects induced by the peptide. Alamethicin was found to alter the state of lipids in the gel state in a way that domains of fluid lipids are formed close to the defects. Differential calorimetry revealed phase characteristics of the lipid mixtures and the effect of small amounts of alamethicin on the phase behavior. It was also shown that the sound velocity profiles of the membranes suspensions can be well calculated from the heat capacity traces of the samples. This result confirms the correlation between the mechanical properties and the specific heat of membrane systems.

Surfactant-DNA complexes in low ionic strength dilute solutions

We have used a combination of optical spectroscopy, fluorescence, isothermal titration calorimetry, surfactant-selective electrode techniques, high-resolution ultrasonic velocity and density measurements to study the binding of the cationic surfactant dodecyltrimethylammonium bromide to short fragments, 200 base pairs, of DNA in dilute solutions at 1 mM NaCl concentration. At the first stage the surfactant forms small aggregates on the DNA surface. This binding is accompanied by a significant change in the DNA secondary structure. At the second stage, we observed the formation of large aggregates of surfactant on the DNA surface.

Excited state complex formation between 3-aminophthalhydrazide and DNA: a fluorescence quenching reaction

Abstract Fluorescence quenching reaction of 3-aminophthalhyrdazide (luminol) due to the interaction with short DNA fragments has been studied employing steady-state and time correlated single photon counting techniques. The interaction of luminol with DNA in the excited state has also been analysed using Stern–Volmer (S–V) mechanism. A very weak ground state interaction is proposed to explain the higher values of the quenching rate constant and is consistent with positive curvatures in the S–V plots. A plausible explanation of the quenching mechanism has been discussed on the basis of hydrogen bonding interaction between luminol and DNA fragment. It is proposed that the phosphate backbone present in DNA is responsible for the interaction with luminol.

High-Resolution Ultrasonic Spectroscopy for Analysis of Industrial Emulsions and Suspensions

This article describes the application of high-resolution ultrasonic spectroscopy (HR-US) for the analysis of industrial emulsions and suspensions. The benefits of HR-US are discussed, including the ability to perform a direct analysis of emulsions and suspensions, which would otherwise be impossible, require significantly more effort in the laboratory, or produce erroneous results. The HR-US 102 spectrometer is also introduced. Manufacture of this laboratory-scale instrument is now possible due to recent technological advances in HR-US. The article outlines the principles of the HR-US technique and illustrates the application of the HR-US 102 spectrometer for analysis of absorption of ligands on the surface of particles, thermal stability, and effects of thermal history on microstructure of emulsions, crystallization, and particle sizing in diluted and concentrated emulsions.