Aggregation Analysis employing Ultrasonic Spectroscopy

Ultrasonic monitoring of polymer collapse in 5% (w/w) PNIPAM solution

Aggregation of carbonic anhydrase in solution, induced by thermal denaturation

Coagulation in calcium fortified milk

 

 

Ultrasonic monitoring of polymer collapse in 5% (w/w) PNIPAM solution

An example of the application of the temperature ramp regime is in the analysis of heat transition in an aqueous solution of poly(N-isopropylacrylamide), a polymer whose applications include thermoresponsive gels. As temperature rises, the polymer coil collapses into a compact globule, and aggregates are formed. Ultrasonic velocity decreases during the process, reflecting the dehydration of the polymer, and the intrinsic elasticity of the globules and aggregates. Attenuation increases as the aggregates cause increased scattering of the ultrasonic waves. As illustrated above, ultrasonic spectrometry can be used to pinpoint the temperature and width of the phase transition and analyse the transformations in the polymer structure, both of which are illustrated by the changes in velocity. The differences in attenuation mean the structure of the aggregates can be characterized.

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Aggregation of carbonic anhydrase in solution, induced by thermal denaturation

Carbonic anhydrase metalloenzymes are involved in critical physiological processes related to the respiration and transport of CO2/HCO3 between metabolizing tissues and the lungs, pH homeostasis and various biosynthetic reactions such as gluconeogenesis, lipogenesis.  One of most important characteristics of the enzymes is thermal stability, which is determined by chemical structure and the conformation of protein molecules. To examine this, 1 mL of freshly prepared enzyme solution (1% w/v) in tris-buffer, (pH 7.4) was loaded into one ultrasonic cell of HR-US 102 spectrometer, and a reference cell was filled with the buffer. The difference in the ultrasonic velocity between two solutions and the change in attenuation in the solution of carbonic anhydrase upon the heating of the samples at heating rate of 0.1 C/min was investigated.  As seen in the figure above, the difference in ultrasonic velocity (solution - buffer) decreases gradually within the temperature range between 30-58C. This decrease in ultrasonic velocity is attributed to the different temperature dependences of density and compressibility of the hydration shell of protein in comparison with bulk water. The beginning of protein aggregation is indicated by the sharp growth in ultrasonic attenuation above 50C. The formation of protein particles and the scattering of ultrasonic wave increase the attenuation. The main heat-induced transition (protein denaturation) is observed between 58C to 64C and is indicated by sharp changes in both ultrasonic parameters (increase in attenuation/decrease in velocity). The decrease in velocity demonstrates the formation of a highly compressible hydrophobic core of protein aggregates in which hydrophobic amino acid residues stick to each another.  This extra compressibility of the core decreases the ultrasonic velocity, similar to what is generally observed in hydrophobic aggregation during the formation of surfactant micelles. The insert shows the ultrasonic temperature profiles in the carbonic anhydrase solution measured after several weeks of the storage of the solution at 4C; the transition temperature is almost the same as that of the fresh sample. However the effect of transition on the ultrasonic parameters is different. The ultrasonic attenuation is nearly twice as high in the aged sample compared with the fresh sample, and the changes in ultrasonic velocity are the opposite.  This indicates that the ageing of the enzyme solution during the storage can be analysed by High-Resolution Ultrasonic Spectroscopy.

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Coagulation in calcium fortified milk

This example shows an application of HR-US spectrometer for monitoring of coagulation in calcium fortified low-fat milks. Adding calcium ions to low fat milk reduces the stability of casein micelles, causing coagulation on heating or when it is added to hot beverages. Additives can be used to stabilise the system, and ultrasonic spectroscopy has been applied to establish the best stabiliser. When the milk coagulates, ultrasonic attenuation increases as a result of scattering of ultrasonic waves by aggregates.  Ultrasonic velocity decreases as the aggregates have a compressible core which slows the waves down. The effect of three different stabilisers on coagulation of milk fortified with 25mM of calcium is shown above. Potassium citrate was shown to be the most effective, followed by sodium carbonate, and sodium acetate was the least effective.

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