Real-Time Monitoring of Heat-Induced Aggregation of β-Lactoglobulin in Aqueous Solutions Using High-Resolution Ultrasonic Spectroscopy

Abstract

High-resolution ultrasonic spectroscopy was applied for real-time monitoring of the heat-induced denaturation and aggregation processes in aqueous solutions of β-lactoglobulin. The temperature profiles for the ultrasonic velocity and attenuation in the frequency range from 4 MHz to 16 MHz were measured during heating and cooling cycles, 35 • C to 120 • C to 35 • C, with different heating and cooling rates. Two processes were identified in the heating profiles: transition to the molten globular state followed by formation of protein aggregates. Both processes are accompanied by a decrease in the ultrasonic velocity and an increase in compressibility. The ultrasonic attenuation did not show a significant change during the transition to the molten globule but increased significantly during aggregation. The diameter of the aggregates (calculated from ultrasonic attenuation) was of the order of 100 nm and depended on the pH and the heating rate. Variation of pH from 6.0 to 7.5 had a pronounced effect on the size of protein aggregates. Some effect of pH on the intrinsic properties of aggregates was also detected.

High-resolution ultrasonic spectroscopy

“…The observed linear decrease in ultrasonic velocity at temperatures before the transition, 50 to 57 • C (not related to denaturation) was extrapolated on the whole temperature range and subtracted as a baseline. aggregation is accompanied by the formation of a "soft", hydrophobic-rich interior of the aggregates, which shall increase the compressibility and decrease the ultrasonic velocity (Ochenduszko and Buckin, 2010). The observed increase in ultrasonic attenuation, as in the case of PNIPAM, can be attributed to the scattering effects on the protein particles.…”

“…High sensitivity of compressibility β S to molecular organization and intermolecular interactions in the medium allows application of the HR-US technique in analysis of a broad range of molecular processes. This includes analysis of conformational transitions of polymers (Ochenduszko and Buckin, 2010;Perinelli et al, 2017;Van Durme et al, 2005;Wang et al, 2006), ligand binding (Jager et al, 2005;Kargerová and Pekař, 2014;Melikishvili et al, 2016), self-assembly and aggregation (Andreatta et al, 2005;Dalgleish et al, 2005;Klučáková and Věžníkova, 2016;Lehmann and Buckin, 2005), crystallisation and gelation (Lawrence et al, 2005;Lehmann et al, 2004;Smyth et al, 2001Smyth et al, , 2004Yuno-Ohta et al, 2016;Dalgleish et al, 2004), analysis of phase transitions and phase diagrams (Hickey et al, 2006(Hickey et al, , 2010, monitoring of chemical and biochemical reactions (Altas et al, 2016;Buckin and Altas, 2017;Resa and Buckin, 2011;Yuelong and Buckin, 2016), and others. Examples of these applications are outlined in Sect.…”

Ultrasonic Monitoring of Biocatalysis in Solutions and Complex Dispersions

“…According to Equations, for dispersions of solid particles of micron and submicron size, the dependence of the scattering contributions α scatt measured at ultrasonic frequencies on the size of the particle has a 'bell' shape profile. The 'bell' has two, normally unresolved, maxima at the particle sizes close to (comparable with) the wavelength of the shear wave in the continuous medium and with the wavelength of the thermal wave in the particle and in the continuous medium (examples of the profiles are given in references [29,31,41]). The scattering contribution to ultrasonic velocity produces an increase of velocity with size following the S-shape profile with plateaus at small and large sizes and the transition sizes around the maxima of ultrasonic attenuation.…”

“…The ultrasonic attenuation profiles for different frequencies are presented in the inset and the profile for frequency 14.5 MHz is plotted in the main frame. This profile was recalculated into the evolution of the 'average' (over ultrasonic scattering profile, attenuation vs. size, see examples of the profiles in [30,41,81]) size of the protein particles during the process using the particle sizing module of HRUS 102 software, which utilized the equations as those discussed in Appendix A.3. The volume fraction of casein micelles was assumed to be constant.…”

High-Resolution Ultrasonic Spectroscopy: Looking at the Interpolyelectrolyte Neutralization from a Different Perspective

“…Therefore, HR-US is a very versatile method that can be used to analyze a broad range of molecular processes. Current applications of this technique include analysis of aggregation, micellization, and gelation phenomena, − phase diagrams of microemulsions, conformational transitions in polymers and biopolymers, interactions between polyelectrolytes and surfactants or amino acids, − stability of emulsions and suspensions, − polymer adsorption onto surfaces, enzymatic hydrolysis of proteins and carbohydrates, ,− and many others. Despite being employed for the characterization of numerous chemical and physical processes, there is a lack in the literature regarding the potential use of HR-US for studying the formation of polyelectrolyte complexes.…”