High-Intensity Ultrasound Mediated Structure-Function Changes of BSA as Affected by pH

The influence of high intensity ultrasound on the structure-function relationship of BSA sonicated at various sonication times at neutral, acidic and basic pH was determined. Protein solutions (3 x 10^-4 M) were treated with high-intensity ultrasound at an ultrasonic intensity of 20 Wcm^-2 with treatment times ranging from 0 to 90 minutes. Changes in structure-function properties of BSA were monitored using a wide variety of biochemical and physicochemical analytical methods including interfacial tensiometry, free sulfhydryl group assays, zeta potential analysis, hydrophobicity assays, circular dichroism spectroscopy, micro DSC and FTIR. The results were attributed to mechanical and chemical changes in the fundamental protein structure due to cavitational events generated by high-intensity ultrasound. Hence, the results of this study contribute to a better understanding of the interaction of high-intensity ultrasound waves with proteins.

The results of the first part of this study showed that application of high intensity ultrasound increased the rate of adsorption of bovine serum albumin (BSA) at the air-water interface both in the short-term and long-term range of the adsorption kinetics. The rate of adsorption increased with increasing sonication duration. Possible structural modifications were monitored using microDSC to investigate phase transitions, Ellman’s assay to determine free sulfhydryl content, zeta-potential analyses to measure surface charge of BSA, photon correlation spectroscopy to determine particle size, blue native PAGE to identify presence of monomeric and/or polymeric units and finally FT-IR spectroscopy to determine the content of secondary structural elements. Results indicated that application of high-intensity ultrasound caused subtle structural changes in BSA and consequently altered its functional properties.

In the second part of the study, the influence of pH on the ultrasound-influenced structure-function modification of BSA was investigated. The interfacial activity of BSA generally increased with increasing sonication time, independently of solvent pH. Nevertheless, solvent pH itself had an influence on interfacial activity of BSA i.e. equilibrium surface tension values and diffusion rates calculated from the short- and long-term solution of the adsorption kinetics model varied with both pH and ultrasonic duration. The changes in the structure-function relationship of native protein upon ultrasonication were related to the pH-dependent isomerization states of BSA. The findings in the pH-sonication experiments supported previous findings that suggest the formation of a modified intermediate structure that enhanced the short-term and long-term surface activity of BSA at the air-solvent interface.

In conclusion, the results indicate that ultrasonication of proteins does not lead to a complete loss of structure of proteins but instead yields a complex, biopolymer and solvent specific alteration of the underlying molecular structure of the treated biopolymer where functionalities may be preserved or even enhanced. As such, the study also offers an explanation as to previously observed changes in enzyme functionality upon ultrasonication.