Effects of ultrasound on the digestive enzyme pepsin A

Kathryn Kapp, Rebecca Hetz


Many medical procedures use high frequency sound waves for both treatment and imaging. Even with regular use of ultrasound, there remains an incomplete understanding of its molecular level effects on biological macromolecules. Ultrasound results in acoustic cavitation, the formation, expansion, and violent, implosive collapse of bubbles within a liquid containing dissolved gas.  High pressure results in shear-stress and high temperatures (up to 5000°C) at the site of bubble collapse can cause thermally-induced breakdown of the water molecule generating reactive free radical species. Both shear-stress, high temperatures, and free radical reactions, may denaturation nearby biomacromolecules.  To more completely understand potential molecular-level effects of ultrasound on biological macromolecules and the operative mechanism of denaturation, the activity of the digestive enzyme pepsin (a 35 kDa protein with primarily beta-sheet secondary structure) was measured as a function of sonication time using a stop-point assay of hemoglobin degradation. After a one-hour sonication, enzyme activities typically decreased by ~40%, suggesting a sonochemically-induced structural change to the enzyme active site. To assess whether free radicals were responsible for loss of enzyme activity, pepsin was sonicated in the presence of the free radical scavenger, n-butanol, up to 0.1 M.  Enzyme activity decreases was significantly less (<5%) in the presence of scavenger which showed that radical reactions that alter the enzyme structure are the principle mechanism for enzyme denaturation. The compact shape and relatively small size of pepsin likely protects the enzyme from significant shear-stress.


sonochemistry, sonication, ultrasound, pepsin, free radicals

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