Action of shear on enzymes: Studies with alcohol dehydrogenase

Abstract

Yeast alcohol dehydrogenase (ADH) solutions (approximately 1 mg/ml, pH 7) were sheared in a coaxial cylindrical viscometer. This was fitted with a lid sealing the contents from the atmosphere and preventing evaporation. At 30 degrees C after a total of 5 hr intermittent shearing at 683 sec-1 no losses of activity were observed. No losses were found after 5 hr continuous shearing and in a no-shear control. At 40 degrees C and 683 sec-1 there were only small activity losses in 5 hr. Shearing at 3440 sec-1 no measurable losses of activity were found with a 1.03 mg/ml solution in 5 hr at 30 degrees C, a 1.03 mg/ml solution in 8 hr at 5 degrees C, and with a 3.89 mg/ml solution in 3 hr at 5 degrees C. In all these cases, however, a white precipitate formed that was not observed in zero shear control experiments. The sheared 3.89 mg/ml solution was clarified by centrifugation. It was shown that there were no ADH aggregates in the supernatant and that the precipitate was less than 2% of the original protein. At 30 degrees C under adverse pH conditions (pH 8.8) there was no significant difference in activity losses of an approximately 1 mg/ml solution sheared at 65 and 744 sec-1. An approximately 0.5 mg/ml ADH solution, pH 7, was agitated in a small reactor with no free air-liquid interface. Peak shear rates near the impeller were estimated to be about 9000 sec-1. Only a small decrease in specific activity was observed until over 15 hr total running at 5 degrees C.

The effects of shear flow on protein structure and function

“…Several studies show that the initial destabilization of the protein structure occurs at the solvent-air or solvent-solid interface, where surface tension forces unfold the native protein. 36,37,64,79 The air-water interfacial force is estimated to be 140 pN, 80 taking the air-water interface to be 2 nm in depth with a 0.07 N/m surface tension. 81 This value is comparable to the 150 pN observed to unfold globular proteins in atomic force microscopy studies.…”

Charge-mediated Fab-Fc interactions in an IgG1 antibody induce reversible self-association, cluster formation, and elevated viscosity

“…15 Moreover, high shear stress during pumping of viscous solutions of self-associated proteins has, in some cases, been shown to also contribute to the formation of irreversible protein aggregates. 16 Such irreversible aggregates can decrease protein activity and stability, and may elicit adverse immunogenic reactions in patients. [17][18][19] In addition, parenteral administration of highly viscous liquids requires thicker gauge needles that may cause more painful injections.…”

Hydrogen exchange mass spectrometry reveals protein interfaces and distant dynamic coupling effects during the reversible self-association of an IgG1 monoclonal antibody

“…13 The increase in solution viscosity also imposes manufacturing challenges including high back-pressure and clogging of membranes, 2 as well as elevated levels of shear stress during pumping. 14 Reversible-self association of mAbs is generally attributed to weak and transient non-covalent interactions (e.g., hydrogen bonding, electrostatic, hydrophobic, p-p, and van der Waals interactions) between antibody molecules. As the protein concentration increases and the intermolecular distances decrease, the extent of non-covalent interactions between molecules rises.…”