Rheo-NMR Studies of an Enzymatic Reaction: Evidence of a Shear-Stable Macromolecular System

Abstract

Understanding the effects of shear forces on biopolymers is key to understanding how biological systems function. Although currently there is good agreement between theoretical predictions and experimental measurements of the behavior of DNA and large multimeric proteins under shear flow, applying the same arguments to globular proteins leads to the prediction that they should only exhibit shear-induced conformational changes at extremely large shear rates. Nevertheless, contradictory experimental evidence continues to appear, and the effect of shear on these biopolymers remains contentious. Here, a custom-built rheo-NMR cell was used to investigate whether shear flow modifies enzyme action compared with that observed quiescently. Specifically, (1)H NMR was used to follow the kinetics of the liberation of methanol from the methylesterified polysaccharide pectin by pectinmethylesterase enzymes. Two different demethylesterifying enzymes, known to have different action patterns, were used. In all experiments performed, Couette flows with shear rates of up to 1570 s(-1) did not generate detectable differences in the rate of methanol liberation compared to unsheared samples. This study provides evidence for a shear-stable macromolecular system consisting of a largely beta-sheet protein and a polysaccharide, in line with current theoretical predictions, but in contrast to some other experimental work on other proteins.

Rheo-NMR studies of a nematic worm-like micelle system in a high-shear-rate regime

“…The rheo-NMR experiments were performed on samples in a custom-built Couette cell which fitted into a standard 500 MHz 5 mm NMR probe head 34 (Fig. 1).…”

Anomalous shear banding revisited with Rheo-NMR and Rheo-USV

“…Therefore, it is likely that even subtle differences in the experimental setups can lead to widely different behavior regarding elastic turbulence. Indeed, subtle differences in shear cells used in different techniques have also been suggested as an explanation of contradictory results regarding the effects of shear on protein structure (Edwards et al 2010).…”

Micro-rheological behaviour and nonlinear rheology of networks assembled from polysaccharides from the plant cell wall

“…0.5 ml of enzyme solution was then mixed into 30 ml of solution, and the solutions were left at 20 • C for a chosen time, depending on the final DM decrease required and on the rate of the de-methylesterification processes. Assuming the enzyme reaction followed a simple Michaelis-Menton law and were in the linear section of the product production curve (which was monitored independently using NMR to follow the liberation of methanol [44]), preliminary experiments were used to determine the rate of DM decrease as 4% per hour for the plant PME de-methylesterification used. Thus, the PME action could be stopped after a pre-requisite time in order to achieve a desired final DM.…”