Measuring Solution Viscosity and its Effect on Enzyme Activity

Abstract

In proteins, some processes require conformational changes involving structural domain diffusion. Among these processes are protein folding, unfolding and enzyme catalysis. During catalysis some enzymes undergo large conformational changes as they progress through the catalytic cycle. According to Kramers theory, solvent viscosity results in friction against proteins in solution, and this should result in decreased motion, inhibiting catalysis in motile enzymes. Solution viscosity was increased by adding increasing concentrations of glycerol, sucrose and trehalose, resulting in a decrease in the reaction rate of the H(+)-ATPase from the plasma membrane of Kluyveromyces lactis. A direct correlation was found between viscosity (eta) and the inhibition of the maximum rate of catalysis (V(max)). The protocol used to measure viscosity by means of a falling ball type viscometer is described, together with the determination of enzyme kinetics and the application of Kramers' equation to evaluate the effect of viscosity on the rate of ATP hydrolysis by the H(+)-ATPase.

Fig. 1

A: Effect of solute concentration on medium viscosity. The viscosity (η) at a given concentration of carbohydrate (expressed as mass fraction, c) was measured in a falling ball viscometer at 20°C as described in methods. B: Solution viscosity (η) inhibits the plasma membrane H⁺-ATPase. The V_max at a given concentration of solute was calculated by measuring the rate of ATP hydrolysis versus ATP concentration and fitting to the Hill equation [eq 2]. The relative V_max (V_max0/V_max) was plotted against the relative viscosity (η/η0). Both, η0 and V_max0 are the solution viscosity and the maximum velocity respectively in the absence of the viscosogenic agent: Glycerol ○; Trehalose ; Sucrose □.

Fig. 2

Effect of temperature on viscosity. Viscosity solution was measured as described in Fig. 1 in the absence of trehalose (○) and in the presence of 0.5 M trehalose (●) at the indicated temperatures.

Fig. 3

Temperature-mediated modulation of the effects of viscosity on the H⁺-ATPase. The V_max was calculated in the presence of different trehalose concentrations and temperatures as described in methods. The relative V_max (V_max0/V_max) was plotted against the relative viscosity (η/η0). η0 is the viscosity of the solution in the absence of trehalose and V_max0 is the V_max in the absence of trehalose. Temperatures (°C): ○ 20, ● 35 and □ 40. The solid lines are linear regressions of the data.

Fig. 4

Drawing of the setting used to measure viscosity. A Kitasatto flask containing the viscometer was filled with water and connected to a circulating bath with controlled temperature. The clamp holding the flask to the support could be rotated 180°C in order to duplicate the measurement of the time of ball descent.