The addition of hydrogen-bonded cosolvents to aqueous solutions of proteins is known to modify both thermodynamic and dynamic properties of the proteins in a variety of ways. Previous studies suggest that glycerol reduces the free volume and compressibility of proteins. However, there is no directly measured evidence for that. We have measured the apparent specific volume (V) and adiabatic compressibility (K) of a number of proteins, sugars, and amino acids in water and in 30% glycerol at pH 7.4 and 30 degrees C. The values of V and K in water and their changes induced by glycerol were extrapolated to the limit of infinite solute size. The main results were the following: (a) glycerol decreases V and K of proteins, but increases it for amino acids; (b) the V and K values of the protein interior in water were found to be 0.784 +/- 0.026 mL/g and (12.8 +/- 2.5) x 10(-6) mL/g x atm, where the glycerol reduces these values by 8 and 32%, respectively; (c) the coefficient of adiabatic compressibility of the structural component of proteins affected by the glycerol is estimated to be (50 +/- 10) x 10(-6) atm(-1), which is comparable to that of water. We propose that the glycerol induces a release of the so-called "lubricant" water, which maintains conformational flexibility by keeping apart neighboring segments of the polypeptide chain. This is expected to lead to the collapsing of the voids containing the water as well as to increase intramolecular bonding, which explains the observed effect.

Extracellular f luid macroviscosity (EFM), modified by macromolecular cosolvents as occurs in body f luids, has been shown to affect cell membrane protein activities but not isolated proteins. In search for the mechanism of this phenomenon, we examined the effect of EFM on mechanical f luctuations of the cell membrane of human erythrocytes. The macroviscosity of the external medium was varied by adding to it various macromolecules [dextrans (70, 500, and 2,000 kDa), polyethylene glycol (20 kDa), and carboxymethyl-cellulose (100 kDa)], which differ in size, chemical nature, and in their capacity to increase f luid viscosity. The parameters of cell membrane f luctuations (maximal amplitude and half-width of amplitude distribution) were diminished with the elevation of solvent macroviscosity, regardless of the cosolvent used to increase EFM. Because thermally driven membrane f luctuations cannot be damped by elevation of EFM, the existence of a metabolic driving force is suggested. This is supported by the finding that in ATPdepleted red blood cells elevation of EMF did not affect cell membrane f luctuations. This study demonstrates that (i) EFM is a regulator of membrane dynamics, providing a possible mechanism by which EFM affects cell membrane activities; and (ii) cell membrane f luctuations are driven by a metabolic driving force in addition to the thermal one.The viscosity of body fluids is determined by the level of macromolecules consisting of proteins, lipoproteins, and polysacharides (1). Accordingly, elevated plasma viscosity has been observed in various diseases associated with increased levels of proteins and lipoproteins, such as diabetes, hyperlipidemia, macroglobulinemia, multiple myeloma, nephrosis, and others (1-5). Various studies have shown that solvent viscosity affects protein dynamics and reactions (6-10). However, in these studies the solvent viscosity was modified by the addition of high concentrations of small cosolvents such as glycerol and sucrose, producing relatively high viscosity levels. This is incompatible with physiological and pathological states, where fluid viscosity is altered by small concentrations of large macromolecules (1). Other studies, in which the viscosity was elevated by macromolecular cosolvents, have shown that extracellular fluid macroviscosity (EFM) is a regulator of cellular processes, such as secretion of renin (11) and lipoproteins (12), phospholipase A 2 activity at the cell membrane (13, 14), and ganglioside metabolism (15). In search of the mechanism of this phenomenon, the effect of macroviscosity, as modified by macromolecules, on isolated proteins in aqueous solutions was examined (16,17). It was found that the effect of solvent viscosity decreases with increasing molecular weight of the cosolvent and is practically diminished when the cosolvent molecular weight exceeds that of the protein. Because the activity of cell membrane enzymes is known to be sensitive to the physical properties of the membrane (18), we considered the possibility that the...

No abstract

Protein-ligand binding and enzyme activity have been shown to be regulated by solvent viscosity, induced by the addition of viscous cosolvents. This was indirectly interpreted as an effect on protein dynamics. However, viscous cosolvents might affect dynamic, e.g., viscosity, as well as thermodynamic properties of the solution, e.g., activity of solution components. This work was undertaken to examine the effect of viscous cosolvent on the structural dynamics of proteins and its correlation with dynamic and thermodynamic solution properties. For this purpose we studied the effect of viscous cosolvent on the specific ultrasonic absorption, delta mu, of bovine serum albumin, at pH = 7.0 and at 21 degrees C, and frequency range of 3-4 MHz. Ultrasonic absorption (UA) directly probes protein dynamics related to energy dissipation processes. It was found that the addition of sucrose, glycerol, or ethylene glycol increased the BSA delta mu. This increase correlates well with the solvent viscosity, but not with the cosolvent mass concentration, activity of the solvent components, dielectric constant, or the hydration of charged groups. On the grounds of these results and previously reported findings, as well as theoretical considerations, we propose the following mechanism for the solvent viscosity effect on the protein structural fluctuations, reflected in the UA: increased solvent viscosity alters the frequency spectrum of the polypeptide chain movements; attenuating the fast (small amplitude) movements, and enhancing the slow (large amplitude) ones. This modulates the interaction strength between the polypeptide and water species that "lubricates" the chain's movements, leading to larger protein-volume fluctuation and higher ultrasonic absorption. This study demonstrates that solvent viscosity is a regulator of protein structural fluctuations.

No abstract