Benjamin Gavish

Monitoring of erythrocyte aggregate morphology under flow by computerized image analysis

The morphology of red blood cell (RBC) aggregates was studied by direct visualization of RBC aggregation at different flow conditions in a computerized image analyzer. The aggregate morphology is expressed by an Aggregate Shape Parameter (ASP), defined as the ratio of the aggregate projected area to its square perimeter. Aggregation was induced by either dextran-70 (m.w. 70,000) or dextran-500 (m.w. 500,000), and compared to that in plasma. It was found that the aggregate morphology is a characteristic of the aggregating agent--in dextran-500, the RBC form rouleau aggregates as in plasma, while in dextran-70, they form clusters. In each system, while maintaining the overall typical morphology, the ASP decreases (i.e., the aggregate becomes longer) as the aggregate size is increased. The distribution of the ASP as a function of the aggregate size remains unchanged when the aggregate size is changed by modulation of the dextran concentration or the shear stress. Stretching of a rouleau aggregate by application of shear stress is reflected by a corresponding change in the ASP. It is suggested that the ASP is a characteristic of intercellular interactions. A theoretical model is proposed for evaluation of the deviation of aggregate shape from that of rouleau structure.

Viscous cosolvent effect on the ultrasonic absorption of bovine serum albumin.

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 chains movements, leading to larger protein-volume fluctuation and higher ultrasonic absorption. This study demonstrates that solvent viscosity is a regulator of protein structural fluctuations.

REDUCTION OF PROTEIN VOLUME AND COMPRESSIBILITY BY MACROMOLECULAR COSOLVENTS : DEPENDENCE ON THE COSOLVENT MOLECULAR WEIGHT

The partial specific volume (V) and adiabatic compressibility (beta) of myoglobin have been shown to be reduced by small cosolvents such as glycerol (A. Priev, A. Almagor, S. Yedgar, B. Gavish, Biochemistry 35 (1996) 2061-2066). To elucidate the effect of the cosolvent size on these protein properties, in the present study we determined V and beta of myoglobin in solutions containing a homologous cosolvent series from sucrose to dextran--500 (M.W. 500,000). It was found that in addition to the expected effect of the cosolvent concentration, V and beta decrease with increasing cosolvent M.W. This suggests that structural properties of the cosolvent contribute to its effect on the protein interior.

Red blood cell aggregability is enhanced by physiological levels of hydrostatic pressure

The effect of hydrostatic pressure of up to 15 bars on the aggregability of rat and human red blood cells (RBC), i.e., their capability to form aggregates, was studied using computerized image analysis. The aggregate size distribution was determined under ambient pressure, following application of hydrostatic pressure for various durations up to 2 h. It was found that RBC aggregability markedly increases, up to three-fold, as the pressure which had been applied was increased. Accordingly, higher shear stress is required for dispersing the aggregates of pressure-treated RBC than those of untreated cells. The median size of human RBC aggregates was about three times higher than that of rat RBC, and this ratio was maintained following pressure treatment. RBC aggregability is a major determinant in blood flow, especially in the microcirculation. Pressure at the levels used in this study occurs in physiological states such as hyperbaric treatment or diving. The enhanced aggregability induced by application of such pressure implies that blood flow in microvessels might be altered under conditions associated with elevated hydrostatic pressure.

Solvent viscosity effects on protein dynamics studied by ultrasonic absorption

Solvent viscosity is known to play an important role in the kinetics of biochemical reactions, and has been suggested to modulate the dynamic structure of proteins. The effect of viscous cosolvents, of various molecular sizes, on the apparent ultrasonic absorption of bovine serum albumin in solution, at 37 degrees, has been measured in attempt to investigate the following phenomena: 1) The predicted modulating effect of viscous cosolvents on the internal friction of proteins, and 2) Possible differences between the microscopic and macroscopic pictures of the solvent viscosity concerning the proposed effect. We have found that A) The absorption of ultrasound (3-17 MHz) by the protein increases with increasing the cosolvent concentration. B) That increase correlates with the solvent viscosity for small cosolvent molecules, but not with macromolecular cosolvents, and C) Dextran solutions with the same concentration by weight, reveal similar ultrasonic absorption, in spite of large differences in their viscosity. A possible explanation is discussed.