Fibrinogen dimensions at an interface: variations with bulk concentration, temperature, and pH

A hydrodynamic technique has been developed to study the thickness of layers of proteins adsorbed on solids. This technique is based on differences in flowrate through a system of capillaries (constructed from the material of interest) before and after adsorption. The parameter obtained is the hydrodynamic thickness (LH) and is based on the resistance to flow offered by the capillary system. Using this technique, various aspects of the layer thickness of human fibrinogen adsorbed on fritted glass filters have been investigated. LH values for adsorption from Tris buffer, pH 7.35 at 25°C were found to vary as a function of fibrinogen concentration (LH isotherm). Values varied from about 50 to 600A˚in the concentration range 10−4 to 0.03% (w/w). These LH values are of the same order of magnitude as the dimensions of fibrinogen molecules. When an adsorbed layer established at 25°C was subjected to variations of temperature, the following changes were observed: 25 to 50°C, sharp decrease in LH; 50 to 70°C gradual increase in LH; 70 to 25°C, gradual decrease. After the initial increase to 70°C, the effect between 70 and 25°C was reversible with no hysteresis. These effects are probably due partly to reversible thermal denaturation of fragment D and partly to thermal expansion/contraction. DSC measurements in the adsorbed state confirmed this interpretation in that the transition expected for denaturation of fragment D around 60°C ± 10°C was observed. A change in pH from 7.35 to 9.25 caused an instantaneous threefold increase in LH followed by a slow desorption (several days). This effect is reversible when the pH is subsequently decreased before substantial desorption has taken place.