Direct measurement of interactions between adsorbed vitronectin layers: the influence of ionic strength and pH

Vitronectin (Vn) is an adhesive protein in the plasma serum and plays an important role in cell attachment, spreading, and proliferation. The interactions between protein bovine vitronectin layers adsorbed onto a silica probe and a mica surface have been investigated with the use of atomic force microscopy (AFM). Adsorption of vitronectin was confirmed by XPS surface analysis. The force-separation curves and pull-off forces were measured as a function of ionic strength and solution pH. The pull-off force (adhesion force) decreased as the salt concentration increased, which suggests that some binding domains of this protein may associate with the ionic species and reduce its binding ability. Discrete jumps, or discontinuities, in the separation force curve were observed to extend to a maximum of 300 nm, evidence that the protein molecules bridge between the surfaces. As a function of pH, the adhesion force on separation of the protein-coated surfaces showed a maximum at pH 5 (i.e.p. of vitronectin), decreasing in magnitude at lower and higher pH values. At pH 5, the approaching curves illustrated a jump-in force; whereas for pH values away from 5, the approaching force curves were repulsive. Correlation of the interaction forces with Vn conformational changes in different pH environments, directly visualized with the use of AFM imaging, was developed. In its i.e.p. region, the Vn molecular conformation appeared to be dense and compact. Significantly, at wounds/injured sites the pH is low (approximately 5) which this study discovered to facilitate adsorption and formation of vitronectin aggregates, known to trigger their subsequent biological functions.