Measurements of the kinetic constants of protein adsorption onto silica particles

The early events pertaining to protein (human serum albumin: HSA) adsorption and desorption onto silica particles were studied employing real time, in situ measurements. The experimental method involved continuous measurements of the outflowing concentration of HSA with a fluorimeter, based on the natural fluorescence of the protein molecules. The adsorption (desorption) took place inside a well-stirred compartment, where particles were brought into contact by injection into a stream of a protein solution of known concentration. Intense mixing and sufficient protein supply rate allowed the process to take place solely under kinetic control. The acquired data were interpreted, according to a kinetic model, in terms of protein binding rates. From the latter, the kinetic association (k(a)) and dissociation (k(d)) constants were determined. To avoid the influence of steric hindrance, only data points obtained within the first 0.5 s of the initialization of the experiments were used. The experiments were performed at different protein concentrations, ranging from 7.25 nM to 14.5 mu M. The real kinetic constants were determined by extrapolating the data obtained to zero protein concentration. Protein concentration effects were found to be pronounced in the determination of the kinetic association constant, producing values underestimated by as much as 30-fold for a 14.5 mu M concentration. The concentration effect on the kinetic dissociation constant was not very significant: it was only of the order of a factor 2. The ratio of favorable to unfavorable protein orientations, also known as von Smoluchowski's factor (f), was found to be 0.064 for the system of silica and HSA. For HSA adsorbing onto silica particles, the following values were found: k(a)= 4.529 x 10(6) l mol(-1) s(-1); k(d)= 0.21 s(-1). To convert from stirred to stationary conditions, both kinetic constants should be reduced by a factor 62.5, decreasing von Smoluchowski's f factor to 0.001. (C) 1999 Elsevier Science B.V. All rights reserved.