Measurement of protein adsorption kinetics by an in situ, “real-time,” solution depletion technique

Abstract

An experimental method, which provides real-time in situ data, was used to study protein adsorption. Proteins (fibrinogen or immunoglobulin G) were radioactively labeled with 125I and added in small amounts to the appropriate solution under study, either single protein in buffer or plasma. This protein solution was injected at a constant flow rate into a well-mixed cell containing a suspension of spherical soda-lime glass beads. The buffer contained in the cell was gradually displaced by the protein solution. NaI(T1) solid scintillation detectors, coupled to a high voltage power supply and a multichannel analyzer, were placed at the exit of the cell to monitor the radioactivity of the bulk solution. Adsorption of the protein was determined via depletion of protein in the bulk solution prior to attainment of steady state. The method may thus be considered a solution depletion technique. For fibrinogen as a single protein in buffer, a period of rapid initial adsorption was observed, followed by slower adsorption and attainment of constant surface coverage. The period of rapid initial adsorption rate decreased and the initial adsorption rate increased with increasing fibrinogen feed concentration. The maximum coverage observed was about 0.35 μ/cm 2. The effects of diffusion and adsorption kinetics on the initial stages of fibrinogen surface binding were simulated using the appropriate form of the diffusion equation coupled with Langmuir adsorption kinetics. Based on comparisons between the simulated and real data it was determined that the principal adsorption rate limitations were due to protein supply and the kinetics of surface binding. Diffusion effects were shown to be negligible in this well-stirred system. The simulation may also be used to design experiments and fit mathematical adsorption models to data for the adsorption of any solute in a similar system. Adsorption of fibrinogen from blood plasma was found to be transient, thus confirming, essentially in real time and in situ, the so-called “Vroman effect,” which has been observed with non- in situ methods. Adsorption maxima occurred at times which depended on the concentration of plasma fed to the cell and which decreased with increasing concentration. This trend is in agreement with accepted mechanisms of the Vroman effect. Adsorption of IgG from plasma was also found to be transient and the peaks occurred at times shorter than those for fibrinogen, again in agreement with the “principles” of competitive adsorption as reflected in the Vroman effect.