Abstract
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 μM. 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 μM 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=4.529×10 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.
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Disclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.