Methods for studying protein adsorption

TIRF occupies a unique niche, providing a noninvasive method for studying protein adsorption in situ and in real time. The TIRF technique, as it is applied in our laboratory, has been used successfully to study both macroscopic and molecular aspects of protein adsorption. The present goal of this laboratory is to elucidate the interactions occurring when a protein adsorbs to a solid surface using the TIRF technique. It is hoped that, eventually, a complete, general description of the protein adsorption process will be attained. 

Hlady, V., Buijs, J., Jennissen, H.P. Methods for studying protein adsorption. Methods Enz5rmology 309,402-429 (1999)... 

ATR spectroscopy in the infrared has been used extensively in protein adsorption studies. Transmission IR spectra of a protein contain a wealth of conformational information. ATR-IR spectroscopy has been used to study protein adsorption from whole, flowing blood ex vivo 164). Fourier transform (FT) infrared spectra (ATR-FTIR) can be collected each 5-10 seconds165), thus making kinetic study of protein adsorption by IR possible 166). Interaction of protein with soft contact lens materials has been studied by ATR-FTIR 167). The ATR-IR method suffers from problems similar to TIRF there is no direct quantitation of the amount of protein adsorbed, although a scheme similar to the one used for intrinsic TIRF has been proposed 168) the depth of penetration is usually much larger than in any other evanescent method, i.e. up to 1000 nm water absorbs strongly in the infrared and can overwhelm the protein signal, even with spectral subtraction applied. 

A major area for new research concerns the structural and functional consequences of adsorption of proteins to surfaces (items 3-5 in Table IV). Measurement of conformational change is still in an early stage of development. Most methods for studying adsorbed protein conformation are restricted to comparison of spectral differences induced by adsorption, without knowledge of the actual type or amount of change these differences reflect. Better methodology, especially on quantitative aspects, is sorely needed in this area. The orientation of adsorbed proteins may prove to be readily explored with the monoclonal antibody method and therefore certainly deserves wider application. Finally, the behavior of enzymes and antibodies at interfaces is not... 

Recent innovations in filtration technology include tangential flow ultrafiltration methods that act as preconcentration methods for studies of marine biopolymers such as proteins (see also the section on Adsorption techniques below). 

Adsorption of putidaredoxin on gold electrodes has been studied using dynamic spectroscopic ellipsometry and differential capacitance measurements [307]. In Ref. 307, a method for the measurement of metal surface optical perturbation during protein adsorption at a constant potential has been described. The method is based on the concept that the charged transition layer develops between the electrode substrate and the adsorbate. 

Another major advantage of molecular graphics is that it allows real time manipulation of several interacting molecules while quantitatively monitoring the stereochemistry and even the interaction energies. These methods are only beginning to be used for studies of interactions between molecules and only in the most preliminary way for the simulation of protein adsorption 29). 

Techniques and methods for the study of protein adsorption have been well reviewed 4). It is now generally recognized that it is not necessarily the type and amount of protein present at the surface which is most important, but rather the orientation and conformational state of those proteins. At present it is virtually impossible to predict the specific conformation of an adsorbed protein at a particular interface. The techniques used in the determination of protein conformation in solution or in the solid state do not usually apply to adsorbed proteins. Hence, the difference between adsorbed and bulk solution protein conformation has to be inferred indirectly. 

A novel capillary electrophoresis method using solutions of non-crosslinked PDADMAC is reported to be effective in the separation of biomolecules [211]. Soil studies conducted with PDADMAC report the minimization of run-off and erosion of selected types of soils [212]. In similar studies, PDADMAC has found to be a good soil conditioner [213]. The use of PDADMAC for the simultaneous determination of inorganic ions and chelates in the kinetic differentiation-mode capillary electrophoresis is reported by Krokhin [214]. Protein multilayer assemblies have been reported with the alternate adsorption of oppositely charged polyions including PDADMAC. Temperature-sensitive flocculants have been prepared based on n-isopropylacrylamide and DADMAC copolymers [215]. A potentiometric titration method for the determination of anionic polyelectrolytes has been developed with the use of PDADMAC, a marker ion and a plastic membrane. The end-point is detected as a sharp potential change due to the rapid decrease in the concentration of the marker due to its association with PDADMAC [216]. 

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