Proteins adsorbed to solid surfaces

Hydrogen Exchange Mass Spectrometry for Proteins Adsorbed to Solid Surfaces, in Frozen Solutions, and in Amorphous Solids... 

The stmctural and conformational analysis of proteins adsorbed to solid surfaces is difficult because most common analytical methods are not compatible with the presence of the interacting solids. With recent developments in instrumentation and techniques, our understanding of protein adsorption behavior has improved considerably [4, 14]. The most commonly used techniques include attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR), radiolabeling techniques, immunofluorescence enzyme-linked immunosorbent assay (ELISA), ellipsometry, circular dichroism (CD) spectroscopy, surface plasmon resonance (SPR), and amide HX with nuclear magnetic resonance (NMR). Atomic force microscopy (AFM) and scanning... 

Over the past 25 years, there has been increasing interest in expanding the use of HX-MS. In this chapter, we have reviewed its development and application for proteins in three different environments proteins adsorbed onto solid surfaces, in frozen solutions, and in lyophilized solids. The results have demonstrated the capability of HX-MS to detect and monitor protein conformation and dynamics with high resolution in these environments that differ from bulk aqueous solution. In addition, HX-MS has provided quantitative and site-specific information, addressing many of the limitations of more established techniques such as FTIR and CD spectroscopy. 

Due to steric considerations, n will generally be a lower number than in the free ligand case. For the adsorption of proteins on a solid surface, the total ligand concentration is usually represented as the actual surface area of the adsorbent. In studies utiliz-... 

Information about the secondary structures (a-helices, /5-sheets, random coil) can be useful for understanding conformation changes of proteins upon the immobilization process. More specifically, circular dichroism (CD) [70] and FT-IR spectroscopy [56, 58, 61, 71-73] have been applied to study the structural characteristics of various proteins adsorbed on mineral surfaces. Kondo and coworkers [70] have studied the modification in a-helix content of proteins adsorbed on ultrafine silica particles with CD and found a decrease upon immobilization. Circular dichroism is not usually used because this technique is applicable only for the study of enzymes immobilized on nano-sized mineral particles due to problems arising from light scattering effects. On the other hand, infrared spectroscopy does not suffer from light scattering perturbations and has thus been used for the study of the conformation of proteins when they are immobilized on solid supports [57, 58]. 

Adsorption at solid/liquid interfaces has some peculiarities as compared with fluid/fluid interfaces. The chemical nature of the solid surface and its properties (charge, hydrophobicity, etc.) determine the mode and strength of binding, as well as, in many cases, the conformational changes in adsorbed protein molecules. The solid surfaces can be easily modified and tuned up for specific types of interactions. Usually, in contrast to fluid surfaces, solid surfaces are not chemically or energetically uniform, and their heterogeneity may result in nonuniform adsorption of protein layers. Finally, adsorption from solutions is always a competitive process, and in the simplest case competition between a protein and a solvent takes place. 

The fact that a film of protein adsorbed onto 1 surface can still adhere to another surface is proved simply by exposing the film to a suspension of solid particles, e.g. of a metal oxide suspension. 

Several reports have pointed out that iodination of proteins may affect their adsorption to solid surfaces and their chromatographic behavior (40-43). Consequently, we have chosen to determine the quantum yield of unlabeled, adsorbed lysozyme via fluorescence lifetimes. We are currently in the process of modifying our fluorescence equipment to obtain such fluorescence lifetimes. 

Proteins generally adsorb onto solid surfaces from solution. This process is of importance in a number of applications. For instance, the composition, conformation, and orientation of adsorbed proteins are believed to influence cell/substrate interactions (1-3). Also, adsorption of serum proteins onto biomaterials is generally recognized as the initial event in the sequence that culminates in thrombus formation (4,5). Consequently, protein behavior at solid-liquid interfaces has been extensively studied (6-11). Many fundamental questions about the protein adsorption phenomenon, however, remain unanswered (12). 

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. 

The behavior of protein molecules at solid surfaces is very complex. The interaction between the surface and the protein is determined both by the nature of the protein, the surface and the medium outside the surface. The situation is further complicated by the fact that exchange reactions between protein molecules of the same or different kinds take place on the surface. Except for these exchange reactions most protein molecules appear to be irreversibly adsorbed. Although the details of the interaction between protein molecules and surfaces are not known it is assumed that general properties of the surface and the protein such as hydrophobicity, charge density, ion binding, hydration etc. are involved. For reviews, see e.g (21.35-37). 

The adsorption of proteins from aqueous solution to solid surfaces is the result of a combination of hydrophobic, steric, and electrostatic interactions between the protein, solid surface, and solution [ 1-3]. Numerous studies have been conducted to identify the driving forces governing protein adsorption and dynamics at liquid-solid interfaces and have been reviewed elsewhere [4—8], In the adsorbed state, protein stmcture is likely to be perturbed (Figure 15.1). The unfolded or partially unfolded protein can then adopt various flexible conformations depending on the natures of the solid surface the protein [1, 4, 9-13]. While this has been exploited for various applications [12], uncontrolled adsorption can cause protein degradation, compromised function, and even life-threatening immunogenic responses. The molecular mechanisms of protein adsorption have not been fully elucidated and remain a current area of research [ 10]. 

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