Proteinic surface immobilization

Capture array Non-protein molecules that interact with proteins are immobilized on the surface. These may be broad capture agents based on surface chemistries such as the Ciphergen Protein Chip, or may be highly specific such as molecular imprinted polymers or oligonucleotide aptamers... 

Protein is immobilized by combining with the surface of the electrode through a covalent bond, which is called covalent bonding of protein. The process requires low temperature (0°C), low ion intensity, and physiological pH conditions. Although covalent bonding onto the surface of an electrode is more difficult than adsorption, it can provide a more stable immobilized protein. 

As already discussed, a covalent immobilization can be performed via different chemical moieties on the protein surface. Because of that, protein molecules are immobilized in random orientation with at least one, but often several, covalent bonds to the matrix. As a result, the active site might be oriented toward the matrix surface and its accessibility to the substrate molecule hence significantly reduced. This results in a decrease of biological activity and consequently in lower binding capacity or decrease of reaction rate in the case of enzymes. 

As a technique for selective surface illumination at liquid/solid interfaces, TIRF was first introduced by Hirschfeld(1) in 1965. Other important early applications were pioneered by Harrick and Loeb(2) in 1973 for detecting fluorescence from a surface coated with dansyl-labeled bovine serum allbumin, by Kronick and Little(3) in 1975 for measuring the equilibrium constant between soluble fluorescent-labeled antibodies and surface-immobilized antigens, and by Watkins and Robertson(4) in 1977 for measuring kinetics of protein adsorption following a concentration jump. Previous rcvicws(5 7) contain additional references to some important early work. Section 7.5 presents a literature review of recent work. 

Conversely, controlled immobilization of enzymes at surfaces to enable high-rate direct electron transfer would eliminate the need for the mediator component and possibly lead to enhanced stability. Novel surface chemistries are required that allow protein immobilization with controlled orientation, such that a majority of active centers are within electrontunneling distance of the surface. Additionally, spreading of enzymes on the surfaces must be minimized to prevent deactivation due to irreversible changes in secondary structure. Finally, structures of controlled nanoporosity must be developed to achieve such surface immobilization at high volumetric enzyme loadings. 

Sapsford, K. E., I. L. Medintz, J. P. Golden, J. R. Deschamps, H. T. Uyeda, and H. Mattoussi. Surface-immobilized self-assembled protein-based quantum dot nanoassemblies. Langmuir 20, 7720-7728 (2004). 

Surface immobilization of the capture molecules follows standard procedures that are commonly practiced in many biosensor applications and some are discussed in the previous section. Layers of carboxymethyl dextran. Protein A or Protein G, streptavidin-coated surface, or EDC [N-ethyl-N-(diethylaminopropyl) carbidimide]/NHS (N-hydroxysuccmimide)-based amine coupling through amide bond are used for protein (antibody, receptor, etc.) cross-linking. 

Protein Chips Proteins, too, can be immobilized on a solid surface and used to help define the presence or absence of other proteins in a sample. For example, researchers prepare an array of antibodies to particular proteins by immobilizing them as individual spots on a solid surface. A sample of proteins is added, and if the protein that binds any of the antibodies is present in the sample, it can be detected by a solid-state form of the ELISA assay (see Fig. 5-28). Many other types and applications of protein chips are being developed. 

Hence, concerning the interaction with plasma proteins, covalently immobilized heparin performs identically to heparin in solution, and this results in the enrichment of the HCP surface with the most thrombogenic plasma components fibrinogen and thrombin. 

Scheme 6 Combined Solid-Phase and Solution Strategy After Chain Assembly, the Linear Template Sequence Is Cleaved from the Resin, Cyclized in Solution and Re-immobilized on a Resin (Steps a-d) Orthogonal Protection Allows for the Construction of Protein Surface Mimetics, TASP Molecules, or Receptor Mimetics 52 53 ...

Fig. 4. Schematic of a single-step array fabrication process for in vivo biotinylated proteins. Step a A cmde lysate containing the desired biotinylated recombinant protein is printed onto a streptavidin-coated surface coderivatized with a polymer that resists nonspecific protein absorption. Step b Unbound proteins are washed away to leave the purified recombinant protein, specifically immobilized and oriented on the array surface via the biotin moiety on the BCCP tag.

In the present paper, we review recent work in our group that aimed at understanding the relationship between quantities that can be measured via SM FRET and the underlying conformational structure and dynamics of freely diffusing and surface-immobilized protein molecules. [54,55,65,66] Our approach differs from that employed by other researchers in order to interpret SM FRET measurements [49-53,67] in the following respects ... 

To summarize, the model used in this paper captures many important features of protein structure and dynamics and is indeed seen to reproduce many of the general trends observed in SM-FRET experiments. At the same time, we have also observed several intriguing discrepancies between the model predictions and the experimental results. One possibility is that these discrepancies originate from shortcomings of the model. For example, the SM-FRET measurements reported in Refs. [30, 33] were performed on a coiled-coil that was immobilized on a positively charged amino-silanized glass surface and involved charged dye molecules. This implies that the protein-surface and donor-acceptor interactions may be dominated by electrostatic forces. Our... 

Immobilization has also been shown to stabilize against solvent dena-turation of enzymes. However, here we presented suggestive data on the mechanisms of this stabilization. Only the CPO immobilized in 200-A sol-gel showed any solvent or temperature stabilization. CPO bound to matrices with pores smaller than the protein showed little or no stabilization effect owing to surface immobilization alone. This supports the concept that steric hindrance to protein unfolding within a pore is part of the stabilization mechanism. An unresolved question for the future applications of this research is to increase the overall enzyme activity or loading. 

Other than proteins, the immobilized enzyme was also used to digest DNA. For instance, the restriction endonuclease enzyme HaeJR was immobilized on an amine-modified PMMA surface to digest DNA [1063]. 

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