Monolayer of proteins

An ordered antibody array has also been assembled on the solid surface by a combination of Langmuir Blodgett (LB) film method and self-assembling method. An ordered monolayer of protein A is deposited on the solid surface by LB method, which is followed by self-assembling of antibody. Individual antigen molecules which are complexed with the antibody array have been quantitated selectively by atomic force microscopy (AFM). 

To prepare an antibody protein array, a monolayer of protein A, which was compressed at a surface pressure of 11 mN m l was transferred to a compartment containing anti-ferritin antibody in 10 mM pH 7.0 phosphate buffer. The antibody molecules were self assembled onto the protein A layer. The protein A/antibody molecular membrane was transfered to a compartment containing ultrapure water for rinsing, and was then transfered onto the surface of an HOPG plate by the horizontal method. AFM measurements were made in a pH 7.0 of 10 mM phosphate buffer solution. 

The nse of self-assembled monolayers of proteins by direct chemical attachment of the peptide to a modified snrface obviates some of the protein orientation difficnlties observed in the construction of LB mono-layers. An initial report of the construction of peptide SAMs on silanized qnartz was pnblished in early 1998 (185). Pilloud et al. have utilized the covalent ligation of protein cysteine thiols to thiol-terminated silanized... 

Fig. 3. A spread monolayer of protein decreases fe for the absorption of CO (from the gas phase at 1 atm. partial pressure) into water (28). The surface is cleaned thoroughly before the experiment, and the contra-rotating stirrers in the liquid are running at 437 r.p.m. The surface is nonrotating in aU the experiments described here, and when fct is reduced to 1.1 X lO"" cm. sec.", all random surface movements are also eliminated.

Fig. 9. Reduction in kt by spread monolayer of protein, due to prevention of transfer of momentum from ethylacetate, and to reduction in turbulence in the aqueous phase near the interface. Experimental data used here are taken from reference (60).

General characteristics of the support to be considered are discussed in chapter 6 and those of relevance for the use of enzymes in non-conventional media are listed in Table 9.2. In addition, the specific surface area of the support is of special importance for the applications in non-conventional media. Inactivation can occur if the surface area is too large in relation to the amount of enzyme (Figure 9.3). In order to avoid inactivation at least a monolayer of enzyme molecules should be formed on the accessible surface (Wehtje, Adlercreutz and Mattiasson, 1993). For enzymes of high purity and high activity, the amount of enzyme needed is sometimes quite small. In such cases the enzyme can be mixed with a protecting protein before immobilisation to achieve at least a monolayer of protein and thereby avoid... 

It has long been established that all cell membranes in the body are composed of a fundamental structure called plasma membrane. This boundary surrounds single cells such as epithelial cells. More complex membranes such as intestinal epithelium and skin, are composed of multiples of this fundamental structure, which has been visualized as a bimolecular layer of lipid molecules with a monolayer of protein adsorbed into each surface. Cell membranes are further interspersed with small pores that can be protein line channels through the lipid layer or, simply, spaces between the lipid molecules. In membranes composed of many cells, the spaces between the cells constimte another kind of membrane pores (2). 

J.T. Davies, Some factors influencing the orientation of e-amino groups in monolayers of proteins and amino acid polymers, Biochem. J. 56 (1954) 509-513. 

Proteins are found either not to desorb or to desorb only with great difficulty from quiescent interfaces. Langmuir and Schaefer (1939) calculated, on the basis of the Gibbs adsorption equation, that compression of a monolayer of protein of molecular weight 35,000 by 15 mN m-1 should increase its solubility by a factor of 1095. This results from the large area occupied by the molecule at the interface and the resultant large pressure increment of solubility. The failure of protein monolayers to desorb readily on compression was thus taken as an indication of irreversibility. 

Previous chapters have discussed comprehensively protein and DNA-based function and bioelectrochemical systems. Single- and multi-functional monolayers of proteins, DNA-based molecules, and enzymes, and of monolayers of amino acids and DNA-bases as their building blocks testify towards bioelectrochemical control at the nanoscale and sometimes singlemolecule levels. In these respects interfacial bioelectrochemistiy is undergoing a process similar to what has been the case in physical... 

Reliable circular dichroism spectra cannot be obtained from crystalline, oriented polypeptide material that is birefringent or scatters light. However, monolayers of protein do not appear to provide anomalous spectra (4,15), probably because of the relative uniformity of the film and the minimal refractive index increment at the protein/water interface. Nevertheless, previous results from this and other laboratories (3,16,17) have shown that albumin and fibrinogen molecules tend to lie flat, that is, with their long axes parallel to the surface, whereas globulins lie perpendicular to the surface. 

If the surface is first saturated with a monolayer of protein exposed to steady-state concentration cQ, and then is exposed to a second treatment at concentration 2c0, a second front emerges. The second profile represents the situation where no net protein is adsorbed and thus, in principle, is representative of the diffusion-shifted flow pattern of the nonadsorbed protein. Figure 7 shows both the initial (cQ) and second (2c0) fronts and the subtraction curve which is very close to the ideal step function. If the data are interpreted as solution-borne molecules passing over an inert surface, then (a) adsorption must be essentially instantaneous and (b) the surface must become covered by exhausting the concentration of solute at the front as it moves down the column. The slope of the difference profile should represent the rate of uptake of material on the column, and that is essentially infinite on the time scale of the experiment. The point of inflection of the subtracted front indicates the slowing of the sorption process due to filling of sites on the surface. 

Interfaces between two separate phases, such as air/water, oil/water, and solid/ water, are potential adsorption sites of biopharmaceuticals. Adsorption often involves simple diffusion of surface-active solute molecules in the bulk to the interface, and hence the rate of adsorption is generally dependent on the solute concentration. At saturation, a close packed monolayer of protein molecules corresponding to 0.1 to 0.5pg/cm is normally formed at the interface [122], and this adsorption behavior is of particular concern for high-potency therapeutic proteins. However, certain proteins do not conform to such saturation-limited adsorption behavior and tend to show increased adsorption with increasing protein concentration, attaining a local protein concentration at the interface 1000 times higher than the initial concentration in the bulk solution [123]. 

Two forms of the casein proteins are used to stabilize oil/Water emulsions. Most siirply, a mixture of the proteins (as in sodium caseinate), or the individual proteins themselves, allows the formation thin layers, if not monolayers, of protein at fat/Water interfaces (1). By contrast, in preparations such as homogenized milk, the entities which bind to and stabilize the fat/Water interface are much larger and more complex, and may be considered as intact or semi-intact casein micelles (2). These particles are highly aggregated complexes of the four caseins ... 

The high sensitivity of the ER method benefits bioelectrochemists in the detection of the redox reaction of the electron transfer proteins. Even for an adsorption monolayer of proteins, the superficial density of the electroactive center is much smaller than that of small molecules, especially when the molecular weight of the protein is several kilo-Daltons. The redox reaction of adsorbed protein buried in the double-layer charging current in the voltammogram can be detected by the ER method. Ikeda and coworkers succeeded in the clear observation of the ER spectrum and ER voltammogram of a heme c in an adsorbed protein (alcohol dehydrogenase) of ca. 140 kD containing hemes and PQQ, while direct redox reaction could not be detected by cyclic voltammetry [90]. 

A monolayer of protein spreads at the air-water interface. The amount of protein spread is equivalent to 0.80 mg m" and gives a surface tension lowering of 0.035 mN mWhat is the molecular weight of the protein ... 

Similarly, electrode surfaces can be modified by monolayers of proteins in enzymatic biosensors. Amine terminal groups from lysine residues of the protein are typically used for the formation of very stable imine bonds to such a scope, electrode surfaces are modified with monolayers containing -COOH tail groups. Different functional groups present in the polypeptide chain can also be exploited, as in the example reported in Fig. 5.20. 

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