Protein desorption

Protein adsorption has been studied with a variety of techniques such as ellipsome-try [107,108], ESCA [109], surface forces measurements [102], total internal reflection fluorescence (TIRE) [103,110], electron microscopy [111], and electrokinetic measurement of latex particles [112,113] and capillaries [114], The TIRE technique has recently been adapted to observe surface diffusion [106] and orientation [IIS] in adsorbed layers. These experiments point toward the significant influence of the protein-surface interaction on the adsorption characteristics [105,108,110]. A very important interaction is due to the hydrophobic interaction between parts of the protein and polymeric surfaces [18], although often electrostatic interactions are also influential [ 116]. Protein desorption can be affected by altering the pH [117] or by the introduction of a complexing agent [118]. 

Fig. 21 A Schematics of albumin-bound poly(N-isopropylacrylamide) (PNIPAM)-grafted surface via albuminated DC iniferter. B Hypothetical action of temperature-dependent switching of protein desorption by squeezing out and mechanical motion...

It is commonly observed that protein desorption can be very slow or even non-existant, but protein exchange can be rapid. This anomaly has been pointed out many times 93,94). Fortunately Jennissen s studies 95 97) and the arguments and work discussed in this chapter lead to a reasonable explanation. 

The now classic studies by Norde and Lyklema and their detailed thermodynamic analysis (Sect. 4.2) have established that the interaction between a protein and a surface increases with increasing hydrophobicity of the surface and increases with increasing hydrophobicity of the protein. Desorption from hydrophobic surface usually does not occur whereas proteins can often be removed from hydrophilic surfaces by exposure to extreme pH, high ionic strength, or by extensive rinsing 61). ... 

The time scale of fat crystallization is much shorter for topping powders than for ice cream mix as presented in Figure 2. This is due to the much higher emulsifier content in topping powder. The induction of fat crystallization in whippable emulsion systems is due to interfacial protein desorption from the fat globules of the emulsion mediated by the emulsifiers. This phenomenon is described in section 3.1. 

Figure 10 Protein desorption from fulsifier (saturated mono-diglyceride), cream mix with (+E) and without (-E) e,n...

Milk protein desorption at low temperature is due to stronger hydrophilic and weaker hydrophobic forces, and is caused mainly by dissociation of beta-casein39. 

The protein ioad increases in the presence of hydrocolloids. In the presence of additional emulsifiers a very effective desorption of protein takes place during whipping and freezing in the ice cream machine. Effective protein desorption is facilitated by the increased viscosity of the mix due to increased surface shear forces, which makes the ice cream continuous freezer work better. 

Figure 12 Transmission electron microscopy study of protein desorption in icecream mix containing emulsifiers and hydrocolloids, (a) Immediately after homogenization the fat globules (0 are stabilized by adsorbed partially dissociated casein micelles (arrows), (b) During ageing the mix at 5°C, the previously adsorbed protein film is released in the form of coherent protein layers (arrows) into the water phase (w). (c) After mechanical treatment in the ice cream freezer, desorbed protein layers are seen more often in the water phase without association to fat giobules (arrows). From reference 48, courtesy of Dr. W.Buchheim, Kiel, Germany.

During ageing of the mix, interfacial milk protein hydration also increases simultaneously with protein desorption from the fat globules. The water content of the isolated cream layers after centrifugation of ice cream mix can be analyzed by Karl Fischer titration. From such analyses, interfacial protein hydration can be calculated (Figure 13). 

The surface is divided into areas of irreversible binding sites (POPS-enriched) and reversible binding sites (POPC-enriched). Protein adsorption takes place on both areas, however, at different rates. Protein desorption is allowed only from the reversible binding sites. Mass transport of the proteins to the surface is considered by using a mean field ansatz of a stagnation point flow in accordance with the experimental setup. The kinetics of reversible... 

Figure 12. Kinetics of protein desorption from germanium crystal for the mixture shown in Figure 10. Desorption is with isotonic saline flowing at 15 mL/min. Key , Amide 11 band (1550 cm-1) and +, 1400-cmband ( x3.0).

Under HPLAC conditions, similar elution curves for AT III and Heparin were observed on PAOM and PSSO stationary phases. Antithrombin III was immediately eluted by 0.1 M sodium chloride solution and no detectable protein desorption was observed at 2 M ionic strength (Figure 3A). Thus the inhibitor was not retained by either PAOM or PSSO, Compared with the findings mentioned above, thi s can be explained in terms of kinetic effects. By decreasing the elution flow-rate and simultaneously overloading the columms, retention of a small amount of AT III ( 1%) was observed at the same ionic strength, i.e. 0.1 M NaCl. 

A video camera with a silicon intensifier target (SIT) tube was used to prepare video tapes which were analyzed one frame at a time (the time between frames is 33 ms). The location and arrival and detachment time of each cell were recorded. Notations were made of the movements of cells to new positions on the surface and a record was made of unoccupied grid spaces which had previously contained cells. The surfaces examined were albumin-, fibronectin- and collagen-precoated glass. Exposure times to flow were kept short, 1-4 min, in order to minimize the effects of protein desorption and exchange. 

SO that in the second batch productivity was severely reduced and conversion yield was significantly lower even at prolonged operation. SDS-PAGE electrophoresis revealed that most of the desorbed protein had a molecular weight of 31,000 Da, which corresponds to that of Alcaligenes faecalis lipase. Most of the activity lost from batch to batch corresponded to protein desorption from the matrix, enzyme inactivation being quite low. Therefore, it made very little sense to use QLG instead of QL, but the free enzyme was not recoverable from the reaction medium and cost estimates indicated that the enzyme should be used at least five times to make the process economically attractive. Therefore, the next goal was to construct an immobilized lipase biocatalyst from soluble QL. The hydrophobic nature of the active site and the requirement of a hydrophobic interface for lipase action made reasonable to use hydrophobic supports however to test the validity of this hypothesis several immobilization systems were tested. The results obtained are summarized in... 

Figure 1.12 Schematics illustrating interfacial interactions between bone tissne or cell and implanted biomaterials viewed from the host tissne perspective (a) protein adsorption from blood and tissue fluids, (b) protein desorption, (c) substrate surface changes and material release, (d) inflammatory and connective tissue cells approach the implant, (e) possible targeted release of matrix proteins and selected adsorption of proteins, (f) formation of lamina limitans and adhesion of osteogenic cells, (g) bone deposition on both the exposed bone and implant surfaces, and (h) remodeling of newly formed bone.

The above measurements on protein monolayers confirm that rates of protein desorption are quite low until high... 

Protein adsorption Washing Protein desorption Washing - (PBS + Protein) (PBS) (SDS + NaOH + PBS) (PBS) ... 

UHMWPE samples were incubated in BSF, and investigated for adsorbed proteins in the range of plasma proteins by means of protein desorption and gel electrophoresis. Bands of adsorbed proteins corresponding to albumin, immunoglobulin heavy chain... 

Welin-Klinstroem et al used a null ellipsometer equipped with an automatic sample scanning device for studies of adsorption and desorption of fibrinogen and IgG at the liquid/solid interface on surface wettability gradients on silicon wafers. To follow the processes along the wettability gradient, off-null ellipsometry was used. The kinetics of adsorption and nonionic-surfactant-induced desorption varied considerably between fibrinogen and IgG. In the hydrophilic region, veiy little protein desorption was seen when a nonionic surfactant was used. 

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