Desorption protein, kinetics

Figure 36 shows that efflux of PNU-78,517 from the apical membrane is facilitated by BSA. Its permeability coefficients increase with BS A concentration the magnitudes of the values (10 6—10 5 cm/min) and trends indicate that the desorption kinetics are membrane-controlled and that BSA acts as a drug acceptor at the membrane interface via protein binding. The initial mass fraction readily... 

Two investigations have combined TIR with FCS thus far. The first(126) adapted TIR/FCS to measure the absolute concentration of virions in solution. The other(127) measured the adsorption/desorption kinetics of immunoglobulin on a protein-coated surface on the millisecond time scale. 

Fig. 13. Most of the kinetic models which might be applicable to protein adsorption (see Refs.70 73)) k is rate constant, subscript a and d are adsorption and desorption respectively, 1 and 2 are adsorption states — usually native and denatured...

The adsorption and desorption kinetics of surfactants, such as food emulsifiers, can be measured by the stress relaxation method [4]. In this, a "clean" interface, devoid of surfactants, is first formed by rapidly expanding a new drop to the desired size and, then, this size is maintained and the capillary pressure is monitored. Figure 2 shows experimental relaxation data for a dodecane/ aq. Brij 58 surfactant solution interface, at a concentration below the CMC. An initial rapid relaxation process is followed by a slower relaxation prior to achieving the equilibrium IFT. Initially, the IFT is high, - close to the IFT between the pure solvents. Then, the tension decreases because surfactants diffuse to the interface and adsorb, eventually reaching the equilibrium value. The data provide key information about the diffusion and adsorption kinetics of the surfactants, such as emulsifiers or proteins. 

Polymer Adsorption. A review of the theory and measurement of polymer adsorption points out succinctly the distinquishing features of the behavior of macromolecules at solid - liquid interfaces (118). Polymer adsoiption and desorption kinetics are more complex than those of small molecules, mainly because of the lower diffusion rates of polymer chains in solution and the "rearrangement" of adsorbed chains on a solid surface, characterized by slowly formed, multi-point attachments. The latter point is one which is of special interest in protein adsoiption from aqueous solutions. In the case of proteins, initial adsoiption kinetics may be quite rapid. However, the slow rearrangement step may be much more important in terms of the function of the adsorbed layer in natural processes, such as thrombogenesis or biocorrosion / biofouling caused by cell adhesion. 

A more detailed kinetic investigation of the Au/Bipy/cytochrome c system was carried out using the rotating ring-disk technique (12). It was found that rate constants for adsorption and desorption of the protein were 3 x cm sec" and 50 sec", respectively. The limiting first-order rate constant within the protein-electrode complex was determined as 50 sec", a reasonable value as compared to that of long-range electron transfer between or within proteins. 

Wall effects, or the adherence of material to the bare silica capillary wall, has been a difficult problem since the early days of HPCE, particularly for large molecules such as proteins. Small molecules can have, at most, one point of attachment to the wall and the kinetics of ad-sorption/desorption are rapid. Large molecules can have multiple points of attachment resulting in slow kinetics. Several solutions have been proposed, including the use of (a) extreme-pH buffers, (b) high-concentration buffers, (c) amine modifiers, (d) dynamically coated capillaries, and (e) treated or functionalized capillaries. 

A new technique for measuring equilibrium adsorption/desorption kinetics and surface diffusion of fluorescent-labelled solute molecules at surfaces was developed by Thompson et al.74). The technique combines total internal reflection fluorescence with either fluorescence photobleaching recovery or fluorescence correlation spectroscopy with lasers. For example, fluorescent labelled protein was studied in regard to the surface chemistry of blood 75). 

The kinetics of adsorption-desorption is rarely slow in preparative chromatography, and most examples of a slow kinetics are foimd in bioaffinity chromatography, or in the separation of proteins. Thus, there are few cases in which a reaction-kinetic model is appropriate. This model is important, however, because there are many cases where it is convenient to model the finite rate of the mass transfer... 

The VERSE method was extended to describe the consequences of protein de-naturation on breakthrough curves in frontal analysis and on elution band profiles in nonlinear isocratic and gradient elution chromatography [45]. Its authors assumed that a unimolecular and irreversible reaction taking place in the adsorbed phase accormts properly for the denaturation and that the rate of adsorption/desorption is relatively small compared with the rates of the mass transfer kinetics and of the reaction. Thus, the assumption of local equilibrium is no longer valid. Consequently, the solid phase concentration must then be related to the adsorption and the desorption rates, via a kinetic equation. A second-order kinetics very similar to the one in Eq. 15.42 is used. 

FiCURE 4.3 Matrix-assisted laser desorption ionization source, (a) Gas phase ions are formed when the nitrogen laser produces photons at 337 nm that strike the solid matrix-analyte sample. The ions above the sample plate are accelerated to 25,000 eV of kinetic energy and fly through a stainless steel tube (not shown) for TOP m/z analysis, (b) Three commonly used matrix molecules for MALDI of peptides and proteins. 

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... 

Fatty acids are transported between organs either as unesterified fatty acids complexed to serum albumin or in the form of triacylglycerols associated with lipoproteins. Triacylglycerols are hydrolyzed outside cells by lipoprotein lipase to yield free fatty acids (Chapter 19). The mechanism by which fatty acids enter cells remains poorly understood despite a number of studies performed with isolated cells from various tissues [4]. Kinetic evidence has been obtained for both a saturable and a non-saturable uptake of fatty acids. The saturable uptake predominates at nanomolar concentrations of fatty acids and is thought to be mediated, or assisted, by proteins. In contrast, the non-saturable uptake that is effective at higher concentrations of fatty acids has been attributed to passive diffusion of fatty acids across the membrane. Several suspected fatty acid transport proteins have been identified [5]. Although their specific functions in fatty acid uptake remain to be elucidated, these proteins may assist in the desorption of fatty acids from albumin and/or function in uptake coupled to the esterification of fatty acids with CoA, in a process referred to as vectorial acylation. 

The size of a molecule is an important feature because proteins form multiple contacts with the surface (e.g., 77 contact points in the case of the albumin molecule and 703 contact points in the case of the fibrinogen molecule adsorbed on silica [10]). Multipoint binding usually causes adsorption irreversibility having a dynamic nature in the absence of irreversible denaturation. The rates of desorption are, as a rule, much lower than those of adsorption, and in many cases it is virtually impossible to attain the equilibrium state desorbing the adsorbed protein [11]. In other words, the formation of one or several bonds with the surface increases the probability of adsorption of neighboring sites of the same molecule. On the other hand, the desorption of a protein molecule requires the simultaneous rupture of a large number of bonds and, for kinetic reasons, equilibrium is not attained [12-14], This corresponds to a considerable difference between the activation energies for the adsorption and desorption processes [15,16],... 

However, adsorption/desorption kinetics are difficult to follow, particularly when simultaneous structural observations are being made. In this chapter we present information pertaining to two different techniques. The first method extends our previous measurements of surface circular dichro-ism, indicating structural changes as a function of contact time between a plane surface and protein solution. The second technique is based on a chromatographic method that allows us direct access to the kinetics of adsorption/desorption with a subsequent assessment of structural damage. 

Application of a quantity of solution, in this case protein solution, to a column filled with appropriate substrate, leads to an exchange between the solute and the surface, and movement with the solvent front such that the emerging solute profile contains complete information concerning the kinetics of adsorption/desorption. Apart from the interaction of solute with surface, concurrent three-dimensional diffusion often occurs, and perhaps occlusion, if the substrate is porous. Nevertheless, the option of deducing interfacial kinetics from the elution profile in a flowing system has a certain elegance and relevance to the performance of blood plasma proteins in vivo. 

Initial Fast Reversible Adsorption. The chromatographic profiles indicate that protein is adsorbed almost instantaneously on coated glass surfaces, and at short adsorption times, the contact points of the protein with the surface are limited and the kinetic energy of colliding solvent molecules is sufficient to cause desorption, that is, the desorption rate is a function of the surface adsorbed concentration. 

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