Surface concentration , protein

Protein-Pak packings are designed for the size exclusion chromatography of proteins and related compounds. They are based on silica, which is deactivated with glycidylpropylsilane. The diol function prevents the interaction of the target analytes with the silica surface. However, because coverage of the silica surface is always incomplete, residual acidic silanols can interact with the analytes. For this reason, most applications are carried out with a salt concentration above 0.2 mol/liter, which eliminates the interaction of analytes with surface silanols. Protein-Pak packings are stable from pH 2 to pH 8. 

The surface concentration of adsorbed albumin was then determined by comparing the radioactivity of an aliquot of solution of known protein concentration as described previously [3.4]. For this purpose, a Beckman Instrument (Palo Alto, CA) y-counter was used. 

In vitro, chlorhexidine can adversely affect gingival fibroblast attachment to root surfaces. Furthermore, protein production in human gingival fibroblasts is reduced at chlorhexidine concentrations that would not affect cell proliferation. Such findings corroborate earlier studies showing delayed wound healing in standardized mucosal wounds after rinsing with 0.5% chlorhexidine solution. 

Stenberg, E., Persson, B., Roos, H., and Urban-iczky, C. (1991) Quantitative determination of surface concentration of protein with surface plasmon resonance using radiolabeled proteins. J. Colloid Interface Sci., 143, 513-526. 

FIGURE 2. Continuous phase liquid holdup profiles (CPLHP) for different centrifugal accelerations for droplet size R = 5jc10 /w, surface concentration r=5jcl0 il g//n, ionic strength m =0.1M,thickness of adsorbed protein layer L, = 12xl(T /n and zeta potential = 12mV. 

FIGURE 7. Continuous phase liquid holdup profiles (CPLHP) for different zeta potentials for droplet size / =50bcl0" /n, centrifugal acceleration Qc = 10 m lsec, ionic strength m=0.1Af, thickness of adsorbed protein layer Lg = 12xl(T m and surface concentration r = 5jc IQT kglnr. 

Fig. 3. Adsorption of FGN from various dilutions of plasma onto (a) PE, (b) SR, (c) FEP, (d) PTMO-PU and (e) PEO-PU. r represents the surface concentration of protein adsorbed (Reproduced from J Biomater Sri Polymer Edn [Ref 21] through the courtesy of VSP-BV)...

The adsorption experiments were carried out by quantifying each of proteins adsorbed on the material from mono-component protein solutions, from four-component protein solutions, and from plasma and diluted plasma. Adsorption profiles of protein were largely different, depending on the aforementioned experimental conditions. For instance, the behavior of any particular protein from diluted plasma varied in response to the extent of plasma dilution. Cooper s results are illustrated in Fig. 3, on fibrinogen adsorption onto five polymer surfaces. It is seen that the adsorption profiles are different one another, being influenced by the different nature of the polymer surfaces. The surface concentrations of adsorbed protein are mostly time-dependent, and maxima in the adsorption profiles were observed. This is interpreted in terms of replacement of adsorbed fibrinogen molecules by other proteins later in time (Vroman effect). Corresponding profiles were also presented for FN and VN. 

Thermodynamically unfavourable interactions between two biopolymers may produce a significant increase in the surface shear viscosity (rf) of the adsorbed protein layer. This change in surface rheological behaviour is a consequence of the greater surface concentration of adsorbed protein. For instance, with p-casein + pectin at pH = 5.5 and ionic strength = 0.01 M (Ay = 2.6 x 10 m3 mol kg-2), the surface shear viscosity at the oil-water interface was found to increase by 20-30%, i.e., rp = 750 75 and 590 60 mN s m-1 in the presence and absence of polysaccharide. These values of rp refer to data taken some 24 hours following initial protein layer formation (Dickinson et al., 1998 Semenova et al., 1999a). 

Figure 8.6 Comparison of the influence of non-ionic Ci2E6 (hexaoxyethyl-ene ft-dodecyl ether) or anionic SDS (sodium dodecyl sulfate) on adsorbed amount of p-lactoglobulin at the air-water interface (0.1 wt% protein, pH = 6, ionic strength = 0.02 M, 25 °C) as determined by neutron reflectivity measurements. Protein surface concentration is plotted against the aqueous phase surfactant concentration ( ) Ci2E6 ( ) SDS. Reproduced from Dickinson (2001) with permission.

Fig. 12. Hypothetical 3-D plot of protein adsorption isotherm (D plotted against [P]B) as a function of surface ligand concentration, [A]s. Note that the system is reversible only up to a critical [A]s and then behaves irreversibly for higher ligand surface concentrations. The right arrows (— ) denote adsorption the left-facing ones ( -), desorption...

Morrissey 53) used transmission infrared spectroscopy to study protein adsorption onto silica particles in a heavy water (DzO) buffer. By observing the shift in the amide I absorption band, he could deduce the fraction of protein carbonyl groups involved in bonding to the silica surface. He found that bovine IgG had a bound fraction of 0.20 at low bulk solution concentrations, but only about 0.02 at high solution concentrations. However, neither prothrombin nor bovine serum albumin exhibited a change in bound fraction with concentration. Parallel experiments with flat silica plates using ellipsometry showed that the IgG-adsorbed layers had an optical thickness of 140 A and a surface concentration of 1.7 mg/m2 at low bulk solution concentration — in concentrated solutions the surface amount was 3.4 mg/m2 with a thickness of 320 A (Fig. 17). 

But what happens if we have two or more proteins in solution Clearly there will be a competition and the resultant surface concentration of the two proteins at some time, t, will be a complex function of protein, surface, and solvent properties. 

Vroman has shown by antibody methods that plasma interactions with solid surfaces result in a hierarchial adsorption process 98). The high concentration proteins dominate the surface at short times due to the higher collision rates. As time passes... 

In TIRF protein adsorption experiments, it is desirable to correlate the intensity of excited fluorescence with excess protein concentration at the interface. Such an adsorbed layer is often in equilibrium with bulk-nonadsorbed protein molecules which are also situated inside the evanescent volume and thus contributing to the overall fluorescence. Various calibration schemes were proposed, using external nonadsorbing standards40,154 , internal standard in a form of protein solution together with a type of evanescent energy distribution calculation 154), and independent calibration of protein surface excess 155). Once the collected fluorescence intensity is correlated with the amount of adsorbed protein, TIRF can be applied in the study of various interactions between surface and protein. 

Changes in surface pressure with time or concentration can be used to measure various fundamental properties. Changes in surface pressure versus protein concentration curves can be used to determine the excess surface concentration (critical micelle concentration), defined as the amount of protein at the surface divided by the surface area. Indicates minimum amount of protein needed to form an emulsion. 

The FRAP apparatus can also be used in a semi-quantitative manner to measure the surface concentration and subsequent competitive displacement of adsorbed labelled species, such as the fluorescent-labelled protein in the adsorbed layer of a/w or o/w thin films [10]. This can be achieved by focusing the low power 488 nm beam on the film and detection of the emitted fluorescence using the FRAP photon counting photomultiplier. The detected fluorescence signal is proportional to the amount of adsorbed protein at the interfaces of the thin film provided that the incident laser intensity is kept constant. Calculations have proved that the contributions from non-adsorbed protein molecules in the interlamellar region of the film are negligible [12],... 

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