Protein mixture adsorption

In many practical applications, e.g., in the food industry or in the creation of biocompatible implants, it is very important to know the adsorption behavior of multicomponent protein mixtures. Adsorption from mixtures usually proceeds as a competitive process, and all the above-mentioned factors influence relative preference in adsorption of various proteins. In general, it was found that certain proteins may be enriched at the interface, relative to the others [8,59-61 J. Mixtures containing two or three proteins have been used as simple models of complex biological fluids, like plasma [59,60,133] or milk... 

The orientation and Frumkin adsorption models have recently been combined to describe adsorption layer for surfactant-protein mixtures in [37] and [38]. 

In case protein mixtures are needed to be fractionated, samples are subjected to subcellular fractionation, electrophoresis in the liquid phase, adsorption chromatography, and selective precipitation or sequential extraction based on solubility prior to fractionation. 

When a biopolymer mixture is either close to phase separation or lies in the composition space of liquid-liquid coexistence (see Figure 7.6a), the effect of thermodynamically unfavourable interactions is to induce biopolymer multilayer formation at the oil-water interface, as observed for the case of legumin + dextran (Dickinson and Semenova, 1992 Tsapkina et al, 1992). Figure 7.6b shows that there are three concentration regions describing the protein adsorption onto the emulsion droplets. The first one (Cprotein< 0.6 wt%) corresponds to incomplete saturation of the protein adsorption layer. The second concentration region (0.6 wt% < 6 proiem < 6 wt%) represents protein monolayer adsorption (T 2 mg m 2). And the third region (Cprotein > 6 wt%) relates to formation of adsorbed protein multilayers on the emulsion droplets. 

These kinetics studies required development of reproducible criteria of subtraction of foe H-O-H bending band of water, which completely overlaps foe Amide I (1650 cm 1) and Amide II (1550 cm"1) bands (98). In addition, correction of foe kinetic spectra of adsorbed protein layers for foe presence of "bulk" unadsorbed protein was described (99). Examination of kinetic spectra from an experiment involving a mixture of fibrinogen and albumin showed that a stable protein layer was formed on foe IRE surface, based on foe intensity of the Amide II band. Subsequent replacement of adsorbed albumin by fibrinogen followed, as monitored by foe intensity ratio of bands near 1300 cm"1 (albumin) and 1250 cm"1 (fibrinogen) (93). In addition to foe total amount of protein present at an interface, foe possible perturbation of foe secondary structure of foe protein upon adsorption is of interest. Deconvolution of foe broad Amide I,II, and m bands can provide information about foe relative amounts of a helices and f) sheet contents of aqueous protein solutions. Perturbation of foe secondary structures of several well characterized proteins were correlated with foe changes in foe deconvoluted spectra. Combining information from foe Amide I and m (1250 cm"1) bands is necessary for evaluation of protein secondary structure in solution (100). 

The performance of commerically available coated and uncoated capillaries utilizing electroosmotic mobilization has been assessed for model protein mixtures [71]. Although successful separations of basic and neutral proteins were achieved on uncoated capillaries, coated capillaries were required for the separation of acidic proteins in order to provide a constant EOF throughout the capillary. Yeo and Regnier [72] have reported novel coatings for CIEF, which are produced by dynamically modifying octade-cylsilane-derivatized capillaries by adsorption of methylcellulose or surfac-... 

Exposing the saliva protein mixture to the brushed surfaces reveals two remarkable features (a) the brush, even at a-1 = 12 nm2, strongly suppresses adsorption, whereas (2) the shift in the zeta potential is still relatively large. These observations suggest that the small amounts of saliva protein deposited at the brushed surface are positively charged and that small proteins (i.e. of the size of that of LSZ) are hardly involved. 

Liquid chromatography is very often the method of choice for adsorption, separation, and purification of antibodies. Chromatographic separations are based on the differential adsorption and migration speed of components of a protein mixture through a column filled with small particles called chromatographic sorbent or solid phase.71 At the level of adsorption, solid phases are... 

Kim, et al., ( ) have utilized radioiodinated (I proteins to measure adsorption of individual proteins and protein mixtures on LTI carbon surfaces. Their results indicate a very rapid adsorption of albumin onto the LTI carbon surface, consistent with Kim s model of blood interactions via a platelet-adhesion mechanism (.8). Microcalorimetric and electrophoretic mobility studies of protein adsorption on LTI carbon surfaces have been done by Chiu, et al., ( 5). The extension of the adsorbed protein layers have been directly measured by Fenstermaker, et al., ( ) and Stromberg et al., 7) at NBS using ellipsometric methods. 

Our studies (19) indicated that proteins were readily adsorbed from aqueous solution onto hydrophobic polymer surfaces with Langmuir type adsorption and that the rate of adsorption toward a plateau surface concentration depends on the polymer nature. In the study of competitive adsorption from a protein mixture solution (20), fibrinogen and y-globulin adsorb onto FEP very rapidly compared with PEUU and SR. Therefore, the FEP surface in contact with blood has more acceptor sites for platelet adhesion than does the PEUU or SR surface. 

Fang, F., Szleifer, I. Competitive adsorption in model charged protein mixtures Equilibrium isotherms and kinetic behavior. J. Chem. Phys. 2003,119,1053-65. 

Adsorption kinetics and isotherms. The rate of protein adsorption onto solids is usually much slower than that predicted from the diffusion theory [85-87]. For various protein-adsorbent systems, the period of time required to obtain maximum adsorption ranges, as a rule, from several tens of minutes [10,12,14,88] to several hours [11,12,14,63,65,66,79,81,84,89,90]. More rarely, the adsorption terminates after several minutes [67,91] or continues for 24 h and longer [92,93], It cannot be excluded, however, that the initial adsorption rates should be transport limited, as has been shown by Norde et al. [94] for adsorption of lysozyme, RNase, and myoglobin on glass. The importance of diffusion is also obvious at the first step of adsorption from protein mixtures [95]. In this case the interface accommodates initially the protein molecules with the largest diffusion coefficients, and afterwards these molecules may be displaced by other molecules with higher affinity to the surface. 

The primary process of lEC involves adsorption and desorption of ionic species from ionogenic groups located in the packing. lEC was the first of the traditional column techniques to be exploited for modern LC, due principally to the need for the fast routine analysis of amino acids and protein mixtures. 

A concerted effort is presently needed to study the mechanisms influencing adsorption behavior in protein mixtures. Does adsorption from mixtures behave as the sum of independent adsorption events determined by specific affinity constants charactertistic of each species Can such a simple explanation suffice to explain the peak in adsorption isotherms seen for fibrinogen from plasma ("the Vroman effect") and also from binary mixtures (56,57) If the differences in adsorption behavior of mixtures compared to single adsorbates are better understood than at present, a greater degree of control of the adsorption process to achieve a desired end (e.g., selection of a desired protein) may be possible. For example, if the Vroman effect is a general feature of all protein mixtures, then clearly there is an optimum concentration for adsorption to achieve the greatest selectivity. 

In all these applications of biomaterials, adsorption occurs from relatively complex solutions containing many different proteins. For this reason, much of the effort in my laboratories has focused on the behavior of protein adsorption as it occurs from protein mixtures. In this paper, a summary of these studies is provided with the... 

The results of studies in the author s laboratory summarized in Table 1 constitute a relatively large amount of information about how adsorption occurs from the protein mixtures typically encountered by biomaterials. Overall, the most important observations made are that variations in surface chemistry, time of adsorption, and protein type are major factors in determining the composition of the adsorbed layer. The adsorbed layer formed from mixtures thus contains a rather complex and changeable combination of proteins. 

HORBETT Adsorption to Biomaterials from Protein Mixtures... 

Competitive Adsorption of Fibrinogen and Albumin. When a foreign surface contacts blood it encounters a complex mixture of plasma proteins. The adsorption rates and surface coverages determined for proteins individually will undoubtedly differ when several proteins challenge the surface simultaneously. Therefore, a study of the adsorption characteristics of multi-component protein solutions has been conducted using TIRF. When extrinsic labeling is employed, TIRF is particularly suitable for studying competitive adsorption. 

This paper summarizes a series of experiments directed to the development of Fourier transform infrared spectroscopic (FT-IR) techniques for monitoring the events that occur when blood contacts the surface of a biomedical device. Special emphasis is placed on the methodology used for quantification and compositional analysis of protein adsorption from complex protein mixtures in aqueous solutions. 

The study of protein adsorption by FT-IR has not been a single project instead it has involved a series of research steps that are generally outlined in flowsheet format in Figure 1. This perspective demonstrates the need to solve certain technical problems before more advanced, and more technically interesting, experiments can be performed and/or interpreted. For example, just as it is necessary to develop appropriate flow cells before kinetic data can be acquired, so also must the approaches to the analysis of protein mixtures from their infrared spectra be learned before software can be optimized for multicomponent analysis. 

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