Surface adsorbed albumin

Figure 4c shows that the amount of adsorbed proteins is rapidly saturated within several minutes of exposing serum-containing medium to a surface. Albumin, the most abundant serum protein, was expected to preferentially adsorb onto the surfaces during early time points. Then, adsorbed albumin was expected to be displaced by cell adhesion proteins. To investigate the effect of preadsorbed albumin displacement on cell adhesion, SAMs were first exposed to albumin then, HUVECs suspended in a serum-supplemented medium were added [21, 42]. Very few cells adhered to hydrophobic SAMs that had been pretreated with albumin, due to the large interfacial tension between water and the hydrophobic surfactant-like surface. Albumin was infrequently displaced by the cell adhesive proteins Fn and Vn. One the other hand, HUVECs adhered well to hydrophilic SAM surfaces that had been preadsorbed with albumin. In that case, the preadsorbed albumin was readily displaced by cell adhesive proteins. 

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. 

T. P. Burghardt and D. Axelrod, Total internal reflection fluorescence study of energy transfer in surface-adsorbed and dissolved bovine serum albumin, Biochemistry 22, 979-985 (1983). 

Adsorbed molecules may diffuse laterally at the interface. Although surface diffusion is well-known in classical surface chemistry 33), data on adsorbed macromolecules is sparse. Burghardt and Axelrod3+) and Michaeli et al. 35) have both demonstrated rapid interface diffusion of adsorbed albumin. 

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 problem with detailed assessment of the structure of surface adsorbed proteins is that the two-dimensional orientation of structural features gives a linear component to the resultant spectrum. Nevertheless, in the surface denaturation of adsorbed albumin, the helices appear to unfold such that the n-TT transition is amplified at the expense of the tt-tt transition. This situation suggests binding of the carbonyl group of the peptide to the surface, or at least orientation of the carbonyl group perpendicular to the surface. 

Thus, we turned to alkylation to modify albumin s affinity for polymer surfaces. Chemical derivatization of the polymer surface was evaluated by its ability to adsorb albumin. We verified by optical microscopy that the surfaces... 

Figure 7 shows that Silastic initially (2 min of blood contact) adsorbed albumin, y-globulin, fibrinogen, and fibronectin in the relative ratios of 100 52 33 3, where the surface concentration of serum albumin was 300... 

When the albumin films are adsorbed to the surface of the peu-425, the formation of a protein film is evident. Some substructure is seen at the 270/sec and 540/sec shear rates. However, the most striking observation is that the hard segment model peu-ppd, which would be expected to adsorb proteins the most readily, adsorbs albumin in a fashion similar to the polystyrene surfaces. The albumin adsorbs to the peu-ppd as distinct molecules at the low flow rates, and at the 270/sec rate the formation of a network is evident. 

The phenomenon of the rapid adsorption of albumin onto a PEUN surface may be associated with hydrophobic and hydrophilic interactions of the PEUN surface with some sequences of relatively hydrophobic amino acid residues in the interior of albumin. An albumin molecule is composed of three-subdomains (15). There are two gaps between the subdomains. One is a hydrophobic pocket with an affinity constant, K3=1.1x10 M for stearic acid the other is an intermediate hydrophobic pocket with K =1.5xl0 M for bilirubin (16). Perhaps the structure of adsorbed albumin in contact with a PEUN surface is composed of hydrophobic and hydrophilic regions corresponding or complementary to those of the PEUN surface, even though the exterior of native albumin is rich in hydrophilic amino acid side chains. 

Thus, our very preliminary results would indicate that adsorption on a germanium surface produces a protein structure similar to the structure in solution, i.e. it is the solution structure that first adsorbs. However, this initial adsorbed structure may not be the most stable structure and thus, rearrangement to a more stable structure may occur for some proteins. It is important to note that these results apply to germanium surfaces. It has already been noted (7) that the structure of adsorbed albumin and adsorbed IgG changes with the nature of the adsorption surface. 

Utilizing time resolved internal reflection spectroscopic technique (Fig. 6), we were able to isolate the tryptophan intrinsic fluorescence and observe its = 20 ns fluorescence lifetime for albumin in bulk and in the surface microenvironment of a hydrophilic quartz material. The pH dependence of bulk albumin fluorescence lifetime served to "calibrate albumin in terms of native ( 7 ns time constant) protein at pH 7.2 and unfolded (c 4 ns) protein at the isoelectric pH 3.8. The fluorescence lifetime data (Tables I/II) supported the hypothesis that the adsorbed albumin exists in two forms on a hydrophilic quartz surface, each with a possibly different structure (] ). A loosely held "layer," consisting of microaggregates, native and partially unfolded albumin molecules with... 

Fig. 13. pH Dependence of zeta-potential for surfaces adsorbed with bovine serum albumin. O glass siliconized glass... 

Typical friction coefficient, p and mass of proteins on differently prepared polymer surfaces adsorbed from solutions with different content of native (N) and irreversibly denatured (D) albumin... 

The friction coefficients observed for CoCrMo sliding against CoCrMo in the three solutions were consistent with those reported in the literature. That no fluorescence image was detected on the CoCrMo surfaces either after incubation in labeled protein or after the CoCrMo-CoCrMo measurements indicated that the fluorescence of the adsorbed albumin was... 

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