Albumin-fibrinogen mixture

More important is a description of the type of information that can be obtained from the spectra of more complex, flowing mixtures, which can be illustrated by consideration of a 1 1 albumin-fibrinogen mixture in saline. Representative spectra of the protein layer obtained by flowing this mixture past the ATR crystal are shown in Figure 10. In the Amide III (1200-1350-em 1) spectral region, three types of spectral changes occur with time of flow. Looking only at the series of infrared bands around 1300 cm-1, we find that the number of bands, the frequencies of the bands, and the shape... 

The spectral changes in the 1300-cm 1 region are like the spectral changes that occur when the concentration of pure albumin solutions are varied, that is, the 1300-cm 1 spectral changes are tracking the behavior of albumin in the protein layer adsorbed from the albumin-fibrinogen mixture. 

Figure 14. The 1 1 mixture of albumin-fibrinogen adsorbing on germanium (water-subtracted ana scale expanded).

In several series of binary mixtures, relative concentration of each protein in solution determined which was adsorbed most (15), but relationships were not simply predictable. For example, judged by optical thickness of deposited antibody, adsorption of fibrinogen in presence of IgG rapidly rose to its maximum as the relative concentration of fibrinogen was increased, while in presence of albumin fibrinogen adsorption increased almost linearly as the proportion of fibrinogen to albumin in solution was increased (15). 

Torbet,J. (1986). Fibrin assembly in human plasma and fibrinogen/albumin mixtures. Biochem. 25, 5309-5314. 

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). 

A commonly employed first separation step is ammonium sulfate precipitation. This technique exploits the fact that the solubility of most proteins is lowered at high salt concentrations. As the salt concentration is increased, a point is reached where the protein comes out of solution and precipitates. The concentration of salt required for this salting out effect varies from protein to protein, and thus this procedure can be used to fractionate a mixture of proteins. For example, 0.8 M ammonium sulfate precipitates out the clotting protein fibrinogen from blood serum, whereas 2.4 M ammonium sulfate is required to precipitate albumin. Salting out is also sometimes used at later stages in a purification procedure to concentrate a dilute solution of the protein since the protein precipitates and can then be redissolved in a smaller volume of buffer. 

Protein samples used to conduct a protein transfer were bovine serum albumin (BSA, 67,000 daltons), chicken egg ovalbumin (Ovalb, 45,000 daltons), and human serum cryo-precipitate. These agents were obtained from plasma containing a mixture of proteins including Fibrinogen (340,000 daltons), human serum albumin (HSA, 67,000 daltons), and immunoglobulin G (IgG, 47,000-56,000 daltons). The cryo-precipitate was diluted with 20 ml of buffer solution prior to separation. 

Adsorption of albumin, y-globulin, and fibrinogen from single solutions onto several hydrophobic polymers was studied using internal reflection IR spectroscopy. The adsorption isotherms have a Langmuir-type form. The calculated rate and amount of protein adsorbed was dependent on the polymer substrate and the flow rate of the solution. Competitive adsorption experiments were also investigated to determine the specific adsorption of each I-labelled protein from a mixture of proteins. Platelet adhesion to these proteinated surfaces is discussed in relation to a model previously proposed. 

The reduced adsorption of fibrinogen from plasma onto Silastic and poly (HEMA)/Silastic compared with that from pure buffered saline solutions could be caused by competition from other proteins for the adsorption sites. Albumin and y-globulin are both present in plasma in relatively high concentrations (about 45 and 10 mg/ml, respectively, compared with ca. 3 mg/ml for fibrinogen), so either might compete effectively with fibrinogen for adsorption. To test this, mixtures of I-fibrinogen... 

Human serum, another mixture that lacks the competing fibrinogen, deposited films under the same experimental conditions. The films were less able to adsorb matter out of anti-albumin serum than was heated plasma neither heated nor unheated serum showed clear ability to convert fibrinogen (data not listed). 

The thrombotic response (the time vs. platelet and fibrin deposition pattern) for uncoated PVC (shown in Figures 2 and 3) is a response to PVC that is coated with a complex mixture of proteins in the initial seconds of blood contact. Therefore, at least part of the thrombotic response on PVC is generated by a complex protein-coated surface composed of many proteins adsorbed in various conformations, including serum albumin, 7-globulin, fibrinogen, and fibronectin. However, these four proteins account for only 75% of the total protein in plasma and, therefore, significant amounts of other proteins not accounted for by our measurements may be adsorbed to the test surface. 

If the intensities of various infrared bands are measured and plotted against time of flow, the kinetics or rate of adsorption can be determined. This is only true if infrared bands that are not sensitive to conformation or structural changes are used the intensity will then be related directly to the total amount of adsorbed material. The two bands that were common to all proteins studied, and that were independent of conformational changes at constant pH are the bands at 1550 cm-1 (Amide II) and 1400 cm-1. These bands are shown for the fibrinogen-albumin mixture in Figure 11, where... 

Several observations of spectral changes for the fibrinogen-albumin mixture that require further explanation are ... 

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