Protein adsorption determination

Harder P, Grunze M, Dahint R, Whitesides G M and Laibinis P E 1998 Molecular conformation in oligo(ethylene glycol)-terminated self-assembled monolayers on gold and silver surfaces determines their ability to resist protein adsorption J. Rhys. Chem. B 102 426-36... 

Proteins may be covalently attached to the latex particle by a reaction of the chloromethyl group with a-amino groups of lysine residues. We studied this process (17) using bovine serum albumin as a model protein - the reaction is of considerable interest because latex-bound antigens or antibodies may be used for highly sensitive immunoassays. The temperature dependence of the rate of protein attachment to the latex particle was unusually small - this rate increased only by 27% when the temperature was raised from 25°C to 35°C. This suggests that non-covalent protein adsorption on the polymer is rate determining. On the other hand. the rate of chloride release increases in this temperature interval by a factor of 17 and while the protein is bound to the latex particle by only 2 bonds at 25°C, 22 bonds are formed at 35°C. 

The first aspect of biocompatibility is a natural immune response. When a foreign object enters the blood stream, it can be attacked by the body s defense system. The first step is protein adsorption on an object surface. It is believed that the amount and type of protein adsorption is one of the most important steps determining whether the object is tolerated or rejected by the body. The next step is cell adhesion, which may cause aggregation and activation of platelets and triggering of the blood coagulation system with resulting thrombus formation. It may not only lead to sensor failure via surface blocking but directly threatens the patient s health. 

The tendency of proteins to adsorb at interfaces is determined by many variables, including the pH, the ionic strength, the properties of the protein molecules and the interfaces, and the nature of the solvent and other components present. The process of protein adsorption is complicated, and despite the great volume of work over the past decades, a unified theory is still far ahead. Yet, some principles may be indicated. 

Since protein adsorption to an anion exchange resin is reversible and does not constitute a classical immobilization, the ability of the resins to retain activity under various conditions must be determined. Macrosorb KAX DEAE bound -D-glucosidase was tested with solutions of primary interest for their final application. Several batch washes of a 1% w/v slurry were required to ensure complete equilibrium elution for a given concentration, as determined from the absence of pNPG units in subsequent washes. Several salt solutions of typical fermentation media components were tested. These included 3 mM to 50 mM solutions of MgSO, KHgPO, NaQ, and sodium acetate. Also, incubations with cellulase solutions were tested to determine if the proteins present in a cellulose hydrolysis would displace the -D-glucosidase. Both of these displacement studies were carried out at 22°C and 40 C. 

Techniques and methods for the study of protein adsorption have been well reviewed 4). It is now generally recognized that it is not necessarily the type and amount of protein present at the surface which is most important, but rather the orientation and conformational state of those proteins. At present it is virtually impossible to predict the specific conformation of an adsorbed protein at a particular interface. The techniques used in the determination of protein conformation in solution or in the solid state do not usually apply to adsorbed proteins. Hence, the difference between adsorbed and bulk solution protein conformation has to be inferred indirectly. 

As in fluidized bed adsorption, proteins are bound to porous particles as well, these parameters will remain important and must be considered when describing protein adsorption to fluidized beds. As mentioned above, fluidizing the adsorbent allows free movement of the particles within the adsorbent bed, so dispersion in the solid phase is another component determining process performance. 

Concentrations of thermally generated meat flavor components are diminished by protein adsorption when soy extenders are added to fresh meat products before heating. The amounts of individual alkyl pyrazines, thermally generated by heating beef diffusate, decreased linearly as the amount of whole soy, soy 7S or soy 11S proteins were increased in a model system. Similar recoveries were obtained when pyrazines were mixed with soy either as chemical standards or from diffusate. Stoichiometry and energetics of interaction were determined for methyl pyrazine congeners with soy proteins at 120° and 145°C. Results of this study suggest that flavorants can be added in readily determined amounts to compensate for losses due to adsorption in meat-soy products. 

Another challenge is the delivery of a biopharmaceutical to its site of action, as the injection of molecules in solution leads to a partitioning of the molecules according to their physicochemical properties. One approach to deliver particles injected intravenously is based on the concept of differential protein adsorption. After injection the particles adsorb blood proteins according to physicochemical surface properties of the particles. The adsorbed proteins determine the cells to which the particles will be directed (Muller and Keck, 2004). 

As determined by XPS analysis, the surface composition and O/C and N/C elemental ratios for 5 W and 15 W PEO-like and Ag/PEO-like films, following incubation in the protein solutions are illustrated in Table 1 for a power of 5 W. The data (Table 1) demonstrate that the PEO-like coatings are effective at reducing and/or preventing protein adsorption. The PEO-like films completely prevented the adsorption of albumin, as XPS did not detect a nitrogen signal. Moreover, the O/C ratio of the film is maintained after albumin exposure. 

Whether protein adsorption or cell adhesion is intended or not, for each particular application the interaction of the bio-particle with the material s surface should be tuned to reach the optimal result. Protein adsorption and bacterial adhesion, further denoted as particle adhesion, may be manipulated in a controlled way if the various types of interaction determining the adsorption and adhesion processes are identified. 

Protein function at solid-liquid interfaces holds a structural and a dynamic perspective [31]. The structural perspective addresses macroscopic adsorption, molecular interactions between the protein and the surface, collective interactions between the individual adsorbed protein molecules, and changes in the conformational and hydration states of the protein molecules induced by these physical interactions. Interactions caused by protein adsorption are mostly non-covalent but strong enough to cause drastic functional transformations. All these features are, moreover, affected by the double layer and the electrode potential at electrochemical interfaces. Factors that determine protein adsorption patterns have been discussed in detail recently, both in the broad context of solute proteins at solid surfaces [31], and in specific contexts of interfacial metalloprotein electrochemistry [34]. Some important elements that can also be modelled in suitable detail would be ... 

The sorption efficiency of MC was determined as the ratio of the quantity of the adsorbed substance to its initial amount (w / w), expressed in % for a certain ratio (w / w) of adsorbent to substance. Optimal ratios of adsorbent to substance equal 15-20 for barbiturates, 20 - 25 for cyanocobalamin and bilirubin, and 40 - 50 for hemoglobin. The initial concentration of absorbed substances was 100 - 200 pg/ml. The substances were incubated for lmin with MC either in physiological solution or in donor plasma and donor blood at room temperature (pH 7.4). The concentration of substances in the solutions was measured by differential visual and UV-spectroscopy. Concentrations of substances in blood and plasma and adsorption of total plasma proteins was determined by thin-layer chromatography with a fluorescent label. 

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