Adsorbed protein analysis

O. D. Sanni, M. S. Wagner, D. G. Briggs, D. G. Castner and J. C. Vickerman, Classification of adsorbed protein static ToF SIMS spectra by principal component analysis and neural networks, Surface and Interface Analysis, 33, 715 728 (2002). 

M. S. Wagner and D. G. Castner, Analysis of adsorbed proteins by static time of flight secondary ion mass spectrometry, Applied Surface Science, 231 232, 366 376 (2004). 

When a polymer is treated with enzymes for surface modification, some of the undesired protein tends to adsorb on the polymer surface, which subsequently creates problems in the surface analysis and causes a slow down in the rate of catalysis. Adsorbed proteins can be removed from the surfaces by washing with large volumes of 1.5% Na2C03 and water (Eischer-Colbrie et al., 2006) as part of a preparation for surface analysis. Protein-resistant molecules such as polyethylene glycol can be used to prevent the nonspecific protein adsorption. Surfaces can be precoated with an inert protein such as bovine serum albumin (Salisbury et al., 2002) for increasing the rate of catalysis. 

Ninhydrin Assay for Adsorbed Proteins. Measurements were made by a colorimetric procedure based on the reaction of ninhydrin with amino acids (25). The films were hydrolyzed in 5 ml of 2.5N NaOH for 2 hrs in capped plastic tubes in a boiling water bath. Then 1.5 ml of glacial acetic acid was added and mixed next I ml of ninhydrin reagent was added and mixed. [The reagent was three times more concentrated in ninhydrin, SnCb, and citrate than prescribed by Moore and Stein (25)]. The tubes were capped and boiled 20 mins more. The solution was clarified by centrifugation, and the absorbance read immediately at 570 nm on a Beckman DB spectrophotometer. If necessary, the sample was diluted with 50-50 2-propanol-water. Calibration curves (absorbance vs. fig of protein) were constructed in the 0-30 and 0-100 fig range with known amounts of each type of protein subjected to this same analysis procedure. 

The capacity of the nanoparticles to adsorb proteins and to activate the complement in vivo after intravenous administration will influence the fate of the carrier and its body distribution. To approach this aspect, in vitro tests have been developed to investigate the profile of the type of serum proteins that adsorbed onto the nanoparticle surface after incubation in serum and to evaluate the capacity of the nanoparticles to induce complement activation. The analysis of the protein adsorbed onto the nanoparticle surface can be performed by 2D-polyacrylamide gel electrophoresis. This technique allows the identification of the proteins that adsorbed onto the nanoparticle surface. To evaluate modifications of the composition of the adsorbed protein with time, a faster method based on capillary electrophoresis can also be used. Finally, the activation of the complement produced by nanoparticles can be evaluated either by a global technique or by a specific method measuring the specific activation... 

Fig. 12 Illustration of the difference between the small-load approximation and the perturbation analysis for a film in liquid. Experimental data obtained with an adsorbed protein layer in buffer. Dashed line fit with third-order perturbation analysis. Fit parameters df = 14 nm, = 13 300 GPa , and P =- 1 gold electrodes as in Fig. 11. The viscosity of the buffer was 0.96 cP. Solid line simulation with the small-load approximation and the same model parameters as the input to the dashed line. There is a systematic differeuce. (Experimeutal data kiudly provided by 1. Reviakiue)...

Figure 10. Analysis of adsorbed proteins with SDS-polyacrylamide gel electrophoresis.

The adsorption of plasma proteins to polymers precedes the interaction of blood cells with the surfaces, and therefore, is likely to be an important initial event in the response of blood to polymers (25, 29). At present, however, little is known about the adsorbed protein layer, even though it has been studied in some detail in recent years (30-36). Because protein adsorption from blood plasma is a competitive process, differences in the adsorbed layer on different polymer substrates could be a primary cause of differences in thrombogenicity. Previous studies of the composition of the adsorbed protein layer have employed 12oI-labeled protein added to plasma (37-39), antibody binding (34) to detect individual proteins, or electrophoretic analysis of detergent-elutable proteins (17, 33, 35). The procedure used in this study does not require the large surface areas used in previous work (35), nor does it rely on incorporation of radiolabels (36) into adsorbed protein. Instead, a staining method at least 100-fold more sensitive than these other techniques has been used. 

There are, unfortunately, no studies to date of the dissolved protein content of microlayer samples. With the recent development of many sensitive techniques for the analysis of amino-acid mixtures in seawater using liquid chromatography and fluorescence detectors (e.g., Dawson and Pritchard, 1978), it should be relatively simple to analyse for combined amino acids after hydrolysis of the microlayer samples. Analyses of free amino acids in the microlayer seem not to have been performed to date either, but since considerable degradation of surface-adsorbed proteins may take place as a result of UV irradiation, this may be a fruitful area for future research. 

Some of the interactions that determine the three-dimensional structure of a protein molecule support a compact conformation, whereas others tend to expand the molecule. In aqueous solution hydrophobic parts of the protein are buried as much as possible in the interior of the molecule but in the adsorbed state the hydrophobic residues may be exposed to the sorbent surface, still shielded from water. Therefore, an expanded structure will be promoted upon adsorption if the compact structure in solution is stabilized by intramolecular hydrophobic bonding. More precisely, whether or not adsorbing protein molecules change their structure depends on the contribution from intramolecular hydrophobic bonding, relative to those from other interactions, to the overall stabilization of the structure in solution. In reference ( ) such an analysis of the structure determining factors has been made for HPA and RNase. It leads to the conclusion that HPA, more than RNase, is able to adapt its structure at sorbent surfaces. 

Due to the fundamental importance of the adsorbed protein film, many methods have been used to characterize its nature. These methods include ellipsometry (3,A), Fourier transform infrared spectroscopy (FTIR) (5,6), multiple attenuated internal reflection spectroscopy (MAIR) (7,8) immunological labeling techniques (9), radioisotope labeled binding studies (j ), calorimetric adsorption studies (jj ), circular dichroism spectroscopy (CDS) (12), electrophoresis (j ), electron spectroscopy for chemical analysis (ESCA) (1 ), scanning electron microscopy (SEM) (15,16,9), and transmission electron microscopy (TEM) (17-19). 

Surface Force Measurements. This technique enables the measurement of the force (10 mN accuracy) versus distance (0.1-0.2 nm accuracy) between two curved mica surfaces. The forces between two solid surfaces across an aqueous solution are highly sensitive to the structure of the solid/liquid interfaces. When such surfaces are covered with adsorbed protein layers, then, the analysis of the force/distance profiles may reveal the formation of protein bridges between the two surfaces, the occurrence of steric interactions, or any possible protein conformation change. 

The history of the use of electrochemical methods to study the properties of proteins, enzymes, and their component structural units is several decades old. Until quite recently, these studies were primarily associated with the use of polarography to solve analytical problems. The polarographic method of analysis has become very popular for determination of proteins, enzymes, and nucleic acids.It has been found that proteins irreversibly adsorb on mercury and in the presence of cobalt salts promote the evolution of hydrogen. In this case wave height is proportional to the amount of adsorbed protein. These studies have been summarized in some reviews and monographs. " "" ... 

Besides direct surface analysis methods, indirect solution depletion methods can be applied to study protein adsorption. Solution depletion methods use solids of known large surface area (usually dispersed solids) that are placed into a solution of known protein concentration. After incubation, the concentration of the protein in the supernatant is measured. From the depletion, the adsorbed protein amount can be calculated. 

Grundke K, Werner C, Poschel K, Jacobasch HJ (1999) Characterization of adsorbed protein laytas by low-rate dynamic liquid-fluid contact angle measurements using axisymmetric drop shape analysis (part II). Colloids Surf A 156 19-31... 

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