Lysozyme protein adsorption

The effect of pH on the protein adsorption on CMK-3 was also investigated [152], The monolayer adsorption capacities obtained under various pH conditions are plotted in Figure 4.12, where the maximum adsorption was observed in the pH region near the isoelectric point of lysozyme (pi of about 11). Near the isoelectric point, the net charges of the lysozyme molecule are minimized and would form the most compact assembly. A similar pH effect was also seen in the adsorption of cytochrome c on CM K-3. Although the nature of the surface of mesoporous silica and... 

The adsorption of positive proteins onto the epoxy capillary surface can be quantitatively evaluated by using the two on-line detector design. From the responses of the two detectors, it is possible to determine the adsorption of proteins on the surface between the two detectors. Zero-percent recoveries have been reported on an uncoated capillary at pH 7 for lysozyme, cytochrome-c, ribonuclease A, and u-chymotrypsinogen. However, most of these proteins had recoveries between 84.4% and 95%, except for lysozyme (55.5%), on an epoxy-coated capillary. Therefore, coating does reduce protein adsorption significantly. 

Barroug, A., Lemaitre, J., and Rouxhet, P.G., Lysozyme on apatites A model of protein adsorption controlled by electrostatic interactions, Colloids Surf, 37, 339, 1989. 

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. 

Lens hazing and protein deposition are common problems for wearers of soft contact lenses. Previous experiments with hydrophobic-hydrophilic copolymers exposed to plasma showed protein adsorption to be minimal at intermediate copolymer compositions. Adsorption of proteins from artificial tear solutions to a series of polymers and copolymers ranging in composition from 100% poly (methyl methacrylate) (PMMA) to 100% poly(2-hydroxyethyl methacrylate) (PH EM A) was measured. The total protein adsorption due to the three major proteins in tear fluid (lysozyme, albumin, and immunoglobulins) was at a minimum value at copolymer compositions containing 50% or less PH EM A. The elution of the adsorbed proteins from these polymers and copolymers with various solutions also was investigated to assess the binding mechanism. 

Recent experiments indicate that polymers that contain a balance of hydrophobic (nonpolar) and hydrophilic (polar) chemical groups show minimal protein adsorption and cell adhesion (6). With the intent of rationally designing a contact lens material that would minimize protein adsorption, the adsorption of lysozyme, albumin, and immunoglobulin G (IgG) to a series of hydrophobic and hydrophilic polymers and copolymers was measured. The polymers ranged from 100% poly(methyl methacrylate) (PMMA) to 100% poly(2-hydroxyethyl methacrylate) (PHEMA). Adsorption varied significantly for each protein, as did the elutability of the proteins from the surfaces. 

Figure 2 shows that similar adsorption phenomena are exhibited by lysozyme and albumin. Related work seems to suggest that polymers that contain a certain balance of hydrophobic and hydrophilic chemical groups show minimized biological interaction (for example, low protein adsorption, low thrombus deposition, and low platelet consumption) (6). The adsorption of radiolabeled IgG, however, was maximal at intermediate copolymers. This result has a number of implications with respect to both the fundamental adsorption mechanism and the biocompatibility of these materials. 

Protein adsorption studies were done by immersing polymer discs for 24 h in room-temperature solutions of radiolabeled gamma-globulin, albumin, and lysozyme. Standard scintillation counting methods were used to quantify the amount of protein bound to the polymer surfaces after a thorough rinse with physiological saline solution (0.9% aqueous NaCl). 

In a schematic elution pattern of some standard proteins, peroxidase was eluted first with saline, BSA came next with glycine buffer at pH 6.6 and hemoglobin and catalase were eluted at a pH of nearly 8.0. Aldolase, lysozyme, chymotrypsinogen A, malate dehydrogenase, and cytochrome c were not eluted under these conditions, but were eluted with 0.1% SDS. The adsorption order does not depend on the isoelectric point, the molecular mass, or the content of basic amino acids. However, adsorption may depend on the o -helix content, and the secondary structure of those proteins may be important. We have also reported on protein adsorption and separation on siliconized glass surfaces (30), and on the adsorption and separation of nucleic acids on those same surfaces (31-35). 

Both the amide I peak at 1638 cm" and the amide III peak at 1247 cm" support CD observations of the presence of some 3-sheet structure. The shift of the latter peak to a lower frequency around 1240 cm in the adsorbed state suggest that an increase in 3-sheet structure may occur upon adsorption. Also the shift in the amide I band from 1642 cm in solution to 1638 cm when adsorbed further substantiates the hypothesis that the 3-sheet content of FN increases upon adsorption. An increase in 3-sheet structure upon adsorption has been previously reported in FTIR/ATR studies of protein adsorption on contact lens materials. Specifically, an a-helix to random and 3-sheet transition has been observed for adsorbed albumin and lysozyme, as well as a random to 3-sheet transition for mucin (22,26,32). However, a decrease in 3-sheet structure has also been observed for adsorbed y-globulin which contains a high content of 3-sheet structure in its native form (33). 

The kinetics of protein adsorption at an interface can be measured by monitoring surface concentration and surface pressure i.e. depression of surface tension (V) as a function of time (3/7). -casein is more surface active than serum albumin or b-Lg and much more so than lysozyme. This reflects not only the rate of diffusion of the native protein to the interface, but also its molecular flexibility and amphipathic nature (15,17,22). 

In-situ ATR FTIR spectroscopy was used to study the interaction between the differently charged model proteins human serum albumin, lysozyme, immunoglobulin G and multilayer assemblies, which were deposited by alternating adsorption of polyethyleneimine and polyacrylic acid onto silicon crystals. Low adsorbed protein amounts were observed if the top polyelectrolyte layer and the protein were equally charged, whereas enhanced protein adsorption occurred for electrostatic attraction between protein and top polyelectrolyte layer. 18 refs. 

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