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Assessing the influence of adsorbed-state conformation on the bioactivity of adsorbed enzyme layers.

ABSTRACT: Systems using immobilized enzymes are attractive for a wide range of industrial and medical applications because they allow for the fabrication of stable, reusable substrates with highly specific functionality. The performance of these systems is greatly dependent upon the orientation and conformation of the adsorbed enzymes. To investigate these relationships, we have developed and applied methods to quantitatively assess the secondary structure of adsorbed enzyme layers on planar surfaces using circular dichroism (CD) spectroscopy and evaluate their bioactivity using colorimetric assays. These combined measurements provide molecular-level insights regarding whether observed changes in adsorbed enzyme bioactivity are due to the adsorbed orientation of an enzyme or adsorption-induced changes in its conformation. Using this approach, we investigated the adsorption behavior of lysozyme (HEWL), xylanase (XYL), and glucose oxidase (GOx) on OH-, CH(3)-, NH(2)-, and COOH-terminated alkanethiol self-assembled monolayer (SAM) surfaces. The bioactivities of small enzymes HEWL and XYL had pronounced variations between the different SAM surfaces despite their structural stability, highlighting the role of adsorbed orientation on bioactivity. In contrast, GOx, which is a much larger enzyme, exhibited wide variations in both its structure and bioactivity after adsorption, with adsorption-induced conformational changes actually enhancing its bioactivity. These results provide new insights into protein-surface interactions at the molecular level and demonstrate that adsorption can either promote or inhibit bioactivity depending on how the surface chemistry influences the orientation and conformational state of the enzyme on the surface.

SUBMITTER: Fears KP

PROVIDER: S-EPMC3833468 | biostudies-literature | 2009 Dec

REPOSITORIES: biostudies-literature

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Assessing the influence of adsorbed-state conformation on the bioactivity of adsorbed enzyme layers.

Fears Kenan P KP Latour Robert A RA

Langmuir : the ACS journal of surfaces and colloids 20091201 24

Systems using immobilized enzymes are attractive for a wide range of industrial and medical applications because they allow for the fabrication of stable, reusable substrates with highly specific functionality. The performance of these systems is greatly dependent upon the orientation and conformation of the adsorbed enzymes. To investigate these relationships, we have developed and applied methods to quantitatively assess the secondary structure of adsorbed enzyme layers on planar surfaces usin ...[more]

PMID: 19499935

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Project description:While protein-surface interactions have been widely studied, relatively little is understood at this time regarding how protein-surface interaction effects are influenced by protein-protein interactions and how these effects combine with the internal stability of a protein to influence its adsorbed-state structure and bioactivity. The objectives of this study were to develop a method to study these combined effects under widely varying protein-protein interaction conditions using hen egg-white lysozyme (HEWL) adsorbed on silica glass, poly(methyl methacrylate), and polyethylene as our model systems. In order to vary protein-protein interaction effects over a wide range, HEWL was first adsorbed to each surface type under widely varying protein solution concentrations for 2h to saturate the surface, followed by immersion in pure buffer solution for 15h to equilibrate the adsorbed protein layers in the absence of additionally adsorbing protein. Periodic measurements were made at selected time points of the areal density of the adsorbed protein layer as an indicator of the level of protein-protein interaction effects within the layer, and these values were then correlated with measurements of the adsorbed protein's secondary structure and bioactivity. The results from these studies indicate that protein-protein interaction effects help stabilize the structure of HEWL adsorbed on silica glass, have little influence on the structural behavior of HEWL on HDPE, and actually serve to destabilize HEWL's structure on PMMA. The bioactivity of HEWL on silica glass and HDPE was found to decrease in direct proportion to the degree of adsorption-induce protein unfolding. A direct correlation between bioactivity and the conformational state of adsorbed HEWL was less apparent on PMMA, thus suggesting that other factors influenced HEWL's bioactivity on this surface, such as the accessibility of HEWL's bioactive site being blocked by neighboring proteins or the surface itself. The developed methods provide an effective means to characterize the influence of protein-protein interaction effects and provide new molecular-level insights into how protein-protein interaction effects combine with protein-surface interaction and internal protein stability effects to influence the structure and bioactivity of adsorbed protein.

Project description:Adsorption isotherms, circular dichroism (CD) spectroscopy, x-ray photoelectron spectroscopy (XPS), and time-of-flight secondary ion mass spectrometry (ToF-SIMS) were used to investigate the adsorption of human osteocalcin (hOC) and decarboxylated (i.e., Gla converted back to Glu) hOC (dhOC) onto various calcium phosphate surfaces as well as silica surfaces. The adsorption isotherms and XPS nitrogen signals were used to track the amount of adsorbed hOC and dhOC. The intensities of key ToF-SIMS amino acid fragments were used to assess changes in the structure of adsorbed hOC and dhOC. CD spectra were used to investigate the secondary structure of OC. The largest differences were observed when the proteins were adsorbed onto silica versus calcium phosphate surfaces. Similar amounts (3-4 at. % N) of hOC and dhOC were adsorbed onto the silica surface. Higher amounts of hOC and dhOC were adsorbed on all the calcium phosphate surfaces. The ToF-SIMS data showed that the intensity of the Cys amino acid fragment, normalized to intensity of all amino acid fragments, was significantly higher (∼×10) when the proteins were adsorbed onto silica. Since in the native OC structure the cysteines are located in the center of three α-helices, this indicates both hOC and dhOC are more denatured on the silica surface. As hOC and dhOC denature upon adsorption to the silica surface, the cysteines become more exposed and are more readily detected by ToF-SIMS. No significant differences were detected between hOC and dhOC adsorbed onto the silica surface, but small differences were observed between hOC and dhOC adsorbed onto the calcium phosphate surfaces. In the OC structure, the α-3 helix is located above the α-1 and α-2 helices. Small differences in the ToF-SIMS intensities from amino acid fragments characteristic of each helical unit (Asn for α-1; His for α-2; and Phe for α-3) suggests either slight changes in the orientation or a slight uncovering of the α-1 and α-2 for adsorbed dhOC. XPS showed that similar amounts of hOC and dhOC were absorbed onto hydroxyapaptite and octacalcium phosphate surfaces, but ToF-SIMS detected some small differences in the amino acid fragment intensities on these surfaces for adsorbed hOC and dhOC.

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Assessing the influence of adsorbed-state conformation on the bioactivity of adsorbed enzyme layers.

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Assessing the influence of adsorbed-state conformation on the bioactivity of adsorbed enzyme layers.

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