Ion implantation treatment of beads for covalent binding of molecules: application to bioethanol production using thermophilic beta-glucosidase.

We have achieved plasma immersion ion implantation (PIII) treatment of beads and powders using a specially designed plasma treatment system. This simple one-step production of functionalized beads provides an attractive alternative to current commercial functional beads, for which proteins must be chemically attached using linkers. Using the enzyme beta-glucosidase as an example we show that PIII treatment of polyethylene beads enables covalent binding with increased activity of the enzyme compared to the untreated beads. Covalent binding was confirmed using detergent washing. The covalently immobilized enzyme has a broader pH range over which it has high activity than the enzyme in solution. The stability of the immobilized molecules was examined using reaction rate as a function of temperature and was shown to be significantly higher on the PIII treated beads compared to untreated beads. We attribute the increased enzyme activity on PIII treated beads to increased protein binding density and better retention of conformation. The results of this work are of significance in the production of ethanol using a flow process. Covalent binding to beads allows more robust attachment for high flow rates, high activity, large surface area and a broad operating pH range. Treatment could be easily adapted for a range of applications such as linking drugs, dyes and proteins to particles of an appropriate size.

The Vroman effect: competitive protein exchange with dynamic multilayer protein aggregates.

The surface immobilization of proteins is an emerging field with applications in a wide range of important areas: biomedical devices, disease diagnosis, biosensing, food processing, biofouling, and bioreactors. Proteins, in Nature, often work synergistically, as in the important enzyme mixture, cellulase. It is necessary to preserve these synergies when utilizing surface immobilized proteins. However, the competitive displacement of earlier adsorbed proteins by other proteins with stronger binding affinities (the "Vroman effect") results in undesired layer instabilities that are difficult to control. Although this nanoscale phenomenon has been extensively studied over the last 40 years, the process through which this competitive exchange occurs is not well understood. This paper uses atomic force microscopy, QCM-D, TOF-SIMS, and in-solution TOF-MS to show that this competitive exchange process can occur through the turning of multilayer protein aggregates. This dynamic process is consistent with earlier postulated "transient complex" models, in which the exchange occurs in three stages: an initial layer adsorbs, another protein layer then embeds itself into the initial layer, forming a "transient complex;" the complex "turns," exposing the first layer to solution; proteins from the first layer desorb resulting in a final adsorbed protein composition that is enriched in proteins from the second layer.

Mechanisms for covalent immobilization of horseradish peroxidase on ion-beam-treated polyethylene.

The surface of polyethylene was modified by plasma immersion ion implantation. Structure changes including carbonization and oxidation were observed. High surface energy of the modified polyethylene was attributed to the presence of free radicals on the surface. The surface energy decay with storage time after treatment was explained by a decay of the free radical concentration while the concentration of oxygen-containing groups increased with storage time. Horseradish peroxidase was covalently attached onto the modified surface by the reaction with free radicals. Appropriate blocking agents can block this reaction. All aminoacid residues can take part in the covalent attachment process, providing a universal mechanism of attachment for all proteins. The native conformation of attached protein is retained due to hydrophilic interactions in the interface region. The enzymatic activity of covalently attached protein remained high. The long-term activity of the modified layer to attach protein is explained by stabilisation of unpaired electrons in sp(2) carbon structures. A high concentration of free radicals can give multiple covalent bonds to the protein molecule and destroy the native conformation and with it the catalytic activity. The universal mechanism of protein attachment to free radicals could be extended to various methods of radiation damage of polymers.

Free radical functionalization of surfaces to prevent adverse responses to biomedical devices.

Immobilizing a protein, that is fully compatible with the patient, on the surface of a biomedical device should make it possible to avoid adverse responses such as inflammation, rejection, or excessive fibrosis. A surface that strongly binds and does not denature the compatible protein is required. Hydrophilic surfaces do not induce denaturation of immobilized protein but exhibit a low binding affinity for protein. Here, we describe an energetic ion-assisted plasma process that can make any surface hydrophilic and at the same time enable it to covalently immobilize functional biological molecules. We show that the modification creates free radicals that migrate to the surface from a reservoir beneath. When they reach the surface, the radicals form covalent bonds with biomolecules. The kinetics and number densities of protein molecules in solution and free radicals in the reservoir control the time required to form a full protein monolayer that is covalently bound. The shelf life of the covalent binding capability is governed by the initial density of free radicals and the depth of the reservoir. We show that the high reactivity of the radicals renders the binding universal across all biological macromolecules. Because the free radical reservoir can be created on any solid material, this approach can be used in medical applications ranging from cardiovascular stents to heart-lung machines.

Effect of low molecular weight additives on immobilization strength, activity, and conformation of protein immobilized on PVC and UHMWPE.

Horseradish peroxidase (HRP) was immobilized onto both plasticized and unplasticized polyvinylchloride (PVC) and ultrahigh molecular weight polyethylene (UHMWPE). Plasma immersion ion implantation (PIII) in a nitrogen plasma with 20 kV bias was used to facilitate covalent immobilization and to improve the wettability of the surfaces. The surfaces and immobilized protein were studied using attenuated total reflection infrared (ATR-IR) spectroscopy and water contact angle measurements. Protein elution on exposure to repeated sodium dodecyl sulfate (SDS) washing was used to assess the strength of HRP immobilization. The presence of low molecular weight components (plasticizer, additives in solvent, unreacted monomers, adsorbed molecules on surface) was found to have a major influence on the strength of immobilization and the conformation of the protein on the samples not exposed to the PIII treatment. A phenomenological model considering interactions between the low molecular weight components, the protein molecule, and the surface is developed to explain these observations.

A new surface for immobilizing and maintaining the function of enzymes in a freeze-dried state.

We describe a new surface produced by plasma treatment for immobilizing proteins in the dry state. The need for surfaces suitable for immobilizing proteins is increasing because of demand for microarray diagnostic services, biosensors, and chemical processing. Storage of surface attached proteins in the dry state offers benefits of long shelf life, protection from proteases, easier transportation and convenient storage. In this work, we produced plasma-modified polyethylene surfaces and tested them using two important enzymes for which convenient functional assays are available, namely, horseradish peroxidase and catalase. Over 80% of the function of horseradish peroxidase is retained after freeze-drying, and this function is unaltered after 4 months of storage at 4 degrees C on the treated polyethylene surface. The factors important for maintenance of surface attached enzyme stability were (1) plasma immersion ion implantation (PIII) treatment of the surface, (2) freeze-drying with sucrose in the buffer solution, (3) dry storage with desiccant, and (4) maintaining the freeze-dried protein at a reduced temperature. Other than sucrose, no other additives are needed.

Covalent immobilisation of tropoelastin on a plasma deposited interface for enhancement of endothelialisation on metal surfaces.

Currently available endovascular metallic implants such as stents exhibit suboptimal biocompatibility in that they re-endothelialise poorly leaving them susceptible to thrombosis. To improve the interaction of these implants with endothelial cells we developed a surface coating technology, enabling the covalent attachment of biomolecules to previously inert metal surfaces. Using horseradish peroxidase as a probe, we demonstrate that the polymerised surface can retain the presentation and activity of an immobilised protein. We further demonstrated the attachment of tropoelastin, an extracellular matrix protein critical to the correct arrangement and function of vasculature. Not only it is structurally important, but it plays a major role in supporting endothelial cell growth, while modulating smooth muscle cell infiltration. Tropoelastin was shown to bind to the surface in a covalent monolayer, supplemented with additional physisorbed multilayers on extended incubation. The physisorbed tropoelastin layers can be washed away in buffer or SDS while the first layer of tropoelastin remains tightly bound. The plasma coated stainless steel surface with immobilised tropoelastin was subsequently found to have improved biocompatibility by promoting endothelial cell attachment and proliferation relative to uncoated stainless steel controls. Tropoelastin coatings applied to otherwise inert substrates using this technology could thus have broad applications to a range of non-polymeric vascular devices.

Attachment of horseradish peroxidase to polytetrafluorethylene (teflon) after plasma immersion ion implantation.

The aim of this work was to investigate the potential of polytetrafluorethylene (PTFE) as a surface for biologically active protein attachment. A plasma immersion ion implantation (PIII) treatment was applied to PTFE to produce an activated surface for the functional attachment of the enzyme, horseradish peroxidase (HRP). Fourier transform infrared-attenuated total reflectance spectra show oxidation and carbonization of the surface layer as a function of ion fluence. The PIII treatment increases by threefold the amount of attached HRP and the activity of HRP on the modified surface is about seven times higher than that on an untreated PTFE surface. This result indicates that the PIII surface modification improves both the polymer's protein binding capacity and its ability to retain the protein in a bioactive state.

The attachment of catalase and poly-l-lysine to plasma immersion ion implantation-treated polyethylene.

Plasma immersion ion implantation (PIII) treatment of polyethylene increased the functional attachment of catalase and increased the retention of enzyme activity in comparison to untreated controls. The attached protein was not removed by SDS or NaOH, while that on the untreated surfaces was easily removed. Poly-l-lysine was found to attach in a similar way to the treated surface and could not be removed by NaOH, while it did not attach to the untreated surface. This indicates that a new binding mechanism, covalent in nature, is introduced by the plasma treatment. Surfaces treated with PIII maintained the catalase activity more effectively than surfaces plasma treated without PIII. The PIII-treated surface was hydrophilic compared to the untreated surface and retained its hydrophilic character better than surfaces subjected to a conventional plasma treatment process. The strong modification of a deeper region of the polymer than for conventional plasma treatments is believed to be responsible for both the enhanced hydrophilic character and for the increase in functional lifetime of the attached protein. The results show that PIII treatment of polymers increases their usefulness for protein microarrays.

Mapping the phosphoinositide-binding site on chick cofilin explains how PIP2 regulates the cofilin-actin interaction.

Cofilin plays a key role in the choreography of actin dynamics via its ability to sever actin filaments and increase the rate of monomer dissociation from pointed ends. The exact manner by which phosphoinositides bind to cofilin and inhibit its interaction with actin has proven difficult to ascertain. We determined the structure of chick cofilin and used NMR chemical shift mapping and structure-directed mutagenesis to unambiguously locate its recognition site for phosphoinositides (PIs). This structurally unique recognition site requires both the acyl chain and head group of the PI for a productive interaction, and it is not inhibited by phosphorylation of cofilin. We propose that the interaction of cofilin with membrane-bound PIs abrogates its binding to both actin and actin-interacting protein 1, and facilitates spatiotemporal regulation of cofilin activity.

Nanosecond responses of proteins to ultra-high temperature pulses.

Observations of fast unfolding events in proteins are typically restricted to <100 degrees C. We use a novel apparatus to heat and cool enzymes within tens of nanoseconds to temperatures well in excess of the boiling point. The nanosecond temperature spikes are too fast to allow water to boil but can affect protein function. Spikes of 174 degrees C for catalase and approximately 290 degrees C for horseradish peroxidase are required to produce irreversible loss of enzyme activity. Similar temperature spikes have no effect when restricted to 100 degrees C or below. These results indicate that the "speed limit" for the thermal unfolding of large proteins is shorter than 10(-8) s. The unfolding rate at high temperature is consistent with extrapolation of low temperature rates over 12 orders of magnitude using the Arrhenius relation.

The N-terminal fragment of gelsolin inhibits the interaction of DNase I with isolated actin, but not with the cofilin-actin complex.

Apoptosis is essential in embryonic development, clonal selection of cells of the immune system and in the prevention of cancer. Apoptotic cells display characteristic changes in morphology that precede the eventual fragmentation of nuclear DNA resulting in cell death. Current evidence implicates DNase I as responsible for hydrolysis of DNA during apoptosis. In vivo, it is likely that cytoplasmic actin binds and inhibits the enzymatic activity and nuclear translocation of DNase I and that disruption of the actin-DNase I complex results in activation of DNase I. In this report we demonstrate that the N-terminal fragment of gelsolin (N-gelsolin) disrupts the actin-DNase I interaction. This provides a molecular mechanism for the role of the N-gelsolin in regulating DNase I activity. We also show that cofilin stabilises the actin-DNase I complex by forming a ternary complex that prevents N-gelsolin from releasing DNase I from actin. We suggest that both cofilin and gelsolin are essential in modulating the release of DNase I from actin.

Cofilin and DNase I affect the conformation of the small domain of actin.

Cofilin binding induces an allosteric conformational change in subdomain 2 of actin, reducing the distance between probes attached to Gln-41 (subdomain 2) and Cys-374 (subdomain 1) from 34.4 to 31.4 A (pH 6.8) as demonstrated by fluorescence energy transfer spectroscopy. This effect was slightly less pronounced at pH 8.0. In contrast, binding of DNase I increased this distance (35.5 A), a change that was not pH-sensitive. Although DNase I-induced changes in the distance along the small domain of actin were modest, a significantly larger change (38.2 A) was observed when the ternary complex of cofilin-actin-DNase I was formed. Saturation binding of cofilin prevents pyrene fluorescence enhancement normally associated with actin polymerization. Changes in the emission and excitation spectra of pyrene-F actin in the presence of cofilin indicate that subdomain 1 (near Cys-374) assumes a G-like conformation. Thus, the enhancement of pyrene fluorescence does not correspond to the extent of actin polymerization in the presence of cofilin. The structural changes in G and F actin induced by these actin-binding proteins may be important for understanding the mechanism regulating the G-actin pool in cells.