Mechanisms for covalent immobilization of horseradish peroxi-dase on ion beam treated polyethylene

The mechanism that provides the observed strong binding of biomolecules to polymer sur-faces modified by ion beams is investigated. The surface of polyethylene (PE) was modified by plasma immersion ion implantation with nitrogen ions. Structure changes including car-bonization and oxidation were observed in the modified surface layer of PE by Raman spec-troscopy, FTIR ATR spectroscopy, atomic force microscopy, surface energy measurement and XPS spectroscopy. An observed high surface energy of the modified polyethylene was attributed to the presence of free radicals on the surface. The surface energy decay with stor-age time after PIII treatment was explained by a decay of the free radical concentration while the concentration of oxygen-containing groups increased with storage time. Horseradish per-oxidase was covalently attached onto the modified PE surface. The enzymatic activity of co-valently attached protein remained high. A mechanism based on the covalent attachment by the reaction of protein with free radicals in the modified surface is proposed. 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 long-term activity of the modified layer to attach protein (at least 2 years) is explained by stabilisa-tion of unpaired electrons in sp2 carbon structures. The native conformation of attached pro-tein is retained due to hydrophilic interactions in the interface region. A high concentration of free radicals on the surface can give multiple covalent bonds to the protein molecule and de-stroy 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

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

Nanosecond Responses of Proteins to Ultra-High Temperature Pulses

Observations of fast unfolding events in proteins are typically restricted to <100°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°C for catalase and ∼290°C for horseradish peroxidase are required to produce irreversible loss of enzyme activity. Similar temperature spikes have no effect when restricted to 100°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

Acetylene plasma polymerized surfaces for covalent immobilization of dense bioactive protein monolayers

Smooth polymerized surfaces, suitable for biochemical and biomedical applications, were deposited using a modified plasma enhanced chemical vapour deposition method with acetylene as a reaction precursor. Horseradish peroxidase (HRP) activity assays showed that the protein immobilized on the plasma polymerized surfaces maintained its biological function for a much longer period of time compared to that on uncoated surfaces. The kinetics of HRP attachment to the plasma polymerized surfaces were analyzed using quartz crystal microbalance with dissipation analysis. Spectroscopic ellipsometry and attenuated total reflection Fourier transform infrared spectroscopy were used to determine the thickness and the quantity of the attached protein. The results showed that the plasma polymerized surfaces provided a high density of attachment sites to covalently immobilize a dense monolayer of proteins.<br /

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

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