How hydrogen peroxide is metabolized by oxidized cytochrome c oxidase.

In the absence of external electron donors, oxidized bovine cytochrome c oxidase (CcO) exhibits the ability to decompose excess H2O2. Depending on the concentration of peroxide, two mechanisms of degradation were identified. At submillimolar peroxide concentrations, decomposition proceeds with virtually no production of superoxide and oxygen. In contrast, in the millimolar H2O2 concentration range, CcO generates superoxide from peroxide. At submillimolar concentrations, the decomposition of H2O2 occurs at least at two sites. One is the catalytic heme a3-CuB center where H2O2 is reduced to water. During the interaction of the enzyme with H2O2, this center cycles back to oxidized CcO via the intermediate presence of two oxoferryl states. We show that at pH 8.0 two molecules of H2O2 react with the catalytic center accomplishing one cycle. In addition, the reactions at the heme a3-CuB center generate the surface-exposed lipid-based radical(s) that participates in the decomposition of peroxide. It is also found that the irreversible decline of the catalytic activity of the enzyme treated with submillimolar H2O2 concentrations results specifically from the decrease in the rate of electron transfer from heme a to the heme a3-CuB center during the reductive phase of the catalytic cycle. The rates of electron transfer from ferrocytochrome c to heme a and the kinetics of the oxidation of the fully reduced CcO with O2 were not affected in the peroxide-modified CcO.

Spectroscopic evidence of the role of an axial ligand histidinate in the mechanism of adrenal cytochrome b561.

Adrenal cytochrome b(561) (AdCytb) is the prototype of a widespread protein family that specializes in delivering electrons donated by ascorbic acid for different processes in eukaryotic cells. AdCytb transports redox equivalents from cytoplasmic ascorbate across the membranes of chromaffin granules to support norepinephrine synthesis within their matrix. The interaction of AdCytb with ascorbate is central to a proposed mechanism of AdCytb's function, and a histidine in the active site of AdCytb was suggested to bind cytoplasmic ascorbate and serve as the acceptor of the proton released during ascorbate oxidation. AdCytb contains high- and low-potential hemes but their orientation relative to the matrix and cytoplasmic interfaces of chromaffin granule membrane is disputed. Using a combination of three spectroscopic methods (UV-vis absorption, near-infrared magnetic circular dichroism, and electron paramagnetic resonance), we find that a histidine residue that serves as an axial ligand to the high-potential heme undergoes deprotonation with a pK of ~8.0 and is thus a good candidate for interaction with cytoplasmic ascorbate. The low-potential heme of AdCytb is found to have a pK of ~10.5, making it an unlikely candidate for accepting a proton at physiological pH. UV-vis spectroscopy reveals an additional proton acceptor group in AdCytb with a pK of ~6.5 that is not observed by the other two techniques; whether it plays a role in the mechanism of AdCytb is unknown. We incorporate these results into an updated mechanism of AdCytb reduction predicated on the high-potential heme's localization on the cytoplasmic interface of the chromaffin granule membrane.

Functional and structural roles of residues in the third extramembrane segment of adrenal cytochrome b561.

Several residues in the third extramembrane segment (EM3) of adrenal cytochrome b(561) have been proposed to be involved in this cytochrome's interaction with ascorbate, but there has been no systematic evaluation of residues in the segment. We used alanine scanning mutagenesis to assess the functional and structural roles of the EM3 residues and several adjacent residues (residues 70-85) in the bovine cytochrome. Each alanine mutant was expressed in a bacterial system, solubilized with detergent, and affinity-purified. The recombinant proteins contained approximately two hemes per monomer and, except for R74A, retained basic functionality (≥ 94% reduced by 20 mM ascorbate). Equilibrium spectrophotometric titrations with ascorbate were used to analyze the α-band line shape and amplitude during reduction of the high- and low-potential heme centers (b(H) and b(L), respectively) and the midpoint ascorbate concentrations for the b(H) and b(L) transitions (C(H) and C(L), respectively). Y73A and K85A markedly narrowed the b(H) α-band peak; other mutants had weaker effects or no effect on b(H) or b(L) spectra. Relative changes in C(H) for the mutants were larger than changes in C(L), with 1.5-2.9-fold increases in C(H) for L70A, L71A, Y73A, R74A, N78A, and K85A. The amounts of functional b(H) and b(L) centers in additional Arg74 mutants, assessed by ascorbate titration and EPR spectroscopy, declined in concert in the following order: wild type > R74K > R74Q > R74T and R74Y > R74E. The results of this first comprehensive experimental test of the proposed roles of EM3 residues have identified residues with a direct or indirect impact on ascorbate interactions, on the environment of the b(H) heme center, and on formation of the native b(H)-b(L) unit. Surprisingly, no individual EM3 residue was by itself indispensable for the interaction with ascorbate, and the role of the segment appears to be more subtle than previously thought. These results also support our topological model of the adrenal cytochrome, which positions b(H) near the cytoplasmic side of the membrane.

Ligand trapping by cytochrome c oxidase: implications for gating at the catalytic center.

Cytochrome c oxidase is a member of the heme-copper family of oxygen reductases in which electron transfer is linked to the pumping of protons across the membrane. Neither the redox center(s) associated with proton pumping nor the pumping mechanism presumably common to all heme-copper oxidases has been established. A possible conformational coupling between the catalytic center (Fe(a3)(3+)-Cu(B)(2+)) and a protein site has been identified earlier from ligand binding studies, whereas a structural change initiated by azide binding to the protein has been proposed to facilitate the access of cyanide to the catalytic center of the oxidized bovine enzyme. Here we show that cytochrome oxidase pretreated with a low concentration of azide exhibits a significant increase in the apparent rate of cyanide binding relative to that of free enzyme. However, this increase in rate does not reflect a conformational change enhancing the rapid formation of a Fe(a3)(3+)-CN-Cu(B)(2+) complex. Instead the cyanide-induced transition of a preformed Fe(a3)(3+)-N(3)-Cu(B)(2+) to the ternary complex of Fe(a3)(3+)-N(3) Cu(B)(2+)-CN is the most likely reason for the observed acceleration. Significantly, the slow rate of azide release from the ternary complex indicates that cyanide ligated to Cu(B) blocks a channel between the catalytic site and the solvent. The results suggest that there is a pathway that originates at Cu(B) and that, during catalysis, ligands present at this copper center control access to the iron of heme a(3) from the bulk medium.

The effects of lubrication on the static frictional resistance of orthodontic brackets.

BACKGROUND: Difficulties are experienced with the collection and storage of freshly harvested human saliva to use as a lubricant for the laboratory testing of the frictional resistance of orthodontic brackets. In order to overcome these difficulties, researchers have suggested the use of saliva substitutes due to their ease of storage and consistency of properties throughout testing. Others have criticized the use of artificial saliva and prefer the dry state. The present study aimed to compare the effects of human saliva and an artificial saliva (Saliva Orthana) with the dry state for the static frictional resistance testing of orthodontic brackets. METHODS: The static frictional resistance and the lubrication effect of human saliva, Saliva Orthana and the dry state were investigated using upper central incisor stainless steel brackets and 0.019 x 0.025 inch stainless steel wires in an Instron Universal Testing Machine. Static frictional resistance was measured 100 times for each lubrication state. The 'wettability' of each lubricant was determined by measuring the contact angle against a stainless steel surface using the CAM 200 Optical Contact Angle Meter. Distilled water acted as a control. The viscosity of each lubricant and their Newtonian or non-Newtonian fluid behaviour under stress was measured using a Brookfield Digital Rheometer Model DV-III+. RESULTS: The differences in static frictional resistance between the three lubricants when examined as a group did not reach statistical significance (p = 0.059). The difference between human saliva and Saliva Orthana was considered to be of weak statistical significance and clinical relevance (Means: 0.917 N; 0.819 N: p = 0.053). Human saliva and the dry state revealed very similar mean frictional values (Means: 0.917 N; 0.875 N: p = 0.932). The contact angle tests indicated a statistically significant difference between the lubricants with Saliva Orthana having the smallest angle and therefore the highest 'wettability'. Human saliva had the highest initial viscosity and behaved as a non-Newtonian fluid, contrasting with Saliva Orthana and distilled water, both of which behaved as Newtonian fluids. CONCLUSION: The current results indicate that artificial saliva is not an ideal alternative to human saliva for friction testing in the laboratory The results therefore support the proposal that, when human saliva is not available, it may be preferable to test orthodontic frictional resistance in the dry state.

Crystal structure of rat haem oxygenase-1 in complex with ferrous verdohaem: presence of a hydrogen-bond network on the distal side.

HO (haem oxygenase) catalyses the degradation of haem to biliverdin, CO and ferrous iron via three successive oxygenation reactions, i.e. haem to alpha-hydroxyhaem, alpha-hydroxyhaem to alpha-verdohaem and alpha-verdohaem to ferric biliverdin-iron chelate. In the present study, we determined the crystal structure of ferrous alpha-verdohaem-rat HO-1 complex at 2.2 A (1 A=0.1 nm) resolution. The overall structure of the verdohaem complex was similar to that of the haem complex. Water or OH- was co-ordinated to the verdohaem iron as a distal ligand. A hydrogen-bond network consisting of water molecules and several amino acid residues was observed at the distal side of verdohaem. Such a hydrogen-bond network was conserved in the structures of rat HO-1 complexes with haem and with the ferric biliverdin-iron chelate. This hydrogen-bond network may act as a proton donor to form an activated oxygen intermediate, probably a ferric hydroperoxide species, in the degradation of alpha-verdohaem to ferric biliverdin-iron chelate similar to that seen in the first oxygenation step.

His92 and His110 selectively affect different heme centers of adrenal cytochrome b(561).

Adrenal cytochrome b(561) (cyt b(561)), a transmembrane protein that shuttles reducing equivalents derived from ascorbate, has two heme centers with distinct spectroscopic signals and reactivity towards ascorbate. The His54/His122 and His88/His161 pairs furnish axial ligands for the hemes, but additional amino acid residues contributing to the heme centers have not been identified. A computational model of human cyt b(561) (Bashtovyy, D., Berczi, A., Asard, H., and Pali, T. (2003) Protoplasma 221, 31-40) predicts that His92 is near the His88/His161 heme and that His110 abuts the His54/His122 heme. We tested these predictions by analyzing the effects of mutations at His92 or His110 on the spectroscopic and functional properties. Wild type cytochrome and mutants with substitutions in other histidine residues or in Asn78 were used for comparison. The largest lineshape changes in the optical absorbance spectrum of the high-potential (b(H)) peak were seen with mutation of His92; the largest changes in the low-potential (b(L)) peak lineshape were observed with mutation of His110. In the EPR spectra, mutation of His92 shifted the position of the g=3.1 signal (b(H)) but not the g=3.7 signal (b(L)). In reductive titrations with ascorbate, mutations in His92 produced the largest increase in the midpoint for the b(H) transition; mutations in His110 produced the largest decreases in DeltaA(561) for the b(L) transition. These results indicate that His92 can be considered part of the b(H) heme center, and His110 part of the b(L) heme center, in adrenal cyt b(561).

Mass spectrometric identification of lysine residues of heme oxygenase-1 that are involved in its interaction with NADPH-cytochrome P450 reductase.

The lysine residues of rat heme oxygenase-1 (HO-1) were acetylated by acetic anhydride in the absence and presence of NADPH-cytochrome P450 reductase (CPR) or biliverdin reductase (BVR). Nine acetylated peptides were identified by MALDI-TOF mass spectrometry in the tryptic fragments obtained from HO-1 acetylated without the reductases (referred to as the fully acetylated HO-1). The presence of CPR prevented HO-1 from acetylation of lysine residues, Lys-149 and Lys-153, located in the F-helix. The heme degradation activity of the fully acetylated HO-1 in the NADPH/CPR-supported system was significantly reduced, whereas almost no inactivation was detected in HO-1 in the presence of CPR, which prevented acetylation of Lys-149 and Lys-153. On the other hand, the presence of BVR showed no protective effect on the acetylation of HO-1. The interaction of HO-1 with CPR or BVR is discussed based on the acetylation pattern and on molecular modeling.

Electrochemical reduction of ferrous alpha-verdoheme in complex with heme oxygenase-1.

The heme oxygenase (HO) reaction consists of three successive oxygenation reactions, i.e. heme to alpha-hydroxyheme, alpha-hydroxyheme to verdoheme, and verdoheme to biliverdin-iron chelate. Of these, the least understood step is the conversion of verdoheme to biliverdin-iron chelate. For the cleavage of the oxaporphyrin ring of ferrous verdoheme, involvement of a verdoheme pi-neutral radical has been proposed. To probe this hypothetical mechanism in the HO reaction, we performed electrochemical reduction of ferrous verdoheme complexed with rat HO-1 under anaerobic conditions. On the basis of the electrochemical spectral changes, the midpoint potential for the one-electron reduction of the oxaporphyrin ring of ferrous verdoheme was found to be -0.47+/-0.01 V vs the normal hydrogen electrode (NHE). Because this potential is far lower than those of both flavins of NADPH-cytochrome P450 reductase, and of NADPH, it is concluded that the one-electron reduction of the oxaporphyrin ring of ferrous verdoheme is unlikely to occur and that the formation of the pi-neutral radical cannot be the initial step in the degradation of verdoheme by HO. Rather, it appears more reasonable to consider an alternative mechanism in which binding of O(2) to the ferrous iron of verdoheme is the first step in the degradation of verdoheme.

Implications of ligand binding studies for the catalytic mechanism of cytochrome c oxidase.

The reaction of oxidized bovine heart cytochrome c oxidase (CcO) with one equivalent of hydrogen peroxide results in the formation of two spectrally distinct species. The yield of these two forms is controlled by the ionization of a group with a pK(a) of 6.6. At basic pH, where this group is deprotonated, an intermediate called P dominates (P, because it was initially believed to be a peroxy compound). At acidic pH where the group is protonated, a different species, called F (ferryl intermediate) is obtained. We previously proposed that the only difference between these two species is the presence of one proton in the catalytic center of F that is absent in P. It is now suggested that the catalytic center of this F form has the same redox and protonation state as a second ferryl intermediate produced at basic pH by two equivalents of hydrogen peroxide; the role of the second equivalent of H(2)O(2) is that of a proton donor in the conversion of P to F. Two chloride-binding sites have been detected in oxidized CcO. One site is located at the binuclear center; the second site was identified from the sensitivity of g=3 signal of cytochrome a to chloride in the EPR spectra of oxidized CcO. Turnover of CcO releases chloride from the catalytic center into the medium probably by one of the hydrophobic channels, proposed for oxygen access, with an orientation parallel to the membrane plane. Chloride in the binuclear center is most likely not involved in CcO catalysis. The influence of the second chloride site upon several reactions of CcO has been assessed. No correlation was found between chloride binding to the second site and the reactions that were examined.

FTIR studies of internal proton transfer reactions linked to inter-heme electron transfer in bovine cytochrome c oxidase.

FTIR difference spectroscopy is used to reveal changes in the internal structure and amino acid protonation states of bovine cytochrome c oxidase (CcO) that occur upon photolysis of the CO adduct of the two-electron reduced (mixed valence, MV) and four-electron reduced (fully reduced, FR) forms of the enzyme. FTIR difference spectra were obtained in D(2)O (pH 6-9.3) between the MV-CO adduct (heme a(3) and Cu(B) reduced; heme a and Cu(A) oxidized) and a photostationary state in which the MV-CO enzyme is photodissociated under constant illumination. In the photostationary state, part of the enzyme population has heme a(3) oxidized and heme a reduced. In MV-CO, the frequency of the stretch mode of CO bound to ferrous heme a(3) decreases from 1965.3 cm(-1) at pH*

Magnesium coated phosphate glass fibers for unidirectional reinforcement of polycaprolactone composites.

Bioresorbable composites have shown much potential for bone repair applications, as they have the ability to degrade completely over time and their degradation and mechanical properties can be tailored to suit the end application. In this study, phosphate glass fiber (from the system 45% P2 O5-16% CaO-24% MgO-11% Na2 O-4% Fe2 O3 (given in mol%)) were used to reinforce polycaprolactone (PCL) with approximately 20% fiber volume fraction. The glass fiber surfaces were coated with magnesium (Mg) through magnetron sputtering to improve the fiber-matrix interfacial properties. The Mg coating provided a rough fiber surface (roughness (Ra) of about 44nm). Both noncoated and Mg-coated fiber-reinforced composites were assessed. The water uptake and mass loss properties for the composites were assessed in phosphate-buffered saline (PBS) at 37°C for up to 28 days, and ion release profiles were also investigated in both water and PBS media. Inhibition of media influx was observed for the Mg-coated composites. The composite mechanical properties were characterized on the basis of both tensile and flexural tests and their retention in PBS media at 37°C was also investigated. A higher retention of the mechanical properties was observed for the Mg-coated composites over the 28 days degradation period.

Composition of the heme centers in chromaffin granule cytochrome b(561).

Electron paramagnetic resonance and circular dichroism spectra of cytochrome b(561) from chromaffin granule membranes indicated a 2:1 stoichiometry of high- and low-potential hemes. Recombinant bovine cytochrome b(561) expressed in a baculovirus system retained native spectroscopic characteristics and represents a promising model for further study.

Hydroxylamine and hydrazine bind directly to the heme iron of the heme-heme oxygenase-1 complex.

We investigated whether or not hydroxylamine (HA) and hydrazine (HZ) interact with heme bound to heme oxygenase-1. Anaerobic addition of either HA or HZ to the ferric heme-enzyme complex produced a low-spin heme species. Titration studies at different pHs revealed that the neutral form of each of HA and HZ selectively binds to the heme with dissociation constants of 9.8 and 1.8 mM, respectively. Electron spin resonance analysis suggested that the nitrogen atom of each amine is coordinated to the ferric heme iron. With a concentrated solution of the heme-enzyme complex, however, another species of HA binding appeared, in which the oxygen atom of HA is coordinated to the iron. This species showed an unusual low-spin signal which is similar to that of the ferric hydroperoxide species in the heme oxygenase reaction.

Mechanical, degradation and cytocompatibility properties of magnesium coated phosphate glass fibre reinforced polycaprolactone composites.

Retention of mechanical properties of phosphate glass fibre reinforced degradable polyesters such as polycaprolactone and polylactic acid in aqueous media has been shown to be strongly influenced by the integrity of the fibre/polymer interface. A previous study utilising 'single fibre' fragmentation tests found that coating with magnesium improved the fibre and matrix interfacial shear strength. Therefore, the aim of this study was to investigate the effects of a magnesium coating on the manufacture and characterisation of a random chopped fibre reinforced polycaprolactone composite. Short chopped strand non-woven phosphate glass fibre mats were sputter coated with degradable magnesium to manufacture phosphate glass fibre/polycaprolactone composites. The degradation behaviour (water uptake, mass loss and pH change of the media) of these polycaprolactone composites as well as of pure polycaprolactone was investigated in phosphate buffered saline. The Mg coated fibre reinforced composites revealed less water uptake and mass loss during degradation compared to the non-coated composites. The cations released were also explored and a lower ion release profile for all three cations investigated (namely Na(+), Mg(2+) and Ca(2+)) was seen for the Mg coated composite samples. An increase of 17% in tensile strength and 47% in tensile modulus was obtained for the Mg coated composite samples. Both flexural and tensile properties were investigated and a higher retention of mechanical properties was obtained for the Mg coated fibre reinforced composite samples up to 10 days immersion in PBS. Cytocompatibility study showed both composite samples (coated and non-coated) had good cytocompatibility with human osteosarcoma cell line.

Reduction of oxaporphyrin ring of CO-bound α-verdoheme complexed with heme oxygenase-1 by NADPH-cytochrome P450 reductase.

Heme oxygenase (HO) catalyses the degradation of heme to biliverdin, carbon monoxide (CO) and ferrous iron via three successive monooxygenase reactions, using electrons provided by NADPH-cytochrome P450 reductase (CPR) and oxygen molecules. For cleavage of the oxaporphyrin ring of ferrous α-verdoheme, an intermediate in the HO reaction, involvement of a verdoheme π-neutral radical has been proposed. To explore this hypothetical mechanism, we performed electrochemical reduction of ferrous α-verdoheme-rat HO-1 complex under anaerobic conditions. Upon binding of CO, an O(2) surrogate, the midpoint potential for one-electron reduction of the oxaporphyrin ring of ferrous α-verdoheme was increased from -0.465 to -0.392 V vs the normal hydrogen electrode. Because the latter potential is close to that of the semiquinone/reduced redox couple of FAD in CPR, the one-electron reduction of the oxaporphyrin ring of CO-bound verdoheme complexed with HO-1 is considered to be a thermodynamically likely process. Indeed the one-electron reduced species, [Fe(II)(verdoheme•)], was observed spectroscopically in the presence of CO in both NADPH/wild-type and FMN-depleted CPR systems under anaerobic conditions. Under physiological conditions, therefore, it is possible that O(2) initially binds to the ferrous iron of α-verdoheme in its complex with HO-1 and an electron is subsequently transferred from CPR, probably via FAD, to the oxaporphyrin ring.

Structural comparisons of arachidonic acid-induced radicals formed by prostaglandin H synthase-1 and -2.

Cyclooxygenase catalysis by prostaglandin H synthase (PGHS)-1 and -2 involves reaction of a peroxide-induced Tyr385 radical with arachidonic acid (AA) to form an AA radical that reacts with O(2). The potential for isomeric AA radicals and formation of an alternate tyrosyl radical at Tyr504 complicate analysis of radical intermediates. We compared the EPR spectra of PGHS-1 and -2 reacted with peroxide and AA or specifically deuterated AA in anaerobic, single-turnover experiments. With peroxide-treated PGHS-2, the carbon-centered radical observed after AA addition was consistently a pentadienyl radical; a variable wide-singlet (WS) contribution from mixture of Tyr385 and Tyr504 radicals was also present. Analogous reactions with PGHS-1 produced EPR signals consistent with varying proportions of pentadienyl and tyrosyl radicals, and two additional EPR signals. One, insensitive to oxygen exposure, is the narrow singlet tyrosyl radical with clear hyperfine features found previously in inhibitor-pretreated PGHS-1. The second type of EPR signal is a narrow singlet lacking detailed hyperfine features that disappeared upon oxygen exposure. This signal was previously ascribed to an allyl radical, but high field EPR analysis indicated that ~90% of the signal originates from a novel tyrosyl radical, with a small contribution from a carbon-centered species. The radical kinetics could be resolved by global analysis of EPR spectra of samples trapped at various times during anaerobic reaction of PGHS-1 with a mixture of peroxide and AA. The improved understanding of the dynamics of AA and tyrosyl radicals in PGHS-1 and -2 will be useful for elucidating details of the cyclooxygenase mechanism, particularly the H-transfer between tyrosyl radical and AA.

Development of remineralizing, antibacterial dental materials.

Light curable methacrylate dental monomers containing reactive calcium phosphate filler (monocalcium phosphate monohydrate (MCPM) with particle diameter of 29 or 90microm) and beta-tricalcium phosphate (beta-TCP) at 1:1 weight ratio in a powder:liquid ratio (PLR) of 1:1 or 3:1 and chlorhexidine diacetate (0 or 5 wt.%), were investigated. Upon light exposure, approximately 90% monomer conversion was gained irrespective of the formulation. Increasing the PLR promoted water sorption by the set material, induced expansion and enhanced calcium, phosphate and chlorhexidine release. Concomitantly, a decline in compressive and biaxial flexural strengths occurred. With a reduction in MCPM particle diameter, however, calcium and phosphate release was reduced and less deterioration in strength observed. After 24h, the remaining MCPM had reacted with water and beta-TCP, forming, within the set materials, brushite of lower solubility. This provided a novel means to control water sorption, component release and strength properties. Measurable chlorhexidine release was observed for 6weeks. Both diffusion rate and total percentage of chlorhexidine release decreased with lowering PLR or by adding buffer to the storage solutions. Higher chlorhexidine release was associated with reduced bacterial growth on agar plates and in a biofilm fermenter. In cell growth media, brushite and hydroxyapatite crystals precipitated on the composite material surfaces. Cells spread on both these crystals and the exposed polymer composite surfaces, indicating their cell compatibility. These formulations could be suitable antibacterial, biocompatible and remineralizing dental adhesives/liners.

An ex vivo laboratory study to determine the static frictional resistance of a variable ligation orthodontic bracket system.

OBJECTIVE: To determine the effects of static frictional resistance on varying the ligation technique in a Delta Force bracket system (Ortho Organizers Ltd, Hampton, UK) and using increasing degrees of bracket/archwire angulation to simulate binding. DESIGN: An ex vivo laboratory investigation using the Instron Universal Testing Machine (Instron Ltd, High Wycombe, UK) to generate sliding forces on an archwire through the Delta Force bracket. The system was lubricated with Saliva Orthana artificial saliva (Nycomed Ltd, Buckinghamshire, UK). SETTING: Biomaterials Laboratory, Eastman Dental Institute, London, UK. MATERIALS AND METHOD: Ninety Delta Force brackets were tested against 0.018-inch stainless steel wire. Three modes of ligation were tested with three different angulations: 0, 5 and 10 degrees to simulate increasing levels of binding. RESULTS: The average static frictional resistance went from 0.20 N, at 0 degrees angulation and minimum ligation, to 2.37 N with 10 degrees angulation and maximum ligation. Results revealed that the ligation pattern was found to be highly statistically significant (P<0.001) in influencing frictional force. The binding angle showed a trend of increasing frictional force with increasing bracket/archwire angulation. Repeatability testing showed no evidence of bias (P=0.171). CONCLUSIONS: These results suggest that the Delta Force variable ligation system does in fact enable friction to be varied, which may have implications in clinical application.