Horse Heart Myoglobin Research Articles

Reductive nitrosylation of ferric cyanide horse heart myoglobin is limited by cyanide dissociation

Cyanide binds to ferric heme-proteins with a very high affinity, reflecting the very low dissociation rate constant ( k off). Since no techniques are available to estimate k off, we report herewith a method to determine k off based on the irreversible reductive nitrosylation reaction to trap ferric myoglobin (Mb(III)). The k off value for cyanide dissociation from ferric cyanide horse heart myoglobin (Mb(III)–cyanide) was determined at pH 9.2 and 20.0 °C. Mixing Mb(III)–cyanide and NO solutions brings about absorption spectral changes reflecting the disappearance of Mb(III)–cyanide with the concomitant formation of ferrous nitrosylated Mb. Since kinetics of reductive nitrosylation of Mb(III) is much faster than Mb(III)–cyanide dissociation, the k off value, representing the rate-limiting step, can be directly determined. The k off value obtained experimentally matches very well to that calculated from values of the second-order rate constant ( k on) and of the dissociation equilibrium constant ( K) for cyanide binding to Mb(III) ( k off = k on × K).

Time Resolved Thermodynamic Studies of Ligand Binding/release to Sol-Gel Encapsulated Horse Heart Myoglobin

The successful confinement of proteins in solid state materials allows for a multitude of applications in the study of protein dynamics, as well as, advances in biotechnologies. The importance of bulk solvent and restricted space on the conformational dynamics of proteins can be identified by encapsulating the biomolecules (peptides, proteins, enzymezes, etc) in environments where both can be regulated. In addition, the affect of encapsulation on ligand binding and preservation of reactivity once encapsulated are of equal importance. With the use of Photoacoustic calorimetry (PAC) and Photothermal beam deflection (PBD) methods along with picosecond-millisecond transient absorption and fluorescence techniques we are investigating enthalpy, molar volume changes, and kinetics associated with with CO dissociation and rebinding to horse heart myoglobin encapsulated in sol-gels. Preliminary PAC data suggest that the dissociation of CO from HHMb results in four kinetic phases: <7ns, ∼125ns, ∼260ns, and ∼600ns. We will discuss the differences in molar volume and enthalpy changes associated with the conformational dynamics of these events in sol-gels relative to native protein in aqueous solution.

Direct Detection of the Oxygen Rebound Intermediates, Ferryl Mb and NO2, in the Reaction of metMyoglobin with Peroxynitrite

Oxygenated hemoproteins are known to react rapidly with nitric oxide (NO) to produce peroxynitrite (PN) at the heme site. This process could lead either to attenuation of the effects of NO or to nitrosative protein damage. Peroxynitrite is a powerful nitrating and oxidizing agent that has been implicated in a variety of cell injuries. Accordingly, it is important to delineate the nature and variety of reaction mechanisms of PN reactions with heme proteins. Here, we present direct evidence that ferrylMb and NO(2) are both produced during the reaction of PN and metmyoglobin (metMb). Kinetic evidence indicates that these products evolve from initial formation of a caged radical intermediate [Fe(IV)=O *NO(2)]. This caged pair reacts mainly via internal return with a rate constant k(r) to form metMb and nitrate in an oxygen rebound scenario. Detectable amounts of ferrylMb are observed by stopped-flow spectrophotometry, appearing at a rate consistent with the rate, k(obs), of heme-mediated PN decomposition. Freely diffusing NO(2), which is liberated concomitantly from the radical pair (k(e)), preferentially nitrates Tyr103 in horse heart myoglobin. The ratio of the rates of in-cage rebound and cage escape, k(r)/k(e), was found to be approximately 10 by examining the nitration yields of fluorescein, an external NO(2) trap. This rebound/escape model for the metMb/PN interaction is analogous to the behavior of alkyl hyponitrites and the well-studied geminate recombination processes of deoxymyoglobin with O(2), CO, and NO. The scenario is also similar to the stepwise events of substrate hydroxylation by cytochrome P450 and other oxygenases. It is likely, therefore, that the reaction of metMb with ONOO(-) and that of oxyMb with NO proceed through the same [Fe(IV)=O *NO(2)] caged radical intermediate and lead to similar outcomes. The results indicate that while oxyMb may reduce the concentration of intracellular NO, it would not eliminate the formation of NO(2) as a decomposition product of peroxynitrite.

Collision Induced Unfolding of Protein Ions in the Gas Phase Studied by Ion Mobility-Mass Spectrometry: The Effect of Ligand Binding on Conformational Stability

Ion mobility spectrometry, with subsequent mass spectrometric detection, has been employed to study the stability of compact protein conformations of FK-binding protein, hen egg-white lysozyme, and horse heart myoglobin in the presence and absence of bound ligands. Protein ions, generated by electrospray ionization from ammonium acetate buffer, were activated by collision with argon gas to induce unfolding of their compact structures. The collisional cross sections (Omega) of folded and unfolded conformations were measured in the T-Wave mobility cell of a Waters Synapt HDMS (Waters, Altrincham, UK) employing a calibration against literature values for a range of protein standards. In the absence of activation, collisional cross section measurements were found to be consistent with those predicted for folded protein structures. Under conditions of defined collisional activation energies partially unfolded conformations were produced. The degree of unfolding and dissociation induced by these defined collision energies are related to the stability of noncovalent intra- and intermolecular interactions within protein complexes. These findings highlight the additional conformational stability of protein ions in the gas phase resulting from ligand binding.

The Mechanism of Oxidative Halophenol Dehalogenation by Amphitrite ornata Dehaloperoxidase Is Initiated by H2O2 Binding and Involves Two Consecutive One-Electron Steps: Role of Ferryl Intermediates

The enzymatic globin, dehaloperoxidase (DHP), from the terebellid polychaete Amphitrite ornata is designed to catalyze the oxidative dehalogenation of halophenol substrates. In this study, the ability of DHP to catalyze this reaction by a mechanism involving two consecutive one-electron steps via the normal order of addition of the oxidant cosubstrate (H(2)O(2)) before organic substrate [2,4,6-trichlorophenol (TCP)] is demonstrated. Specifically, 1 equiv of H(2)O(2) will fully convert 1 equiv of TCP to 2,6-dichloro-1,4-benzoquinone, implicating the role of multiple ferryl [Fe(IV)O] species. A significant amount of heterolytic cleavage of the O-O bond of cumene hydroperoxide, consistent with transient formation of a Compound I [Fe(IV)O/porphyrin pi-cation radical] species, is observed upon its reaction with ferric DHP. In addition, a more stable high-valent Fe(IV)O-containing DHP intermediate [Compound II (Cpd II) or Compound ES] is characterized by UV-visible absorption and magnetic circular dichroism spectroscopy. Spectral similarities are seen between this intermediate and horse heart myoglobin Cpd II. It is also shown in single-turnover experiments that the DHP Fe(IV)O intermediate is an active oxidant in halophenol oxidative dehalogenation. Furthermore, reaction of DHP with 4-chlorophenol leads to a dimeric product. The results presented herein are consistent with a normal peroxidase order of addition of the oxidant cosubstrate (H(2)O(2)) followed by organic substrate (TCP) and indicate that the enzymatic mechanism of DHP-catalyzed oxidative halophenol dehalogenation involves two consecutive one-electron steps with a dissociable radical intermediate.

Method to Characterize the Global Dynamics of Proteins by Temperature Dependent Phosphorescence Lifetime Measurements. Application for the Allosteric Effect in Hemoglobin

At the glass transition temperature of proteins, large scale, anharmonic motions of the conformation become activated. It has been shown that in this temperature range the phosphorescence lifetime of Trp residues that is long (several s) at cryogenic temperatures become efficiently quenched. We report the elaboration of a systematic method based on this observation with the purpose to use this effect for characterizing the global conformational dynamics of proteins. In this method, special conditions were determined under which the onset temperature of phosphorescence quenching can be clearly separated from the slaving effect of fluctuations activated in the solvent, and the relevance of this temperature for the global dynamics of the protein has been verified by parallel experiments on human alpha-oxyFe-betaZn-Hemoglobin, w.t. horse heart Myoglobin and Trp mutant forms of a two-domain protein, yeast phosphoglycerate kinase. The method was used to characterize the role of global dynamics in the allosteric response of human hemoglobin to the binding of effectors as Cl, IHP, DPG, BZF. The quenching effect showed a two state behavior with a characteristic onset temperature sensitive to the presence and quality of allosteric effectors. The data points were fit by a simple two state model and the thermodynamic parameters - enthalpy and entropy changes characteristic for the quenching effect were discussed in terms of oxygen hopping models. The study on hemoglobin verifies the concept that in allosteric regulation, the global dynamics of the protein tetramer plays important role.Acknowledgements: the authors thank Prof. T. Yonetani of University of Pennsylvania for kindly providing Zn-PP- substituted human Hb for the studies. J. Fidy thank the Hung. Ministry of Health for support by grant no. ETT 512/2006.

Effect of zinc and cadmium ions on structure and function of myoglobin

Laser flash photolysis technique was used to study zinc and cadmium ion effects on bimolecular and nanosecond geminate molecular oxygen (O 2) rebinding to horse heart myoglobin. Time courses for geminate recombination are analyzed in terms of a three-step, side path model. In the presence of metal ions, the greatest changes are observed in the rate constant of the O 2 rebinding from within the primary docking site and the rate constant of the O 2 migration from the primary site to the secondary xenon docking sites. The study revealed that modulation of the myoglobin affinity for O 2 by zinc and cadmium occurs at the level of the innermost barrier controlling O 2 rebinding from within the primary docking site. Sets of the calculated rate constants provide a basis for an interpretation of metal ion effects on the myoglobin structure. Overall, the results demonstrate that the metal ions binding to myoglobin gives rise to an increase in the population of the “open” distal pocket protein conformation.

Ligand Binding to Truncated Hemoglobin N from Mycobacterium tuberculosis Is Strongly Modulated by the Interplay between the Distal Heme Pocket Residues and Internal Water

The survival of Mycobacterium tuberculosis requires detoxification of host *NO. Oxygenated Mycobacterium tuberculosis truncated hemoglobin N catalyzes the rapid oxidation of nitric oxide to innocuous nitrate with a second-order rate constant (k'(NOD) approximately 745 x 10(6) m(-1) x s(-1)), which is approximately 15-fold faster than the reaction of horse heart myoglobin. We ask what aspects of structure and/or dynamics give rise to this enhanced reactivity. A first step is to expose what controls ligand/substrate binding to the heme. We present evidence that the main barrier to ligand binding to deoxy-truncated hemoglobin N (deoxy-trHbN) is the displacement of a distal cavity water molecule, which is mainly stabilized by residue Tyr(B10) but not coordinated to the heme iron. As observed in the Tyr(B10)/Gln(E11) apolar mutants, once this kinetic barrier is lowered, CO and O(2) binding is very rapid with rates approaching 1-2 x 10(9) m(-1) x s(-1). These large values almost certainly represent the upper limit for ligand binding to a heme protein and also indicate that the iron atom in trHbN is highly reactive. Kinetic measurements on the photoproduct of the *NO derivative of met-trHbN, where both the *NO and water can be directly followed, revealed that water rebinding is quite fast (approximately 1.49 x 10(8) s(-1)) and is responsible for the low geminate yield in trHbN. Molecular dynamics simulations, performed with trHbN and its distal mutants, indicated that in the absence of a distal water molecule, ligand access to the heme iron is not hindered. They also showed that a water molecule is stabilized next to the heme iron through hydrogen-bonding with Tyr(B10) and Gln(E11).

Myoglobin Modification by Enzyme‐Generated Dopamine Reactive Species

The generation of reactive quinone species (DAQ) from oxidation of dopamine (DA) is involved in neurodegenerative pathologies like Parkinson's disease (A. Borta, G. U. Höglinger, J. Neurochem. 2007, 100, 587-595). The oxidation of DA to DAQ can occur either in a single two-electron process or in two consecutive one-electron steps, through semiquinone radicals, giving rise to different patterns of reactions. The former type of reaction can be promoted by tyrosinase, the latter by peroxidases in the presence of H(2)O(2), which can be formed under oxidative stress conditions. Both enzymes were employed for the characterization of the thiol-catechol adducts formed by reaction of DA and cysteine or glutathione, and for the identification of specific amino acid residues modified by DAQs in two representative target proteins, human and horse heart myoglobin. Our results indicate that the cysteinyl-DA adducts are formed from the same quinone intermediate independently of the mechanism of DA oxidation, and that the hallmark of a radical mechanism is the formation of the cystine dimer. The reactivity of quinone species also controls the DA-promoted derivatization of histidine residues in proteins. However, for the modification of the cysteine residue in human myoglobin, a radical intramolecular mechanism has been proposed, in which the protein acts both as the catalyst and target of the reaction. Most importantly, the modification of myoglobins through DAQ linkages, and in particular by DA oligomers, has dramatic effects on their stability, as it induces protein unfolding and incorporation into insoluble melanic precipitates.

Enrichment of phosphopeptides using bare magnetic particles

Magnetic iron(II, III) oxide (magnetite, Fe(3)O(4)) nanoparticles were used to selectively enrich phosphopeptides from tryptic digests of bovine beta-casein and from tryptic digest mixtures containing bovine beta-casein, cytochrome c, bovine serum albumin, and horse heart myoglobin. The magnetic property of the particles permits an easy and speedy enrichment process. No enrichment of phosphopeptides was observed from ferric magnetic iron(III) oxide (Fe(2)O(3)) nanoparticles. These data collectively demonstrate that the enrichment of phosphopeptides using magnetic iron(II, III) oxide nanoparticles is a practical method for the selective analysis of phosphopeptides and could be helpful in isolating and analyzing phosphorylated peptides from complex biological samples.

N-terminal labeling of proteins by the Pictet–Spengler reaction

The Pictet–Spengler reaction was applied to the N-terminal labeling of horse heart myoglobin. This was performed in the following two steps: (1) conversion of the N-terminal glycine residue to an α-keto aldehyde by a transamination reaction and (2) condensation of the resulting activated myoglobin with tryptamine analogues by the Pictet–Spengler reaction. Ultraviolet (UV)/visible (vis) absorption and circular dichroism (CD) spectral data revealed that the tertiary structure of myoglobin was not altered by the Pictet–Spengler reaction.

Interfacial electron transfer kinetics of myoglobins chemically modified with succinic anhydride at an indium oxide electrode

We modified native horse heart myoglobin (Mb or Mb F1) with succinic anhydride to introduce negative charge(s) onto the surface of the protein, and obtained four kinds of Mbs in which the modification site (i.e. local charge) was different (Mb(F2)–Mb(F5)). The chemical modification significantly affected the electron transfer kinetics at an In 2O 3 electrode in a bis–tris buffer solution (pH 6.8). The heterogeneous electron transfer rate constant ( k 0′) of Mb decreased depending on the modification site for succinic anhydride. The calculated k 0′ values were 1.8 (±0.3) × 10 −4, 1.6 (±0.3) × 10 −4, 5.5 (±0.5) × 10 −5, 4.5 (±0.5) × 10 −5 and 1.5 (±0.7) × 10 −5 cm s −1 for native Mb (or Mb(F1)), Mb(F2), Mb(F3), Mb(F4) and Mb(F5), respectively, which showed a clear reciprocal relationship to the rate of photoinduced electron transfer reactions of Zn–Mbs(F2–F5) with methyl viologen. This effect was also confirmed using cyano-forms of modified Mbs. The present results indicate that the local charge of the Mb molecule is important for its electron transfer kinetics at an electrode surface.

The Nitric Oxide Reductase Activity of Cytochrome c Nitrite Reductase from Escherichia coli

Cytochrome c nitrite reductase (NrfA) from Escherichia coli has a well established role in the respiratory reduction of nitrite to ammonium. More recently the observation that anaerobically grown E. coli nrf mutants were more sensitive to NO. than the parent strain led to the proposal that NrfA might also participate in NO. detoxification. Here we describe protein film voltammetry that presents a quantitative description of NrfA NO. reductase activity. NO. reduction is initiated at similar potentials to NrfA-catalyzed reduction of nitrite and hydroxylamine. All three activities are strongly inhibited by cyanide. Together these results suggest a common site for reduction of all three substrates as axial ligands to the lysine-coordinated NrfA heme rather than nonspecific NO. reduction at one of the four His-His coordinated hemes also present in each NrfA subunit. NO. reduction by NrfA is described by a K(m) of the order of 300 microm. The predicted turnover number of approximately 840 NO. s(-1) is much higher than that of the dedicated respiratory NO. reductases of denitrification and the flavorubredoxin and flavohemoglobin of E. coli that are also proposed to play roles in NO. detoxification. In considering the manner by which anaerobically growing E. coli might detoxify exogenously generated NO. encountered during invasion of a human host it appears that the periplasmically located NrfA should be effective in maintaining low NO. levels such that any NO. reaching the cytoplasm is efficiently removed by flavorubredoxin (K(m) approximately 0.4 microm).

Adsorption and Conformational Change of Myoglobin on Biomimetic Hydroxyapatite Nanocrystals Functionalized with Alendronate

The chemical conjugation of bisphosphonates (BPs), specifically alendronate, to hydroxyapatite could be an effective means to impart to it fine-tuned bioactivity. Horse heart myoglobin (Mb), a well-characterized protein, has been adsorbed onto biomimetic hydroxyapatite nanocrystals (nHA) and onto the nHA/alendronate conjugate powdered samples. The obtained materials have potential use in bone implantation and as prospective drug-delivery devices. The kinetic absorption of Mb onto nHA is dramatically affected by its functionalization with alendronate. The covering of the nHA surface by alendronate inhibits the adsorption of myoglobin. The adsorption mechanisms of the protein were studied by spectroscopic techniques (UV-vis and surface-enhanced Raman spectroscopy). The results indicate that the protein changes conformation upon adsorption on the inorganic substrate. In particular, the interaction with nHA alters the coordination state of the iron in the heme through the formation of a hexacoordinated low-spin Mb heme, possibly involving the distal histidine. Instead, the covering of the nHA surface by alendronate does not adsorb the protein but preserves the coordination state of the heme moiety. This study could be of significance either in the field of biomaterials science, in particular, to fine tune a bone-specific drug delivery device and to test nHA as a new support for heterogeneous catalysis, improving the understating of enzyme immobilization.

Specific electrochemical iodination of horse heart myoglobin at tyrosine 103 as determined by Fourier transform ion cyclotron resonance mass spectrometry

The iodination of proteins remains a useful tool in biochemistry for radiolabelling. However, chemical or enzymatic iodination is difficult to control and can give deleterious polyiodination. Previously, we have shown that electrooxidation with nitrite is a rapid method for the selective nitration of tyrosine residues in proteins. In principle, it should be possible to substitute a number of electrooxidisable anions into the tyrosine phenol ring.Electrochemical iodination is more difficult to control than nitration because the rapid anodic oxidation of I− leads to persistent formation of the iodinating triiodide anion. However, application of pulsed electrooxidation and reduction cycles is shown to be an effective procedure for the selective mono and double-iodination of myoglobin, which may have general application to other proteins in controlling of the level of iodination.Mono- and double-iodination of myoglobin by this method was confirmed by electrospray FT-ICR mass spectrometry. Infrared multiphoton dissociation (IRMPD) enabled localization of the site of mono-iodination to be restricted to either His97 or Tyr103. More extensive sequence coverage was obtained with electron capture dissociation (ECD), allowing unambiguous assignment of the site of iodination to Tyr103.

Identifying the site of spin trapping in proteins by a combination of liquid chromatography, ELISA, and off-line tandem mass spectrometry

An off-line mass spectrometry method that combines immuno-spin trapping and chromatographic procedures has been developed for selective detection of the nitrone spin trap 5,5-dimethyl-1-pyrroline- N-oxide (DMPO) covalently attached to proteins, an attachment which occurs only subsequent to DMPO trapping of free radicals. In this technique, the protein–DMPO nitrone adducts are digested to peptides with proteolytic agents, peptides from the enzymatic digest are separated by HPLC, and enzyme-linked immunosorbent assays (ELISA) using polyclonal anti-DMPO nitrone antiserum are used to detect the eluted HPLC fractions that contain DMPO nitrone adducts. The fractions showing positive ELISA signals are then concentrated and characterized by tandem mass spectrometry (MS/MS). This method, which constitutes the first liquid chromatography–ELISA–mass spectrometry (LC-ELISA-MS)-based strategy for selective identification of DMPO-trapped protein residues in complex peptide mixtures, facilitates location and preparative fractionation of DMPO nitrone adducts for further structural characterization. The strategy is demonstrated for human hemoglobin, horse heart myoglobin, and sperm whale myoglobin, three globin proteins known to form DMPO-trappable protein radicals on treatment with H 2O 2. The results demonstrate the power of the new experimental strategy to select DMPO-labeled peptides and identify sites of DMPO covalent attachments.

Quantitative, Multiplexed Assays for Low Abundance Proteins in Plasma by Targeted Mass Spectrometry and Stable Isotope Dilution

Biomarker discovery produces lists of candidate markers whose presence and level must be subsequently verified in serum or plasma. Verification represents a paradigm shift from unbiased discovery approaches to targeted, hypothesis-driven methods and relies upon specific, quantitative assays optimized for the selective detection of target proteins. Many protein biomarkers of clinical currency are present at or below the nanogram/milliliter range in plasma and have been inaccessible to date by MS-based methods. Using multiple reaction monitoring coupled with stable isotope dilution mass spectrometry, we describe here the development of quantitative, multiplexed assays for six proteins in plasma that achieve limits of quantitation in the 1-10 ng/ml range with percent coefficients of variation from 3 to 15% without immunoaffinity enrichment of either proteins or peptides. Sample processing methods with sufficient throughput, recovery, and reproducibility to enable robust detection and quantitation of candidate biomarker proteins were developed and optimized by addition of exogenous proteins to immunoaffinity depleted plasma from a healthy donor. Quantitative multiple reaction monitoring assays were designed and optimized for signature peptides derived from the test proteins. Based upon calibration curves using known concentrations of spiked protein in plasma, we determined that each target protein had at least one signature peptide with a limit of quantitation in the 1-10 ng/ml range and linearity typically over 2 orders of magnitude in the measurement range of interest. Limits of detection were frequently in the high picogram/milliliter range. These levels of assay performance represent up to a 1000-fold improvement compared with direct analysis of proteins in plasma by MS and were achieved by simple, robust sample processing involving abundant protein depletion and minimal fractionation by strong cation exchange chromatography at the peptide level prior to LC-multiple reaction monitoring/MS. The methods presented here provide a solid basis for developing quantitative MS-based assays of low level proteins in blood.

Exploring the Cooperativity of the Fast Folding Reaction of a Small Protein Using Pulsed Thiol Labeling and Mass Spectrometry

It has been difficult to obtain directly residue-specific information on side chain packing during a fast (ms) protein folding reaction. Such information is necessary to determine the extent to which structural changes in different parts of the protein molecule are coupled together in defining the cooperativity of the overall folding transition. In this study, structural changes occurring during the major fast folding reaction of the small protein barstar have been characterized at the level of individual residue side chains. A pulsed cysteine labeling methodology has been employed in conjunction with mass spectrometry. This provides, with ms temporal resolution, direct information on structure formation at 10 different locations in barstar during its folding. Cysteine residues located on the surface of native barstar, at four different positions, remain fully solvent-accessible throughout the folding process, indicating the absence of any ephemeral nonnative structure in which these four cysteine residues get transiently buried. For buried cysteine residues, the rates of the change in cysteine-thiol accessibility to rapid chemical labeling by the thiol reagent methyl methanethiosulfonate appear to be dependent upon the location of the cysteine residue in the protein and are different from the rate measured by the change in tryptophan fluorescence. But the rates vary over only a 3-fold range. Nevertheless, a comparison of the kinetics of the change in accessibility of the cysteine 3 thiol with those of the change in the fluorescence of tryptophan 53, as well as of their denaturant dependences, indicates that the major folding reaction comprises more than one step.

Peroxynitrite-mediated oxidation of ferrous carbonylated myoglobin is limited by carbon monoxide dissociation

Peroxynitrite-mediated oxidation of ferrous nitrosylated myoglobin (Mb(II)–NO) involves the transient ferric nitrosylated species (Mb(III)–NO), followed by NO dissociation and formation of ferric myoglobin (Mb(III)). In contrast, peroxynitrite-mediated oxidation of ferrous oxygenated myoglobin (Mb(II)–O 2) involves the transient ferrous deoxygenated and ferryl derivatives (Mb(II) and Mb(IV) O, respectively), followed by Mb(III) formation. Here, kinetics of peroxynitrite-mediated oxidation of ferrous carbonylated horse heart myoglobin (Mb(II)–CO) is reported. Values of the first-order rate constant for peroxynitrite-mediated oxidation of Mb(II)–CO ( i.e., for Mb(III) formation) and of the first-order rate constant for CO dissociation from Mb(II)–CO ( i.e., for Mb(II) formation) are h = (1.2 ± 0.2) × 10 −2 s −1 and k off(CO) = (1.4 ± 0.2) × 10 −2 s −1, respectively, at pH 7.2 and 20.0 °C. The coincidence of values of h and k off(CO) indicates that CO dissociation represents the rate limiting step of peroxynitrite-mediated oxidation of Mb(II)–CO.

Surface interaction of well‐defined, concentrated poly(2‐hydroxyethyl methacrylate) brushes with proteins

AbstractThe interaction of concentrated polymer brushes with proteins was chromatographically investigated. By the use of surface‐initiated atom transfer radical polymerization, a low‐polydispersity poly(2‐hydroxyethyl methacrylate) (PHEMA) was densely grafted onto the inner surfaces of silica monoliths with mesopores of about 50 and 80 nm in mean size. The graft density reached 0.4–0.5 chains/nm2. The 80‐nm‐mesopore monolithic column with the concentrated PHEMA brush was characterized through the elution of low‐polydispersity pullulans with different molecular weights, clearly showing two modes of size exclusion, that is, one by the mesopores and the other by the brush phase. The latter mode gave a sharp separation with a critical molecular weight (size‐exclusion limit) of about 1000. This molecular size of pullulan was comparable to the distance between the nearest‐neighbor graft points. The elution behaviors of five proteins of different sizes (bovine serum thyroglobulin, bovine serum immunoglobulin G, bovine serum albumin, horse heart myoglobin, and bovine serum aprotinin) were studied with this PHEMA‐grafted column. The smallest protein, aprotinin, with a pullulan‐reduced molecular weight slightly larger than the critical value of 1000, was eluted much behind the corresponding pullulan, and this indicated that it barely got into the brush layer, suffering from a strong affinity interaction within the brush. On the other hand, the other four larger proteins were eluted at the same elution volumes as the equivalent pullulans, and this meant that they were perfectly excluded from the brush layer and separated only in the size‐exclusion mode by the mesopores without an affinity interaction with the brush surface. This excellent inertness of the concentrated brush in the interaction with the large proteins should afford the system long‐term stability against biofouling. © 2007 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 45: 4795–4803, 2007