Radiolytic Oxidation of Two Inverse Dipeptides, Methionine-Valine and Valine-Methionine: A Joint Experimental and Computational Study.

The two inverse peptides methionine-valine (Met-Val) and valine-methionine (Val-Met) are investigated in an oxidative radiolysis process in water. The OH radical yields products with very different absorption spectra and concentration effects: Met-Val yields one main product with a band at about 400 nm and other products at higher energies; there is no concentration effect. Val-Met yields at least three products, with a striking concentration effect. Molecular simulations are performed with a combination of the Monte Carlo, density functional theory, and reaction field methods. The simulation of the possible transients enables an interpretation of the radiolysis: (1) Met-Val undergoes an H atom uptake leaving mainly a neutral radical with a 2-center-3-electron (2c-3e) SN bond, which cannot dimerize. Other radicals are present at higher energies. (2) Val-Met undergoes mainly an electron uptake leaving a cation monomer with a (2c-3e) SO bond and a cation dimer with a (2c-3e) SS bond. At higher energies, neutral radicals are possible. This cation monomer can transfer a proton toward a neutral peptide, leaving a neutral radical.

Covalent and Oriented Surface Immobilization of Antibody Using Photoactivatable Antibody Fc-Binding Protein Expressed in Escherichia coli.

Fc-specific antibody binding proteins (FcBPs) with the minimal domain of protein G are widely used for immobilization of well-oriented antibodies onto solid surfaces, but the noncovalently bound antibodies to FcBPs are unstable in sera containing large amounts of antibodies. Here we report novel photoactivatable FcBPs with photomethionine (pMet) expressed in E. coli, which induce Fc-specific photo-cross-linking with antibodies upon UV irradiation. Unfortunately, pMet did not support protein expression in the native E. coli system, and therefore we also developed an engineered methionyl tRNA synthetase (MRS5m). Coexpression of MRS5m proteins successfully induced photoactivatable FcBP overexpression in methionine-auxotroph E. coli cells. The photoactivatable FcBPs could be easily immobilized on beads and slides via their N-terminal cysteine residues and 6xHis tag. The antibodies photo-cross-linked onto the photoactivatable FcBP-beads were resistant from serum-antibody mediated dissociation and efficiently captured antigens in human sera. Furthermore, photo-cross-linked antibody arrays prepared using this system allowed sensitive detection of antigens in human sera by sandwich immunoassay. The photoactivatable FcBPs will be widely applicable for well-oriented antibody immobilization on various surfaces of microfluidic chips, glass slides, and nanobeads, which are required for development of sensitive immunosensors.

Effects of gamma-ray-induced free radicals on the metal content and amino acid composition of human metallothionein-1.

Metallothioneins (MTs), a low-mass class of metalloproteins, are characterized by a high thiolate sulphur and metal content. MTs are involved in metal homeostasis and heavy metal detoxification, and are efficient scavengers of free radicals. This article describes zinc release from human MT-1 and modification of its amino acid composition when subjected to free radicals generated during gamma ray radiolysis. The effect of gamma ray radiolysis of untreated and metal-depleted human MT-1 was tested under multiple aerobic and anaerobic conditions at increasing irradiation doses. Under all conditions, a rapid increase of serine in the early stages of irradiation was observed. Irradiation for longer times led to cysteic acid formation, except under argon atmosphere. Several other amino acid concentrations gradually decreased. Formation of limited amounts of hydroxyproline, hydroxylysine and ornithine as well as some less common derivatives such as cystathionine occurred as side-effects.

X-ray spectroscopic approaches to the investigation and characterization of photochemical processes.

Despite a wealth of studies exemplifying the utility of the 2-5 keV X-ray range in speciation and electronic structure elucidation, the exploitation of this energy regime for the study of photochemical processes has not been forthcoming. Herein, a new endstation set-up for in situ photochemical soft X-ray spectroscopy in the 2-5 keV energy region at the Stanford Synchrotron Radiation Lightsource is described for continuous photolysis under anaerobic conditions at both cryogenic and ambient temperatures. Representative examples of this approach are used to demonstrate the potential information content in several fields of study, including organometallic chemistry, biochemistry and materials chemistry.

Radiation-induced oxidative damage to the DNA-binding domain of the lactose repressor.

Understanding the cellular effects of radiation-induced oxidation requires the unravelling of key molecular events, particularly damage to proteins with important cellular functions. The Escherichia coli lactose operon is a classical model of gene regulation systems. Its functional mechanism involves the specific binding of a protein, the repressor, to a specific DNA sequence, the operator. We have shown previously that upon irradiation with gamma-rays in solution, the repressor loses its ability to bind the operator. Water radiolysis generates hydroxyl radicals (OH* radicals) which attack the protein. Damage of the repressor DNA-binding domain, called the headpiece, is most likely to be responsible of this loss of function. Using CD, fluorescence spectroscopy and a combination of proteolytic cleavage with MS, we have examined the state of the irradiated headpiece. CD measurements revealed a dose-dependent conformational change involving metastable intermediate states. Fluorescence measurements showed a gradual degradation of tyrosine residues. MS was used to count the number of oxidations in different regions of the headpiece and to narrow down the parts of the sequence bearing oxidized residues. By calculating the relative probabilities of reaction of each amino acid with OH. radicals, we can predict the most probable oxidation targets. By comparing the experimental results with the predictions we conclude that Tyr7, Tyr12, Tyr17, Met42 and Tyr47 are the most likely hotspots of oxidation. The loss of repressor function is thus correlated with chemical modifications and conformational changes of the headpiece.

Photo-leucine and photo-methionine allow identification of protein-protein interactions in living cells.

Protein-protein interactions are the key to organizing cellular processes in space and time. The only direct way to identify such interactions in their cellular environment is by photo-cross-linking. Here we present a new strategy for photo-cross-linking proteins in living cells. We designed two new photoactivatable amino acids that we termed photo-methionine and photo-leucine based on their structures and properties closely resembling the natural amino acids methionine and leucine, respectively. This similarity allows them to escape the stringent identity control mechanisms during protein synthesis and be incorporated into proteins by the unmodified mammalian translation machinery. Activation by ultraviolet light induces covalent cross-linking of the interacting proteins, which can be detected with high specificity by simple western blotting. Applying this technology to membrane protein complexes, we discovered a previously unknown direct interaction of the progesterone-binding membrane protein PGRMC1 with Insig-1, a key regulator of cholesterol homeostasis.

Photofragmentation of C,N-protected alpha-amino acids: comparing tert-leucine with sulfur-containing amino acids methionine and cysteine.

The photochemical fingerprint for the N-acetyl methyl ester of the aliphatic amino acid tert-leucine 1 was investigated. This reaction path was compared with the electron transfer active amino acids methionine (N-acetyl methyl ester derivative 2a as well as the methyl ester derivative 2b) and the cysteine derivatives 3a and 3b (penicillamine derivative). Photofragmenation of the ester group dominated the photolysis of 1, whereas loss of methylmercaptane was observed for 2a and 3a. Vinylglycine derivatives 11 and 14 were formed to a minor extent. The gem-dimethylated compound 3b gave a solvent-characteristic product pattern with photoelimination products 18 and 19 in acetonitrile and loss of methylmercaptane to give 20 in methanol.

Efficient sensitized photooxygenation in water by a porphyrin-cyclodextrin supramolecular complex.

[reaction: see text] The 2:1 inclusion complex between (2,3,6-tri-O-methyl)-beta-cyclodextrin (TMbetaCD) and 5,10,15,20-tetrakis(4-sulfonatophenyl)-porphyrin (TPPS(4)) behaves as a supramolecular sensitizer in water providing photooxygenation with turnover numbers up to 30,000 with a very minor sensitizer bleaching (<10%). The protocol, which employs only 4 equiv of the cyclodextrin additive with respect to the porphyrin sensitizer (5 x 10(-7) M), leads to high yield oxidation of model biomolecules such as l-methionine methyl ester and uracil and is also effective for phenol degradation in aqueous solution.

Photo-oxidation of methionine-containing peptides by the 4-carboxybenzophenone triplet state in aqueous solution. Competition between intramolecular two-centered three-electron bonded (S...S)+ and (S...N)+ formation.

Quantum yields for the formation of transients were measured following the quenching of triplet 4-carboxy-benzophenone (3CB*) by methionine-containing peptides in aqueous solutions. Ketyl radicals (CBH.), ketyl radical anions (CB.-) and various sulfur radical cations were identified following the triplet-quenching events. The presence of these intermediates indicated that the triplet-quenching mechanism can be characterized as mainly electron-transfer in nature. The quenching rate constants were of the order of 2 x 10(9) M-1 s-1. There were small, but significant, differences in the triplet-quenching rate constants, and these trends indicate the existence of multiple sulfur targets in the quenchers. The absorption of the transient products was followed in detail by using spectral-resolution analysis. From the absorption data, quantum yields were estimated for the formation of the various transients. There were differences found in the yields of the transient products between the experiments, where the quenchers were the "mixed" stereoisomers of methionylmethionine (L,D and D,L) and experiments where the quenchers were L,L and D,D stereoisomers. Triplet-quenching data from several other methionine-containing small oligopeptides were analyzed in an analogous manner. Systematic variations were observed, and these patterns were discussed in terms of competitive donation of protons to the CB.- within the charge-transfer complex. The competition was between protons on carbons adjacent to the sulfur-radical center and protons on the protonated amino groups of the radical cation. In addition, there was a competition between the two intramolecular two-centered, three-electron bonded species (S therefore S)+ and (S therefore N)+ that play roles in the secondary kinetics.

Reduction of selected oligopeptides containing methionine induced by hydrated electrons.

Bimolecular rate constants for the reaction of the hydrated electron with zwitterionic forms of several linear oligopeptides containing methionine were determined using the pulse radiolysis technique. The rate constants were found to vary in the range of (0.2-1.2) x 10(9) M(-1) s(-1). The reactivity of the peptides toward e(aq)- is governed by the pKa of the N-terminal amino group and the number of peptide bonds. At a fixed number of peptide bonds, the reactivity increases with the pKa, and for a given N-terminal amino acid residue, it increases with the number of peptide bonds. For the linear peptides the observed transient spectra are assigned to deaminated oligopeptide radicals, *CHRCONH approximately, obtained due to rapid deamination of the corresponding electron adducts formed initially. The radicals derived from oligopeptides containing methionine at the N-terminus absorb at approximately 400 nm with extinction coefficients of approximately 1300+/-150 M(-1) cm(-1). Their absorption maxima are shifted hypsochromically by approximately 30 nm with respect to those derived from oligopeptides with glycine at the N-terminus. The e(aq)- reacts with cyclic Met-Met, k = 2.0 x 10(9) M(-1) s(-1), probably by addition to the carbonyl bond, forming an adduct absorbing below 250 nm with epsilon250 = 2300+/-250 M(-1) cm(-1).

Influence of DL methionine and sodium metabisulphite on the photostability of vitamin k1.

On exposure to UV light at 254 nm in a borosilicate glass actinometer, vitamin k1 decomposed rapidly with a degradation rate constant of 7.63 day-1. Under UV irradiation, DL methionine and sodium metabisulphite were found to have some photostabilizing effect for vitamin k1 formulations. The degradation rates were reduced by 43.4% and 60.4% in the presence of 0.1% w/v of DL methionine and 0.2% w/v of sodium metabisuphite, respectively. When vitamin k1 formulations were stored at room temperature in clear glass bottles and exposed to the weaker light source of both room fluorescent light and natural daylight, vitamin k1 decomposed at a much slower degradation rate constant of 0.31 day-1. Despite the slower degradation rate, DL methionine and sodium metabisuphite failed to protect vitamin k1 from degradation on exposure to this weaker light source. When vitamin k1 was stored in white plastic bottles under the same testing conditions, the photo-protective effect of these photostabilizers reappeared. The protection provided extended the shelf-life from one day to 2.5 days. When amber glass bottles replaced white plastic bottles as storage containers, vitamin k1 was found stable on an open bench for at least 30 days. Such stability was also observed when vitamin k1 was stored in a refrigerator in either white plastic bottles or amber bottles. Findings in this study showed that package and storage conditions that shield the products from light are still the most efficient ways to maintain the photostability of vitamin k1. However, photostabilizers such as DL methionine and sodium metabisulphite may provide additional protection to vitamin k1 when it is stored in less effective light-resistant containers.

Oxidation of human insulin-like growth factor I in formulation studies. 3. Factorial experiments of the effects of ferric ions, EDTA, and visible light on methionine oxidation and covalent aggregation in aqueous solution.

The influence of ferric ions, EDTA, and visible light on the oxidation of methionine and the covalent reducible and nonreducible dimerization in human Insulin-like Growth Factor I (hIGF-I) in aqueous (1 mM) phosphate buffer solution were studied. A reduced factorial experiment with two levels of each factor was used. Regression models for the three responses were constructed with partial least square (PLS) analysis. The hIGF-I variants were quantified by reversed-phase high-performance liquid chromatography (RP-HPLC), gel filtration, and reduced sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The formation of the hIGF-I variants in aqueous solution at pH 6.1 exhibited different dependencies on the variables studied. The oxidation of methionine was affected mainly by visible light and the combination of 10 ppm ferric ions and 20 ppm EDTA, whereas ferric ions alone had no significant effect. The covalent dimerization of hIGF-I was correlated to visible light and ferric ions. The interaction effects of ferric ions with either visible light or EDTA were also significant on the dimerization rates. Both reducible and nonreducible soluble covalent dimers were formed, with the reducible dimer being the most prominent. The oxidation of methionine 59 in hIGF-I is catalyzed by light and by ferric ions in combination with EDTA. The covalent dimerization of hIGF-I is mainly affected by light and by ferric ions. Both reducible and nonreducible dimerization increased by oxidative conditions. Human IGF-I appears to dimerize covalently by both disulfide scrambling and by a radical-promoted nondisulfide pathway. EDTA is necessary for ferric ions to be active in the oxidation of methionine in hIGF-I but not for the covalent dimerization.

Photolysis (193 nm) of aliphatic dipeptides in aqueous solution.

The photodecomposition of four aliphatic dipeptides containing valine and methionine was studied in neutral solution upon excitation at 193nm. The quantum yields of ionization and decomposition in Ar-saturated aqueous solution are phi(e)- = 0.06-0.16 and phi(d) = 0.06-0.16, respectively. Phi(d) is enhanced (0.08-0.28) under N2O, where the hydrated electrons are converted into OH radicals. A comparison of the phi(d) values of dipeptides with those of monomeric amino acids indicates efficient decomposition of the peptide bond (bond scission).

[The effect of gamma radiation on the methionine and tryptophan content of soy protein products]. / Einfluss der Gamma-Bestrahlung auf den Methionin- und Tryptophangehalt in Sojaproteinprodukten.

Effect of gamma irradiation on the content of methionine and tryptophan in soya protein products were investigated. Irradiation with doses of 1, 3 and 5 kGy caused significant (p = 0.05) decrease of methionine contents. Parallel with the decrease in methionine also the relative quality of proteins is lowered. Tryptophan content were significantly reduced (p = 0.05) at irradiation doses of 3 and 5 kGy. The relative decrease of methionine and tryptophan contents are significantly correlated (p = 0.05) with relative increase in amount of products by the irradiation induced oxidation of lipids; correlation coefficients were 0.576 and 0.715.

Application of methionine as a detector molecule for the assessment of oxygen radical generation by human neutrophils and endothelial cells.

Diverse cell types can generate reactive oxygen species (ROS) which are implicated in many disease processes and are ascribed both beneficial and deleterious roles. In vitro studies of this phenomenon indicate that properties of the microenvironment in culture influence the cells' behaviour with regard to ROS generation in vivo. To date, however, the assessment of cellular ROS generation has been limited to techniques which are invasive of the culture environment, or require cells to be in suspension. This study describes the application of NMR spectroscopy to the detection of ROS generation, a technique which is non-invasive of the cell culturing environment.

Formation of three-electron bonds in one-electron oxidized methionine dipeptides: a pulse radiolytic study.

One electron oxidation of methionine dipeptides (Met-X and X-Met, where X = Gly or Ser) was carried out using the pulse radiolysis technique. It was apparent that the mode of oxidative action of OH radicals on methionine dipeptides was governed by the sequence of amino acids. Spectral evidence suggests that an intramolecular three-electron bond between nitrogen and sulphur atoms is not formed if these two atoms are separated by a peptide bond.

OH radical-induced crosslinks of methionine peptides.

Reactions of radiation-generated OH radicals with methionine (Met) and its homopeptides, L-Met-L-Met and tri-L-Met, were investigated through reaction products. Samples of irradiated Met and HCl-hydrolysates of its irradiated homopeptides were trimethylsilylated and analysed by capillary gas chromatography-mass spectrometry. Mass spectra taken revealed the formation of three products, 2-amino-4-(methyldithio)butanoic acid, 4,4'-thiobis(2-aminobutanoic acid) and homocystine. G values of these products were determined to be 0.10, 0.16 and 0.30, respectively.

Assessment of electron irradiation damage to biomolecules by electron diffraction and electron energy-loss spectroscopy.

Electron radiation damage is one of the most severe problems in high resolution electron microscopy by biomolecules. The techniques of electron diffraction and electron energy-loss spectroscopy were applied to gain a better understanding of radiation damage in amino acids and nucleic acid bases. The results when compared with G-values for the release of ammonia and hydrogen sulphide from amino acids seem to indicate that bond scission is an important cause of radiation damage at moderate doses of irradiation. High resolution structural disorder in nucleic acid bases was found to involve loss of atoms peripheral to the main ring structure.