Hydroxyl Group Of Tyrosine Residue Research Articles

In situ green synthesis and characterization of sericin-silver nanoparticle composite with effective antibacterial activity and good biocompatibility

Silver nanoparticle has been widely applied to a variety of fields for its outstanding antimicrobial activity. However, the stability of silver nanoparticle limits its application under certain conditions. Thus, improving the stability of silver nanoparticle via biosynthesis is a promising shortcut to expand its application. Sericin from silkworm cocoon has good hydrophilicity, reaction activity, biocompatibility and biodegradability. In this study, we developed a novel, simple, one-step biosynthesis method to prepare sericin-silver nanoparticle composite in situ in solution. Sericin served as the reductant of silver ion, the dispersant and stabilizer of the prepared sericin-silver nanoparticle composite. Natural light was the only power source used to catalyze the synthesis of silver nanoparticle in situ in solution. The novel sericin-silver nanoparticle composite was characterized by ultraviolet-visible and fluorescence spectroscopy, X-ray diffraction, transmission electron microscopy and fourier transform infrared spectroscopy. The results showed silver nanoparticle could be synthesized through the reduction of AgNO3 by the phenolic hydroxyl group of tyrosine residues of sericin under the catalysis of natural light. The synthesized silver nanoparticle had good crystalline, size distribution and long-term stability at room temperature. Light irradiation was essential for the preparation of sericin-silver nanoparticle composite. The antibacterial activity assay showed 25mg/L and 100mg/L were the minimum concentrations of sericin-silver nanoparticle composite required to inhibit the growth of Staphylococcus aureus and kill this bacterium, respectively. The cytotoxicity assay showed cell viability and cell growth were almost not affected by sericin-silver nanoparticle composite under the concentration of 25mg/L. Our study suggested the preparation of sericin-silver nanoparticle composite was environmentally friendly and energy conservation, and the prepared sericin-silver nanoparticle composite had long-term stability, effective antibacterial activity and good biocompatibility. This novel sericin-silver nanoparticle composite has shown great potentials for biomedical application such as antibacterial agent and wound care.

The Unexpected and Exceptionally Facile Chemical Modification of the Phenolic Hydroxyl Group of Tyrosine by Polyhalogenated Quinones under Physiological Conditions.

The phenolic hydroxyl group of tyrosine residue plays a crucial role in the structure and function of many proteins. However, little study has been reported about its modification by chemical agents under physiological conditions. In this study, we found, unexpectedly, that the phenolic hydroxyl group of tyrosine can be rapidly and efficiently modified by tetrafluoro-1,4-benzoquinone and other polyhalogenated quinones, which are the major genotoxic and carcinogenic quinoid metabolites of polyhalogenated aromatic compounds. The modification was found to be mainly due to the formation of a variety of fluoroquinone-O-tyrosine conjugates and their hydroxylated derivatives via nucleophilic substitution pathway. Analogous modifications were observed for tyrosine-containing peptides. Further studies showed that the blockade of the reactive phenolic hydroxyl group of tyrosine in the substrate peptide, even by very low concentration of tetrafluoro-1,4-benzoquinone, can prevent the kinase catalyzed tyrosine phosphorylation. This is the first report showing the exceptionally facile chemical modification of the phenolic hydroxyl group of tyrosine by polyhalogenated quinones under normal physiological conditions, which may have potential biological and toxicological implications.

Mechanisms of catalytic action and chemical modifications of endonucleases WEN1 and WEN2 from wheat seedlings

Hydrolysis of DNA catalyzed by wheat endonucleases WEN1 and WEN2 is pronouncedly processive. A correlation has been revealed between appearance of new products of DNA hydrolysis with different length and conformational changes in the enzymes. The first conformational conversion of the endonucleases is associated with appearance of large fragments of DNA hydrolysis with length longer than 500 bp, and the second conversion is associated with formation of oligonucleotides with length of 120-140 bp, and the third conversion is associated with formation of short oligonucleotides and mononucleotides. Formation of the DNA-enzyme complex is accompanied by appearance of fluorescence at λ = 410-440 nm. The intensity, positions, and numbers of maximums of the fluorescence spectra of DNA-WEN1 and DNA-WEN2 complexes are different and depend on the methylation status of the DNA and on the presence of Mg2+. The endonucleases hydrolyze DNA by two mechanisms: one is metal-independent, and the other depends on one or two Mg2+ ions. One Mg2+ ion is located inside the catalytic center of WEN1, whereas the WEN2 center contains two Mg2+ ions. The first (site-specific) stage of DNA hydrolysis does not depend on Mg2+. Mg2+ ions evoke changes in the site specificity of the endonuclease action (WEN1) and abolish their ability to recognize the methylation status of DNA. Products of DNA hydrolysis by endonucleases WEN1 and WEN2 in the presence of Mg2+ are similar in length (120-140 bp). The endonucleases have at least two centers (domains) - catalytic and substrate-binding. Two histidine and apparently two lysine plus two dicarboxylic amino acid residues are present inside the catalytic center of WEN1. The catalytic center of WEN2 contains at least one histidine residue and apparently two residues of aspartic or glutamic acid, which are involved in coordination of the metal ions. The catalytic centers of WEN1 and WEN2 seem to be formed, respectively, by HD/E(D/EK)KH and HD/ED/E amino acid residues. The site-specificity of the endonuclease action is due to the DNA-binding domain. This domain contains dicarboxylic amino acid residues, which seem to be responsible for sensitivity of the enzymes to the methylation status of DNA. The hydroxyl groups of tyrosine residues in the enzymes also seem to contribute to recognizing methylated bases in DNA.

Current Perspectives on Sunitinib Targeted Therapy for Tumors

This review highlights therapeutic agents from recent cancer therapeutic trials showing the greatest potential for further clinical use for sunitinib in the near future. In fact, sunitinib is one of multi-tyrosine kinase inhibitors; tyrosine kinases are enzymes, which transfer phosphate groups from ATP to the hydroxyl group of tyrosine residues on signal transduction molecules. Phosphorylation of signal transduction molecules, in turn, induces dramatic changes in tumor growth, including activation of angiogenesis and DNA synthesis. Therefore, sustain efforts have been directed for developing inhibitors for angiogenesis, which is the marginal process for tumor growth and development through targeting TKs. Almost if not all angiogenesis inhibitors target the vascular endothelial growth factor (VEGF) signaling pathway.

Selective Enrichment and Mass Spectrometric Identification of Nitrated Peptides Using Fluorinated Carbon Tags

Protein tyrosine nitration (PTN) is a post-translational modification that is related to several acute or chronic diseases. PTN introduces a nitro group in the ortho position of the phenolic hydroxyl group of tyrosine residues. PTN has been shown to be involved in the pathogenesis of inflammatory responses, cancers, and neurodegenerative and age-related disorders. Furthermore, it has been proposed that PTN regulates signal cascades related to nitric oxide (NO·) production and NO-mediated processes. Although nitrated proteins as markers of oxidative stress are confirmed by immunological assays in various affected cells or tissues, it is not known how many different types of proteins in living cells are nitrated. Since protein nitration is a low-abundance post-translational modification, development of an effective enrichment method for nitrated proteins is needed to detect nitrated peptides or proteins from the limited amount of pathophysiological samples. In the present study, we developed an enrichment method using specific chemical tagging. Nitroproteome profiling using chemical tagging and mass spectrometry was validated by model proteins. Furthermore, we successfully identified numerous nitrated proteins from the Huh7 human hepatoma cell line.

Purification and characterization of a novel biosurfactant produced by Bacillus licheniformis MS3

The physical properties and chemical structure of a new biosurfactant (licheniformin) produced by Bacillus licheniformis MS3 were investigated. The purified biosurfactant was identified as a lipopeptide with amino acid sequence of Gly, Ala, Val, Asp, Ser, Gly, Tyr and a lactone linkage between the carboxyl group of Aspargine and hydroxyl group of Tyrosine residue. The fatty acid moiety was attached to N-terminal amino acid residue through an amide bond. The purified licheniformin could lower the surface tension of water from 72 to 38 mN/m at concentrations higher than 15 μg/mL and its relative emulsion volume (EV%) was equal to 36%. It also showed stable surface activity over a wide range of temperature (45–85°C) and pH (3–11).

Mass Spectrometric Kinetic Analysis of Human Tyrosylprotein Sulfotransferase-1 and −2

Protein tyrosine O-sulfation, a widespread post-translational modification, is mediated by two Golgi enzymes, tyrosylprotein sulfotransferase-1 and -2. These enzymes catalyze the transfer of sulfate from the universal sulfate donor 3'-phosphoadenosine-5'-phosphosulfate (PAPS) to the hydroxyl group of tyrosine residues to form tyrosine O-sulfate ester and PAP. More than 60 proteins have been identified to be tyrosine sulfated including several G protein-coupled receptors, such as CC-chemokine receptor 8 (CCR8) that is implicated in allergic inflammation, asthma, and atherogenesis. However, the kinetic properties of purified tyrosylprotein sulfotransferase (TPST)-1 and -2 have not been previously reported. Moreover, currently there is no available quantitative TPST assay that can directly monitor individual sulfation of a series of tyrosine residues, which is present in most known substrates. We chose an MS-approach to address this limitation. In this study, a liquid chromatography electrospray ionisation mass spectrometry (LC/ESI-MS)-based TPST assay was developed to determine the kinetic parameters of individual TPSTs and a mixture of both isozymes using CCR8 peptides as substrates that have three tyrosine residues in series. Our method can differentiate between mono- and disulfated products, and our results show that the K(m,app) for the monosulfated substrate was 5-fold less than the nonsulfated substrate. The development of this method is the initial step in the investigation of kinetic parameters of the sequential tyrosine sulfation of chemokine receptors by TPSTs and in determining its catalytic mechanism.

Identification of surrogate markers for determining drug activity using proteomics.

In a high proportion of human carcinomas overexpression of the EGFR (epidermal growth factor receptor), a receptor tyrosine kinase, represents a potential target for cancer treatment. EGFR is induced to dimerize through ligand binding which activates the tyrosine kinase activity of the receptor. This catalyses the transfer of ATP's gamma-phosphate to hydroxyl groups of tyrosine residues on the receptor, creating binding sites that recruit downstream signalling proteins. New drugs, SMTKIs (small-molecule tyrosine kinase inhibitors), have been designed to inhibit the tyrosine kinase activity of the receptor, producing an anti-tumour effect. The development of surrogate markers to determine the drug activity of these new inhibitors would be of great benefit in drug evaluation and in the subsequent management of patient disease. This review describes current treatments of cancer using tyrosine kinase inhibitors and the use of proteomic analysis to identify possible markers of activity of these new drugs.

Characterization and use of monoclonal antibodies for isolation of phosphotyrosyl proteins from retrovirus-transformed cells and growth factor-stimulated cells.

Protein kinases that phosphorylate the hydroxyl group of tyrosine residues of proteins have been implicated in cell transformation by some retroviruses and in regulation of normal cell growth by some polypeptide growth factors. To facilitate the identification of tyrosine kinase substrates, we developed monoclonal antibodies to the hapten azobenzylphosphonate. One of these antibodies, MA-2G8, proved to be especially attractive in that it bound a derivative of aminophenylphosphate, a close phosphotyrosine analog, with higher affinity than it bound the corresponding derivative of aminobenzylphosphonate; however, its affinity for phosphoserine was negligible. In this paper we describe the optimal conditions for using this antibody to isolate phosphotyrosine proteins, emphasizing particularly that its interaction with phosphotyrosyl proteins is sensitive to ionic detergents and to antibody density on the immunosorbent matrix. The antibody also bound ATP citrate lyase; this enzyme lacks phosphotyrosine but contains phosphohistidine, which is similar structurally to phosphotyrosine. By attaching the antibody at high density to Sepharose beads and omitting ionic detergents from the buffers, it was possible by microbatch immunoadsorption (followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) to isolate the 120,000-dalton transforming protein and several other phosphotyrosyl proteins from cells transformed by Abelson murine leukemia virus. Under the same conditions, phosphotyrosyl proteins were also isolated from human epidermal carcinoma cells (A431) that had been stimulated with epidermal growth factor; most prominent among these proteins was the 170,000-dalton receptor for epidermal growth factor.

Characterization and use of monoclonal antibodies for isolation of phosphotyrosyl proteins from retrovirus-transformed cells and growth factor-stimulated cells.

Protein kinases that phosphorylate the hydroxyl group of tyrosine residues of proteins have been implicated in cell transformation by some retroviruses and in regulation of normal cell growth by some polypeptide growth factors. To facilitate the identification of tyrosine kinase substrates, we developed monoclonal antibodies to the hapten azobenzylphosphonate. One of these antibodies, MA-2G8, proved to be especially attractive in that it bound a derivative of aminophenylphosphate, a close phosphotyrosine analog, with higher affinity than it bound the corresponding derivative of aminobenzylphosphonate; however, its affinity for phosphoserine was negligible. In this paper we describe the optimal conditions for using this antibody to isolate phosphotyrosine proteins, emphasizing particularly that its interaction with phosphotyrosyl proteins is sensitive to ionic detergents and to antibody density on the immunosorbent matrix. The antibody also bound ATP citrate lyase; this enzyme lacks phosphotyrosine but contains phosphohistidine, which is similar structurally to phosphotyrosine. By attaching the antibody at high density to Sepharose beads and omitting ionic detergents from the buffers, it was possible by microbatch immunoadsorption (followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) to isolate the 120,000-dalton transforming protein and several other phosphotyrosyl proteins from cells transformed by Abelson murine leukemia virus. Under the same conditions, phosphotyrosyl proteins were also isolated from human epidermal carcinoma cells (A431) that had been stimulated with epidermal growth factor; most prominent among these proteins was the 170,000-dalton receptor for epidermal growth factor.

Radioimmunoassay of dermorphin-like peptides in mammalian and non-mammalian tissues

A selective RIA for D-Ala 2-Dermorphin (Der), a natural peptide extracted from amphibian skin, has been developed using an antibody raised in rabbits against Der which has been coupled to BSA through its phenolic hydroxyl groups of tyrosine residues with 2,4-Dichloro-6-methoxy-1,3,5-triazine. The cross-reactivity of this antibody with dermorphin analogs, C- and N-terminal fragments of dermorphin molecule, some opioid and gastrointestinal peptides was tested. Der-like immunoreactivity has been identified in tissue extracts of rats, frog and cephalopoda. Der-like peptides were purified by passing methanol extracts of the tissues through a Sephadex G 25 column ( 16×100 cm ) eluted with 0.1 M acetic acid at 4°C. Der-like immunoreactivity from neural tissue of Dosidicus gigas, Eledone moscata, and rat brain showed a good agreement with an authentic sample of synthetic dermorphin.

Protected natural peptides as intermediates for preparing semisynthetic peptides and protein analogs Selective acylation of ϵ-amino groups in cyanogen bromide peptides of cytochrome c using azidoformates

Treatment of CNBr peptides 66–80, 81–104 and 66–104 from cytochrome c with t-butyloxycarbonyl azide leads to selective acylation of the ϵ-amino groups of lysine residues and the phenolic hydroxyl groups of tyrosine residues with less than 25% acylation of the α-amino groups. Similar selectivity was obtained for reactions of benzyloxycarbonyl azide, p-nitrobenzyloxycarbonyl azide and p-methoxybenzyloxycarbonyl azide with peptide 81–104. All of these protective groups can be removed under mild conditions, and thus, the partially protected natural peptides are desirable intermediates for the preparation of semisynthetic peptides. Model condensation reactions of N α t-butyloxycarbonyl methionine N-hydroxysuccinimide ester with Z-protected peptide 81–104 produced a peptide corresponding to residues 80 to 104 of cytochrome c in 94% yield.

Photooxidation of bovine neurophysin II in the presence of rose bengal.

In order to elucidate the role of some amino acid residues in bovine neurophysin II (NP-II) for its binding ability for oxytocin and vasopressin, NP-II has been photooxidized in the presence of rose bengal and oxygen, and the relationships between the loss of hormonesbinding ability of modified protein and the destruction of photosusceptible amino acids were examined. Photooxidation of NP-II causes rapid oxidation of single methionine residue to corresponding sulphoxide followed by a slow destruction of single tyrosine residue. More prolonged irradiation causes also slight decomposition of cystine residues. The hormonesbinding ability of NP-II is almost completely retained even when the methionine residue was completely photooxidized, but is gradually decreased with the progress of the photodegradation of the tyrosine residue. The decrease in binding ability of the photooxidized protein proceeds almost identically for oxytocin and [8-arginine] vasopressin as the ligand. The binding ability is decreased to about 70% of the original when 80% of tyrosine were degraded and to about 30% of the original when the tyrosine was completely photooxidized and about 6% of cystine residues were degraded. The influence of the destruction of tyrosine residue for the loss of hormones-binding ability of protein seemed to be amplified by the subsequent photooxidation of cystine residues since there is no direct correlation between the photodestruction of cystine residues and the decrease in the binding ability. The pH binding profiles of photooxidized NP-II are found to be essentially identical with those of native protein, indicating the non-essential role of phenolic hydroxyl group of tyrosine residue of NP-II in hormones-binding process. O-Acetylation of the tyrosine residue of NP-II with N-acetylimidazole gives no significant effect on the binding ability for oxytocin or [8-arginine] vasopressin. These findings suggest that the single methionine residue of NP-II has no contribution to the binding process, while the single tyrosine residue, particularly its aromatic ring, of NP-II may participate with the binding process of the protein to both oxytocin and vasopressin with similar contribution.

The pH Jump Study of Enzyme Proteins

Rapid conformational changes due to pH jump were studied kinetically at 25 degrees mainly by the stopped-flow method using liquefying alpha-amylase from Bacillus subtilis [EC 3.2.1-.1, liquefying]. First, the conformational change due to a pH jump produced by mixing with alkali was monitored as a function of time at 245 nm through the ionization of phenolic hydroxyl groups of tyrosine residues which were originally buried and finally become exposed due to the pH jump. Three distinct phases of conformational change were clearly recognized by this method by varying the final pH values. Each phase involved the exposure of an essentially definite number of tyrosine residues, whose rate constant was crucially dependent on pH. Second, these phases of conformational change were subjected to examination in terms of the optical rotation change at 411 nm and the reversibility upon reverse pH jump with respect to conformational reconstitution, as observed through the protonation ofphenolic hydroxyl groups of ionized tyrosine residues and the enzyme activity. The first phase, which occurs above pH 12.5, involves no change in the optical rotation and is reversible as observed by the above two monitoring methods. In contrast, the other two phases, which are observed above pH 12.7, are accompanied by an optical rotation change and no appreciable reversibility was detected by these methods.

On the action of carboxypeptidase a on ester substrates in alkaline solution

Summary Kinetic measurements on the pH dependency of the rate parameter k cat /K M for the Mn ++ -carboxypeptidase A δ -catalyzed hydrolysis of O -( trans -cinnamoyl)-L-β-phenyllactate have shown that an ionizing function in the enzyme with a pK a value of 9. 3 is involved in this reaction. Since experiments on the acetylation of the native γ-form of the enzyme appear to indicate that the phenolic hydroxyl groups of tyrosine residues in carboxypeptidase do not play a crucial role in the enzymatic catalysis of the hydrolysis of O -( trans -cinnamoyl)-L-β-phenyllactate, it is suggested that the group of pK a = 9. 3 is probably the active site Mn ++ -H 2 O complex.

Studies on chemically modified cytochrome c. I. The acetylated cytochrome c.

When cytochrome cpurified from bovine heart muscle was acetylated with acetic anhydride under specific conditions, the acetylated cytochrome cpreparations were obtained, in which 1, 2 and 3 acetyl groups had incorporated to the ɛ-amino groups of lysine residues. They were purified chromatographically to homogeneous states. With these preparations it was found that the enzymatic activity of cytochrome cin the cytochrome oxidase [EC 1.9.3.1] reaction decreased gradually and linearly with the decrease in its basicity. The acetylation of 6 ɛ-amino groups in cytochrome cmolecule led the protein to complete loss of the activity. From the amino acid analysis of these preparations of the acetylated cytochrome c, it was found that the 22nd lysine residue from the amino terminus of the protein was most reactive toward acetic anhydride. When cytochrome cwas treated with a large excess amount of acetic anhydride in the absence of sodium acetate, not only its ɛ-amino group of lysine residue but also the hydroxyl group of tyrosine residue was acetylated completely. With this preparation, a drastic change was observed in the visible and ultraviolet absorption spectra of the oxidized form, while that of the reduced form remained unchanged. The heme of this preparation combined with CO and oxygen. It was suggested that the heme moiety of this preparation had less interaction with protein moiety than that of the native one.

STUDIES ON THE CHEMICAL MODIFICATION OF SILK

Deterioration and yellowing of silk fibroin were hitherto controlled by methylation with diazomethane, but in this paper it is shown that the shrinkages of silk by neutral salts and mineral acid solutions may be remarkably diminished. The causes of the shrinkage control of silk by methylation with diazomethane are also discussed on the bases of our experiments and many data attributing to the methylation of phenolic hydroxyl groups of tyrosine residues are proposed. Tyrosine residues of silk fibroin are, it is concluded, responsible for the shrinkage phenomena of silk by salts and acids; this conclusion coincides with the preceding study by R. Murase.