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Chemical & Pharmaceutical Bulletin 2022Protein bioconjugation has become an increasingly important research method for introducing artificial functions in to protein with various applications, including... (Review)
Review
Protein bioconjugation has become an increasingly important research method for introducing artificial functions in to protein with various applications, including therapeutics and biomaterials. Due to its amphiphilic nature, only a few tyrosine residues are exposed on the protein surface. Therefore, tyrosine residue has attracted attention as suitable targets for site-specific modification, and it is the most studied amino acid residue for modification reactions other than lysine and cysteine residues. In this review, we present the progress of our tyrosine chemical modification studies over the past decade. We have developed several different catalytic approaches to selectively modify tyrosine residues using peroxidase, laccase, hemin, and ruthenium photocatalysts. In addition to modifying tyrosine residues by generating radical species through single-electron transfer, we have developed a histidine modification method that utilizes singlet oxygen generated by photosensitizers. These highly reactive chemical species selectively modify proteins in close proximity to the enzyme/catalyst. Taking advantage of the spatially controllable reaction fields, we have developed novel methods for site-specific antibody modification, detecting hotspots of oxidative stress, and target identification of bioactive molecules.
Topics: Biocatalysis; Coordination Complexes; Hemin; Humans; Laccase; Molecular Structure; Peroxidase; Protein Processing, Post-Translational; Tyrosine
PubMed: 35110442
DOI: 10.1248/cpb.c21-00915 -
The American Journal of Pathology May 2015Myeloperoxidase and eosinophil peroxidase are heme-containing enzymes often physically associated with fibrotic tissue and cancer in various organs, without any direct...
Myeloperoxidase and eosinophil peroxidase are heme-containing enzymes often physically associated with fibrotic tissue and cancer in various organs, without any direct involvement in promoting fibroblast recruitment and extracellular matrix (ECM) biosynthesis at these sites. We report herein novel findings that show peroxidase enzymes possess a well-conserved profibrogenic capacity to stimulate the migration of fibroblastic cells and promote their ability to secrete collagenous proteins to generate a functional ECM both in vitro and in vivo. Mechanistic studies conducted using cultured fibroblasts show that these cells are capable of rapidly binding and internalizing both myeloperoxidase and eosinophil peroxidase. Peroxidase enzymes stimulate collagen biosynthesis at a post-translational level in a prolyl 4-hydroxylase-dependent manner that does not require ascorbic acid. This response was blocked by the irreversible myeloperoxidase inhibitor 4-amino-benzoic acid hydrazide, indicating peroxidase catalytic activity is essential for collagen biosynthesis. These results suggest that peroxidase enzymes, such as myeloperoxidase and eosinophil peroxidase, may play a fundamental role in regulating the recruitment of fibroblast and the biosynthesis of collagen ECM at sites of normal tissue repair and fibrosis, with enormous implications for many disease states where infiltrating inflammatory cells deposit peroxidases.
Topics: Animals; Blotting, Western; Cell Movement; Cells, Cultured; Collagen; Enzyme-Linked Immunosorbent Assay; Eosinophil Peroxidase; Extracellular Matrix; Female; Fibroblasts; Fluorescent Antibody Technique; Humans; Peroxidase; Rats; Rats, Sprague-Dawley; Real-Time Polymerase Chain Reaction; Sus scrofa
PubMed: 25759268
DOI: 10.1016/j.ajpath.2015.01.013 -
Yakugaku Zasshi : Journal of the... 2018The chemical labeling of proteins with synthetic probes is a key technique used in chemical biology, protein-based therapy, and material science. Much of the chemical... (Review)
Review
The chemical labeling of proteins with synthetic probes is a key technique used in chemical biology, protein-based therapy, and material science. Much of the chemical labeling of native proteins, however, depends on the labeling of lysine and cysteine residues. While those methods have significantly contributed to native protein labeling, alternative methods that can modify different amino acid residues are still required. Herein we report the development of a novel methodology of tyrosine labeling, inspired by the luminol chemiluminescence reaction. Tyrosine residues are often exposed on a protein's surface and are thus expected to be good targets for protein functionalization. In our studies so far, we have found that 1) hemin oxidatively activates luminol derivatives as a catalyst, 2) N-methyl luminol derivative specifically forms a covalent bond with a tyrosine residue among the 20 kinds of natural amino acid residues, and 3) the efficiency of tyrosine labeling with N-methyl luminol derivative is markedly improved by using horseradish peroxidase (HRP) as a catalyst. We were able to use molecular oxygen as an oxidant under HRP/NADH conditions. By using these methods, the functionalization of purified proteins was carried out. Because N-methyl luminol derivative is an excellent protein labeling reagent that responds to the activation of peroxidase, this new method is expected to open doors to such biological applications as the signal amplification of HRP-conjugated antibodies and the detection of protein association in combination with peroxidase-tag technology.
Topics: Catalysis; Heme; Hemeproteins; Luminescence; Luminol; Peroxidase; Tyrosine
PubMed: 29311464
DOI: 10.1248/yakushi.17-00186-1 -
BMC Microbiology Apr 2023Environmental contamination by phenol has been reported in both aquatic and atmospheric environments. This study aimed to separate and purify the peroxidase enzyme from...
Environmental contamination by phenol has been reported in both aquatic and atmospheric environments. This study aimed to separate and purify the peroxidase enzyme from bacteria that degrade phenol from wastewater sources. An enrichment culture of MSM was used to screen 25 bacterial isolates from different water samples for peroxidase production, six of the isolates exhibited high levels of peroxidase enzyme activity. Qualitative analysis of peroxidase revealed that isolate No. 4 had the highest halo zones (Poly-R478: 14.79 ± 0.78 mm, Azure B: 8.81 ± 0.61 mm). The promising isolate was identified as Bacillus aryabhattai B8W22 by 16S rRNA gene sequencing with accession number OP458197. As carbon and nitrogen sources, mannitol and sodium nitrate were utilized to achieve maximum peroxidase production. A 30-h incubation period was used with pH 6.0, 30 °C, mannitol, and sodium nitrate, respectively, for maximal production of peroxidase. Purified peroxidase enzyme showed 0.012 U/mg specific activity, and SDS-PAGE analysis indicated a molecular weight of 66 kDa. The purified enzyme exhibits maximum activity and thermal stability at pH values of 4.0 and 8.0, respectively, with maximum activity at 30 °C and complete thermal stability at 40 °C. In the purified enzyme, the Km value was 6.942 mg/ml and the Vmax value was 4.132 mol/ml/hr, respectively. The results demonstrated that Bacillus aryabhattai B8W22 has promising potential for degrading phenols from various phenol-polluted wastewater sources.
Topics: Phenol; Peroxidase; Wastewater; RNA, Ribosomal, 16S; Phenols; Peroxidases; Hydrogen-Ion Concentration
PubMed: 37120512
DOI: 10.1186/s12866-023-02850-9 -
Journal of the American Society of... Aug 2022
Topics: Anti-Neutrophil Cytoplasmic Antibody-Associated Vasculitis; Antibodies, Antineutrophil Cytoplasmic; Glomerulonephritis; Humans; Peroxidase; T-Lymphocytes
PubMed: 35906086
DOI: 10.1681/ASN.2022060668 -
Molecules (Basel, Switzerland) Jun 2023The sensitive and accurate determination of glyphosate (Glyp) is urgently demanded because it is closely correlated with human health and environmental safety. In this...
The sensitive and accurate determination of glyphosate (Glyp) is urgently demanded because it is closely correlated with human health and environmental safety. In this work, we proposed a sensitive and convenient colorimetric assay by employing copper ion peroxidases for the detection of Glyp in the environment. Free Cu(II) ions displayed high peroxidase activity and can catalytically oxidize the colorless 3,3',5,5'-tetramethylbenzidine (TMB) into blue oxTMB, resulting in an obviously visible discoloration reaction. Once the Glyp is added, the ability of copper ions to mimic peroxidase can be largely suppressed because of the generation of Glyp-Cu chelate. The favorable selectivity and sensitivity were demonstrated in the colorimetric analysis of Glyp. Furthermore, this rapid and sensitive method was successfully applied in the accurate and reliable determination of glyphosate in the real sample, holding promising applications in pesticide determination in the environment.
Topics: Humans; Colorimetry; Peroxidase; Copper; Oxidoreductases; Peroxidases; Ions; Hydrogen Peroxide; Glyphosate
PubMed: 37375185
DOI: 10.3390/molecules28124630 -
International Journal of Molecular... Dec 2022Implantation of scaffolds causes a local inflammatory response whereby the early recruitment of neutrophils is of great importance not only for fighting the infection,...
Implantation of scaffolds causes a local inflammatory response whereby the early recruitment of neutrophils is of great importance not only for fighting the infection, but also for facilitating effective regeneration. We used luminol-dependent chemiluminescence, flow cytometry, ELISA, and confocal microscopy to assess the responses of neutrophils after the exposure to the scaffold-decellularized bovine pericardium (collagen type I) crosslinked with genipin (DBPG). We demonstrated that DBPG activated neutrophils in whole blood causing respiratory burst, myeloperoxidase (MPO) secretion, and formation of neutrophil extracellular trap-like structures (NETs). In addition, we studied platelets, another important player of the immediate immune host response. We found that platelets triggered redox-activation of isolated neutrophils by the pericardium scaffold, and likely participate in the NETs formation. Free radicals generated by neutrophils and hypochlorous acid produced by MPO are potent oxidizing agents which can oxidatively degrade biological structures. Understanding the mechanisms and consequences of redox activation of neutrophils by pericardium scaffolds is important for the development of new approaches to increase the efficiency of tissue regeneration.
Topics: Cattle; Animals; Neutrophils; Extracellular Traps; Peroxidase; Oxidation-Reduction; Respiratory Burst; Blood Platelets
PubMed: 36555108
DOI: 10.3390/ijms232415468 -
ACS Applied Bio Materials Feb 2024In biosensor development, silk fibroin is advantageous for providing transparent, flexible, chemically/mechanically stable, biocompatible, and sustainable substrates,...
In biosensor development, silk fibroin is advantageous for providing transparent, flexible, chemically/mechanically stable, biocompatible, and sustainable substrates, where the biorecognition element remains functional for long time periods. These properties are employed here in the production of point-of-care biosensors for resource-limited regions, which are able to display glucose levels without the need for external instrumentation. These biosensors are produced by photopatterning silk films doped with the enzymes glucose oxidase and peroxidase and photoelectrochromic molecules from the dithienylethene family acting as colorimetric mediators of the enzymatic reaction. The photopatterning results from the photoisomerization of dithienylethene molecules in the silk film from its initial uncolored opened form to its pink closed one. The photoisomerization is dose-dependent, and colored patterns with increasing color intensities are obtained by increasing either the irradiation time or the light intensity. In the presence of glucose, the enzymatic cascade reaction is activated, and peroxidase selectively returns closed dithienylethene molecules to their initial uncolored state. Color disappearance in the silk film is proportional to glucose concentration and used to distinguish between hypoglycemic (below 4 mM), normoglycemic (4-6 mM), and hyperglycemic levels (above 6 mM) by visual inspection. After the measurement, the biosensor can be regenerated by irradiation with UV light, enabling up to five measurement cycles. The coupling of peroxidase activity to other oxidoreductases opens the possibility to produce long-life reusable smart biosensors for other analytes such as lactate, cholesterol, or ethanol.
Topics: Silk; Colorimetry; Peroxidases; Biosensing Techniques; Peroxidase; Glucose
PubMed: 38270977
DOI: 10.1021/acsabm.3c00872 -
Plant Physiology Apr 2023Reactive oxygen species are produced in response to pathogens and pathogen-associated molecular patterns, as exemplified by the rapid extracellular oxidative burst...
Reactive oxygen species are produced in response to pathogens and pathogen-associated molecular patterns, as exemplified by the rapid extracellular oxidative burst dependent on the NADPH oxidase isoform RESPIRATORY BURST OXIDASE HOMOLOG D (RBOHD) in Arabidopsis (Arabidopsis thaliana). We used the H2O2 biosensor roGFP2-Orp1 and the glutathione redox state biosensor GRX1-roGFP2 targeted to various organelles to reveal unsuspected oxidative events during the pattern-triggered immune response to flagellin (flg22) and after inoculation with Pseudomonas syringae. roGFP2-Orp1 was oxidized in a biphasic manner 1 and 6 h after treatment, with a more intense and faster response in the cytosol compared to chloroplasts, mitochondria, and peroxisomes. Peroxisomal and cytosolic GRX1-roGFP2 were also oxidized in a biphasic manner. Interestingly, our results suggested that bacterial effectors partially suppress the second phase of roGFP2-Orp1 oxidation in the cytosol. Pharmacological and genetic analyses indicated that the pathogen-associated molecular pattern-induced cytosolic oxidation required the BRI1-ASSOCIATED RECEPTOR KINASE (BAK1) and BOTRYTIS-INDUCED KINASE 1 (BIK1) signaling components involved in the immune response but was largely independent of NADPH oxidases RBOHD and RESPIRATORY BURST OXIDASE HOMOLOG F (RBOHF) and apoplastic peroxidases peroxidase 33 (PRX33) and peroxidase 34 (PRX34). The initial apoplastic oxidative burst measured with luminol was followed by a second oxidation burst, both of which preceded the two waves of cytosolic oxidation. In contrast to the cytosolic oxidation, these bursts were RBOHD-dependent. Our results reveal complex oxidative sources and dynamics during the pattern-triggered immune response, including that cytosolic oxidation is largely independent of the preceding extracellular oxidation events.
Topics: Arabidopsis Proteins; Peroxidase; Hydrogen Peroxide; Arabidopsis; NADPH Oxidases; Peroxidases; Plant Immunity; Mitochondria; Oxidation-Reduction; Oxidative Stress; Reactive Oxygen Species; Protein Serine-Threonine Kinases
PubMed: 36582183
DOI: 10.1093/plphys/kiac603 -
Sensors (Basel, Switzerland) Sep 2021Hypochlorous acid (HOCl) generates from the reaction between hydrogen peroxide and chloride ions via myeloperoxidase (MPO)-mediated in vivo. As very important reactive... (Review)
Review
Hypochlorous acid (HOCl) generates from the reaction between hydrogen peroxide and chloride ions via myeloperoxidase (MPO)-mediated in vivo. As very important reactive oxygen species (ROS), hypochlorous acid (HOCl)/hypochlorite (OCl) play a crucial role in a variety of physiological and pathological processes. However, excessive or misplaced production of HOCl/OCl can cause variety of tissue damage and human diseases. Therefore, rapid, sensitive, and selective detection of OCl is very important. In recent years, the fluorescent probe method for detecting hypochlorous acid has been developed rapidly due to its simple operation, low toxicity, high sensitivity, and high selectivity. In this review, the progress of recently discovered fluorescent probes for the detection of hypochlorous acid was summarized with the aim to provide useful information for further design of better fluorescent probes.
Topics: Fluorescent Dyes; Humans; Hydrogen Peroxide; Hypochlorous Acid; Peroxidase
PubMed: 34640646
DOI: 10.3390/s21196326