Citrulline ureidase (CTU, EC3.5.1.20) degrades citrulline into ornithine, carbon dioxide, and ammonia. Here, we present the report on expression of recombinant CTU in Escherichia coli. The soluble and active recombinant CTU was expressed in the periplasmic space with the vector pET-22b and the His-tagged CTU was purified with Ni-Affinity Chromatography. The yield of soluble recombinant protein was significantly increased when 1% sorbitol was supplemented in medium. By using phenylisothiocyanate (PITC) pre-column derivatization HPLC, the enzyme activity of recombinant CTU was determined via measuring of the substrate citrulline and the corresponding products. Our results could be useful in the study of CTU biochemical characteristics, enzymatic preparation of ornithine and development of an enzymatic detection method of citrulline.
- MeSH
- Bacteria MeSH
- Bacteriological Techniques methods utilization MeSH
- Chromatography, Affinity methods utilization MeSH
- Citrulline * isolation & purification metabolism MeSH
- Enzyme Assays methods utilization MeSH
- Escherichia coli enzymology immunology isolation & purification MeSH
- Francisella tularensis * enzymology isolation & purification metabolism MeSH
- Hydrolases isolation & purification MeSH
- Ornithine * isolation & purification metabolism MeSH
- Recombinant Proteins genetics isolation & purification metabolism MeSH
- Sorbitol diagnostic use MeSH
- Statistics as Topic MeSH
- Tularemia diagnosis etiology microbiology MeSH
- Urease isolation & purification metabolism MeSH
- Chromatography, High Pressure Liquid methods utilization MeSH
- Publication type
- Research Support, Non-U.S. Gov't MeSH
The genus Bothrops is responsible for approximately 90% of snakebites in Brazil. In the present study biomarkers of oxidative stress (OS) were evaluated in the blood of victims of snakebites from Bothrops jararaca and Bothrops jararacussu. Patient monitoring started from the emergency entrance at the hospital up to 30 days, groups divided as follows: time 0 (t0), 24 hours (t24h), 7 days (t7d) and 30 days (t30d). The activities of catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx), glutathione reductase (GR), glutathione S-transferase (GST), and myeloperoxidase (MPO), as well as the contents of reduced glutathione (GSH), vitamin E, lipid peroxidation (TBARS), protein carbonyls (PC) were examined in blood. Initial determinations revealed increased CAT, GR and GPx activities and decreased SOD and GST activities together with the depletion of GSH contents, while markers of oxidative damage showed increased TBARS levels and decreased PC concentrations in victims of snakebite compared to controls (blood donors). Regarding the temporal effect, no statistical differences among the groups were detected for the distinct parameters analyzed. The responses obtained in OS biomarkers in victims of snakebite compared to healthy subjects indicate that Bothrops envenomation promoted a pronounced and persistent systemic OS in the blood of those subjects.
- MeSH
- Antioxidants therapeutic use MeSH
- Biomedical Research methods MeSH
- Bothrops MeSH
- Enzyme Assays methods utilization MeSH
- Glutathione immunology isolation & purification blood MeSH
- Protein Carbonylation immunology drug effects MeSH
- Humans MeSH
- Oxidative Stress * immunology drug effects MeSH
- Lipid Peroxidation immunology drug effects MeSH
- Statistics as Topic MeSH
- Case-Control Studies MeSH
- Snake Bites * MeSH
- Vitamin E immunology isolation & purification blood MeSH
- Inflammation * enzymology immunology MeSH
- Check Tag
- Humans MeSH
- Publication type
- Research Support, Non-U.S. Gov't MeSH
- Geographicals
- Brazil MeSH
- MeSH
- Blood Group Antigens isolation & purification MeSH
- Anemia, Hemolytic, Autoimmune diagnosis etiology MeSH
- Blood Transfusion, Autologous methods utilization MeSH
- Blood Donors MeSH
- Diagnostic Techniques and Procedures standards utilization MeSH
- Enzyme Assays methods utilization MeSH
- Erythrocytes MeSH
- Hematologic Tests methods standards utilization MeSH
- Humans MeSH
- Blood Specimen Collection MeSH
- Polymerase Chain Reaction methods utilization MeSH
- Reference Standards MeSH
- Quality Control MeSH
- Societies, Medical MeSH
- Statistics as Topic MeSH
- Check Tag
- Humans MeSH
- Publication type
- Practice Guideline MeSH
- MeSH
- Chromatography history methods utilization MeSH
- Enzyme Assays methods utilization MeSH
- Phenylalanine isolation & purification metabolism MeSH
- Phenylketonuria, Maternal * diagnosis genetics MeSH
- Genetic Testing MeSH
- Pregnancy Complications diagnosis MeSH
- Humans MeSH
- Mass Screening * MeSH
- Prenatal Diagnosis MeSH
- Statistics as Topic MeSH
- Amino Acid Metabolism, Inborn Errors * diagnosis pathology MeSH
- Check Tag
- Humans MeSH
- MeSH
- Chromatography MeSH
- Cystathionine metabolism MeSH
- Cystine metabolism MeSH
- Enzyme Assays methods utilization MeSH
- Phenylalanine isolation & purification blood MeSH
- Phenylketonurias * diagnosis MeSH
- Histidine metabolism MeSH
- Homocystinuria * blood metabolism urine MeSH
- Aminoisobutyric Acids blood metabolism urine MeSH
- Leucine metabolism MeSH
- Humans MeSH
- Methionine metabolism MeSH
- Mass Screening MeSH
- Proline metabolism MeSH
- Statistics as Topic MeSH
- Tyrosinemias * blood metabolism MeSH
- Valine blood metabolism MeSH
- Amino Acid Metabolism, Inborn Errors diagnosis pathology prevention & control MeSH
- Check Tag
- Humans MeSH
- Publication type
- Review MeSH
