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Catalase Activity
Published in Robert A. Greenwald, CRC Handbook of Methods for Oxygen Radical Research, 2018
The following modification of the method of Gregory and Fridovich6 has been successfully employed in the analysis and purification of the catalase isozymes from E. coli B.2,3 Polyacrylamide gels11 (4%) are pre-electrophoresed in 0.188 M bicine-imidazole, pH 7.8, and a pH 5.5 stacking gel12 is added. Ten units of catalase activity are then applied to each gel, and samples are subjected to electrophoresis in the bicine-imidazole buffer at 2 to 4°C. Gels are removed from glass tubes at the end of the run and incubated individually for 45 min in the dark in a solution of 0.43 mg/mℓ 3,3′-diaminobenzidine·4 HCl (Sigma Chemical Co.) and 43 μg/mℓ horseradish peroxidase (Worthington Biochemical Co., HPOD grade) in 50 mM imidazole-HCl, pH 7.4. Following this incubation, gels are rinsed in deionized water and soaked in a solution of 20 mM H2O2 in the imidazole-HCl buffer at 25°C. Within minutes, the achromatic bands of catalase activity are visible against the colored brown background. Gels are again rinsed well and stored in sealed tubes containing deionized water.
Events of Tumor Progression Associated with Carcinogen Treatment of Epithelial and Fibroblast Compared with Mutagenic Events
Published in George E. Milo, Bruce C. Casto, Charles F. Shuler, Transformation of Human Epithelial Cells: Molecular and Oncogenetic Mechanisms, 2017
George E. Milo, Bruce C. Casto
Determination and quantification of carcinogen-DNA adducts were conducted using the 32P-postlabeling assay method.44–46 Control or carcinogen-modified DNA (1 μg) was digested with 2 μg each of micrococcal endo-nuclease and spleen exonuclease in 10 μl of 20 mM sodium succinate and 10 mM CaCl2, pH 6.0, at 38°C for 2 h. The resulting deoxyribonucleoside-3’-monophosphates were then converted to (5’-32P)deoxyribonucleoside-3’,5’-bisphosphates by T4 polynucleotide kinase-catalyzed (32P) phosphate transfer from [γ-32P]ATP as follows. A 10-μl aliquot of DNA digest was added to a solution prepared by mixing 1.5 μl of 0.1 M Bicine-NaOH, 0.1 M MgCl2, 0.1 M dithiothreitol, and 10 mM spermidine at pH 9.0; 5.0 μl of [γ-32P]-ATP; and 1.0 μl of T4 polynucleotide kinase (3.0 U/μl). The solution was incubated at 38°C for 2 h.
Microbiota in a cooling-lubrication circuit and an option for controlling triethanolamine biodegradation
Published in Biofouling, 2018
Thomas Klammsteiner, Heribert Insam, Maraike Probst
A strong negative correlation (r = –0.97) between TEA (7.85–6.57 mg l−1) and bicine concentration (1.16–1.76 mg l−1) was found. In the CLC, bicine concentrations were monitored to serve as a proxy for cooling liquid decay and bacterial contamination since plant operators observed a correlation between bacterial abundance and bicine concentration. Several mechanisms for bicine formation have been proposed before, mainly in connection with gas treatment (Fytianos et al. 2016). Oxidation of TEA or the influence of heat on methyldiethanolamine or diethanolamine can accelerate bicine formation (Lawson et al. 2003). In the CLC, bicine concentrations showed a delayed rise compared to decreases in TEA. The pathways of microbial TEA degradation under anaerobic conditions were already published (Frings et al. 1994; Knapp et al. 1996; Speranza et al. 2006), but no information on bacterial bicine production from TEA is available in the literature. If bicine was solely produced by TEA oxidation, a positive correlation would be expected between O2 and bicine concentrations. However, this correlation was weak and not significant, indicating other sources of bicine production than TEA oxidation. Based on the above-mentioned observations in earlier work and on the data from the present study, a connection between bacterial bicine production and TEA degradation is likely.