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Method of Fractionating Subcellular Particles to Determine Intracellular Localization of Radiotracers
Published in Lelio G. Colombetti, Principles of Radiopharmacology, 2019
H. Orii, K. Samezime, K. Komine
NAD diaphorase (E.C.I.6.4.3.): The method of Edelhoch and Mahler,6,7 slightly modified, was used. The following were mixed: 0.1 mℓ of 6 mM reduced NAD (PL Biochemicals Inc.), 0.1 mℓ of 1.2 mM sodium 2,6 = dichlorophenolindophenol, 0.3 mℓ of 0.2 M tris buffer (pH 7.5), 2.4 mℓ water, 0.1 mℓ of Tween 20 (5 mg/mi) and 0.1 mℓ sample, and the decrease in absorbance at 660 nm was recorded.
Glycerine Analysis
Published in Eric Jungermann, Norman O.V. Sonntag, Glycerine, 2018
Pardue and Frings [15] added a blue dye (2,6-dichlorophenolindophenol) to the solution. As NADH is generated, it reduces the blue dye to a colorless form. A spectrometer monitors this process. Reaction rates are then monitored and the glycerine levels are calculated based on the reaction rate during about the first minute of the reaction. They used spiked blood plasma samples and found good accuracy (within 2% relative of expected results) in the 50–200 part per million (ppm) range, with relative standard deviations of 1–2%.
Ferrihemoglobin in Normal Blood
Published in Manfred Kiese, Methemoglobinemia: A Comprehensive Treatise, 2019
Kajita et al.186 isolated three distinct fractions with ferrihemoglobin reductase activity from human red cells by chromatography on DEAE cellulose. Flavin was not found in the preparations. They all reacted with NADH and NADPH and reduced ferrihemoglobin in the presence of methylene blue, NADPH showing an about 20 times lower Km and NADH a four times higher Vmax. With a modified purification procedure, which removes hemoglobin by filtration through Sephadex® G-75 and separates the enzymes by chromatography on CM-Sephadex, Niethammer and Huennekens187 obtained two enzymes from human red cells, which differed in their absorption spectrum. The enzymes had similar molecular weights and catalytic activities and transferred electrons to methylene blue from NADH three times more rapidly than from NADPH. Later the authors188 reported that the only difference between the two forms of enzyme is the presence of a tightly, but noncovalently, bound mole of NADP per mole of protein. With starch gel electrophoresis of hemolysates or with extracts of hemolysates partially purified by adsorption on DEAE cellulose, Hsieh and Jaffé189 found only one band with ferrihemoglobin reductase NAD activity and two bands with ferrihemoglobin reductase NADP activity in normal human red cells. Using a different technique, isoelectric focusing, Sonnet et al.190 obtained similar results. Hegesh and Avron191 isolated from human red cells an enzyme which was similar to or identical with the ferrihemoglobin reductase NAD I of Scott et al.171 It reduced ferrihemoglobin very rapidly if ferrocyanide was added to the solution. From the results of a comparison of the test used by Hegesh and Avron191,211 (ferrihemoglobin reduction in the presence of ferrocyanide) and the diaphorase test with 2,6-dichlorophenolindophenol used by Scott192 and other authors, Scott193 concluded that both methods determine the same enzyme.
Inhalation of PM2.5 from diesel exhaust promote impairment of mitochondrial bioenergetics and dysregulate mitochondrial quality in rat heart: implications in isoproterenol-induced myocardial infarction model
Published in Inhalation Toxicology, 2022
Bhavana Sivakumar, Gino A. Kurian
Mitochondria from hearts were isolated by differential centrifugation as mentioned elsewhere (Barrientos et al. 2009). Mitochondrial bioenergetics was assessed via measuring the mitochondrial ETC enzyme activity for NADH-oxidoreductase (NQR), succinate decylubiquinone DCPIP reductase (SQR), Ubiquinol cytochrome c reductase (QCR), and cytochrome c oxidase (COX) using a Synergy H1 multimode reader (BioTek, Winooski, VT). The oxidation of NADH by complex I was recorded using decylubiquinone as the electron acceptor and the decrease in absorption was measured at 340 nm using molar absorption coefficient ε = 6.2 mM−1cm−1. Complex II activity was measured as the rate of reduction of 2,6-dichlorophenolindophenol (DCIP) at 600 nm (ε = 21 mM−1cm−1). Complex III activity or the activity of cytochrome c reductase was assessed as the rate of reduction of cytochrome c at 550 nm (ε = 18.5 mM−1cm−1). Complex IV activity was determined by monitoring the rate of cytochrome c oxidation at 550 nm (ε = 19.6 mM−1cm−1). ATP estimation was done using ATPlite (Perkin Elmer, Waltham, MA) system in the isolated mitochondria of all the experimental groups. Briefly, 50 µl of mitochondria was added to the lysis buffer, shaken for 5-10mts. After which the substrate buffer was added and the mitochondria were incubated in dark for 20mts followed by luminescence detection (Sivakumar and Kurian 2021a, 2021b).
Current Status of Thalassemia in Lao People’s Democratic Republic
Published in Hemoglobin, 2022
Alongkone Phengsavanh, Sourideth Sengchanh, Chanthala Souksakhone, Boupalisone Souvanlasy, Vanphanom Sychareun
Simple screening protocols and examination of the prevalence and molecular basis of thalassemia in pregnant Lao women were conducted on 307 pregnant women attending the Mother and Child Health Hospital, Vientiane, Lao PDR. Initial screening was performed locally, applying a combined osmotic fragility (OF) and dichlorophenolindophenol (DCIP) test. Erythrocyte counts were recorded. The remaining blood specimens were transferred to Thailand for further Hb and DNA analyses. Subjects were divided into four groups according to the results of the screening tests. Among 307 participants examined, 154 (50.2%) had negative results on both tests [–/–], 58 (18.8%) were positive on the OF test but not the DCIP test [+/–], 22 (7.1%) were negative on the OF test but positive on the DCIP test [–/+], and 73 (23.7%) were positive on both tests [+/+]. As many as 25 thalassemia genotypes including various complex syndromes were observed. Three clinically important forms of thalassemia including α0-, β-thal and Hb E were identified in 39 (12.7%), 11 (3.6%), and 93 (30.2%) subjects, respectively. The performance characteristics of the initial screening for these three types of thalassemia were determined. The sensitivity, specificity, positive and negative predictive values were found to be 99.2, 85.5, 83.0 and 99.4%, respectively [5].
Using hydrogen peroxide to prevent antibody disulfide bond reduction during manufacturing process
Published in mAbs, 2018
Cheng Du, Yunping Huang, Ameya Borwankar, Zhijun Tan, Anthony Cura, Joon Chong Yee, Nripen Singh, Richard Ludwig, Michael Borys, Sanchayita Ghose, Nesredin Mussa, Zheng Jian Li
An example of a redox indicator is 2,6-dichlorophenolindophenol (DCPIP, Figure 2A). Its color changed in mAb 2 purified DS, which had high percentage of LMW (Figure 2B), and its color can be completely or partially or not changed in CB with high, medium and low level of free thiols, respectively (Figure 2C). To determine the DCPIP color change range, the DCPIP stock solution was added to a series dilution of mAb 2 cell lysates with known free thiol concentrations (Figure 2D). DCPIP underwent a color change in mAb 2 CB with 3–5% cell lysate and free thiol concentrations of 80–100 μM range. Therefore, when a CB sample had a free thiol concentration higher than 100 μM, the DCPIP changed color and this feature makes DCPIP a rapid and easy-to-use method to forecast the risk of disulfide bond reduction during manufacturing.