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The Modification of Histidine Residues
Published in Roger L. Lundblad, Chemical Reagents for Protein Modification, 2020
Saluja and McFadden45 have explored the reaction of diethylpyrocarbonate with spinach ribulose bisphosphate carboxylase. One interesting observation is that the plot of half-inactivation time vs. the reciprocal of diethylpyrocarbonate concentration suggested that saturation kinetics existed consistent with the “affinity” binding of reagent prior to protein modification. The solid line shows the change in absorbance at 242 nm (Figure 15) of the carboxylase upon reaction with diethylpyrocarbonate (the magnitude of the increase is consistent with the modification of 2.4 histidine residues per combination of small subunit and large subunit). This change is completely reversed on treatment with hydroxylamine. Also shown is the change in absorbance at 242 nm on the reaction of N-acetylcysteine with diethylpyrocarbonate in 0.1 M succinate pH 6.4. This spectral change occurs only in carboxylate buffers and is comparatively transient when compared to the protein reaction product. The study of Bloxham46 on the reactivity of the active site histidine in lactate dehydrogenase is particularly fascinating. The rate of reaction of the histidine residue in the native enzyme was compared to the thiomethyl derivative (prepared by reaction with methyl methanethiosulfonate) as shown in Figure 16. There is a substantial decrease in the nucleophilic character of the active-site histidine (histidine-195). Cromartie47 has examined the modification of alcohol oxidase with diethylpyrocarbonate in 0.050 M sodium phosphate, pH 7.5, at 0°C.
Analytical Toxicology of Vitreous Humor
Published in Steven H. Y. Wong, Iraving Sunshine, Handbook of Analytical Therapeutic Drug Monitoring and Toxicology, 2017
In keeping with current desires for more rapid and inexpensive analyses, Penttila et al29 investigated the use of the Alcoscan test strip for rapid screening of samples for the presence of ethanol. This study involved saliva from living subjects and urine and vitreous humor from deceased individuals. The test is based on the reaction between ethanol and alcohol oxidase in the presence of an indicator dye system to form an indamine-type dye with a stable blue color. Visual comparison with a color chart or, as done in this study, use of reflectance photometry (Glucoscan), would provide quantitation.
Preclinical developments of enzyme-loaded red blood cells
Published in Expert Opinion on Drug Delivery, 2021
Luigia Rossi, Francesca Pierigè, Alessandro Bregalda, Mauro Magnani
Alcohol oxidase (AlOx) from Pichia pastoris (a methylotrophic yeast) has a much higher affinity for methanol than for ethanol. Based on this observation, we investigate the potential use of this enzyme for the treatment of methanol intoxication [77]. In fact, in humans, methanol is metabolized to formaldehyde by alcohol dehydrogenase and formaldehyde is metabolized to formic acid by acetaldehyde dehydrogenase, leading to cytochrome C oxidase inhibition. AlOx was encapsulated into human and murine erythrocytes up to 2 units/ml of packed cells. Enzyme‐loaded erythrocytes showed an increased rate of the hexose‐monophosphate‐shunt activity and a significant methemoglobin production resulting from the intracellular generation of H2O2. However, the in vivo survival of these cells did not seem to be significantly affected by methanol catabolism. In vivo, mice receiving AlOx‐loaded erythrocytes were able to keep the blood methanol concentrations below values that were about 50% of those found in control mice who received similar amounts of methanol. Thus, AlOx‐loaded erythrocytes may add an important contribution to the detoxification protocol against methanol poisoning. Formic acid can eventually be further degraded by encapsulate formate dehydrogenase [78].
Alcohol quantification: recent insights into amperometric enzyme biosensors
Published in Artificial Cells, Nanomedicine, and Biotechnology, 2018
Vinita Hooda, Vikas Kumar, Anjum Gahlaut, Vikas Hooda
Alcohol oxidase (AOX, alcohol oxidoreductase, EC 1.1.3.13) is the chief enzyme for alcohol metabolism in Hansenula polymorpha, Pichia pastoris, Candida boidinni and some other yeast species. The initial step of alcohol catabolism in the cells of yeast, to be specific its oxidation to acetaldehyde, with corresponding generation of hydrogen peroxide is done by the enzyme AOX as per the reaction represented in equation.