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The Modification of Cysteine
Published in Roger L. Lundblad, Chemical Reagents for Protein Modification, 2020
2-Chloromercuri-4-nitrophenol is a compound related to the organic mercurial described above. It has proved useful as a “reporter” group in the study of microenvironmental changes in the modified protein.41,42 An excellent example of this is provided from the studies of Marshall and Cohen90 on the properties of ornithine transcarbamylase modified with 2-chloromercuri-4-nitrophenol. The enzyme from S. faecalis was modified in 0.1 M MOPS, 0.1 M KC1, pH 7.5 using changes in absorbance at 403 nm to follow the extent of modification. The pH dependence of the spectrum of the modified S. faecalis enzyme is shown in Figure 48. The bovine enzyme is carboxamido methylated on a nonessential sulfhydryl group before reaction with the organic mercurial. Modification of the bovine enzyme with 2-chloromercuri-4-nitrophenol is performed in 0.020 M MOPS, 0.1 M KC1, pH 7.11 at 25°C. The modification was followed by the change in absorbance at 405 nm. The effect of pH on the spectrum of the modified bovine enzyme is shown in Figure 49. Baines and Brocklehurst91 have reported the synthesis and characterization of 2-(2’-pyridylmercapto)-mercuri-4-nitrophenol, a reagent which does have certain advantages. In particular, the spectral changes occurring on modification (Figure 50) permit the more facile in situ determination of the extent of reaction.
Biochemical Methods of Studying Hepatotoxicity
Published in Robert G. Meeks, Steadman D. Harrison, Richard J. Bull, Hepatotoxicology, 2020
Prasada Rao S. Kodavanti, Harihara M. Mehendale
Pipet 0.02 ml of serum or plasma into a cuvette. To this, add 0.5 ml of 2-amino-2-methyl-propanediol buffer followed by 0.4 ml of metal ion buffer and 0.1 ml of 4-nitrophenyl phosphate. Monitor the reaction at 30°C over a period of 10 min in a spectrophotometer at A405. During this reaction, colorless 4-nitrophenyl phosphate is hydrolyzed by alkaline phosphatase to form brilliantly yellow 4-nitrophenol. The rate at which the color is generated is proportional to the enzyme activity.
Vibrio
Published in Dongyou Liu, Handbook of Foodborne Diseases, 2018
V. vulnificus consists of one flagellar (H) serotype and seven somatic (O) serotypes. Based on biochemical characteristics (e.g., indole reaction, ornithine decarboxylation reaction, and o-nitrophenol production), V. vulnificus is classified into three biotypes. Biotype 1 (positive for indole and ornithine decarboxylase [ODC] reactions) occurs in salt or brackish water worldwide and is predominantly responsible for the entire disease spectrum in humans, with a fatality rate of >50%. Biotype 2 (negative for indole and ODC reactions) is found in saltwater in eastern and western Europe and primarily infects eel (Anguilla) and occasionally humans. Biotype 3 (negative for o-nitrophenol production) appears to be a hybrid of biotypes 1 and 2. Identified from persons handling freshwater fish (Tilapia) in Israel, biotype 3 may cause serious infections requiring amputation, with a mortality rate of <8%. Use of 16S rRNA, capsular polysaccharide (CPS) allele, virulence-correlated gene, BOX-A1R-based repetitive extragenic palindromic-PCR (BOX-PCR) genomic fingerprinting, and multilocus sequence typing analyses permits improved discrimination among V. vulnificus isolates [6,7].
Development of reliable quantitative structure–toxicity relationship models for toxicity prediction of benzene derivatives using semiempirical descriptors
Published in Toxicology Mechanisms and Methods, 2023
Ayushi Singh, Sunil Kumar, Archana Kapoor, Parvin Kumar, Ashwani Kumar
The sign of the coefficient of LogKow and HF descriptors in the above-mentioned equations is positive, indicating that increasing the value of these descriptors enhances the toxicity of NB derivatives toward the T. pyriformis. Negative coefficients of TE and CV show their indirect relationship with toxicity. The standardized coefficient of LogKow, CV, HF, and TE for Model 3 and 4 are 0.5512, −0.3774, 0.4464, −1.0234, and 0.5234, −0.4324, 0.5291, −1.2031, respectively, indicating that total energy is the maximum contributor to NB derivatives toxicity followed by the almost equal positive contribution of LogKow and heat of formation. When comparing the toxicity of various NB derivatives as shown in Table S2, the value of TE for NB is −1578.012 whereas the value for 1,4-dinitrobenzene is −2338.29. These greater negative total energy for 1,4-dinitrobenzene makes it more toxic. Likewise, 2-nitrophenol possesses total energy of −1873.55 while for 2-nitroaniline the value of TE is −1778.12. Therefore, 2-nitrophenol is more toxic. Similarly, these descriptors can be used to easily determine the toxicity of different NB derivatives. The plots for Model 3 are shown in Figure 3 here and Figures S6 and S7 of Supplementary Information.
HPLC-DAD profiling and inhibitory potentials of ethylacetate and aqueous extracts of Talinum triangulare on key enzymes linked to type-2 diabetes (α-amylase and α-glucosidase) and oxidative stress (monoamine oxidase)
Published in Egyptian Journal of Basic and Applied Sciences, 2019
Olakunle B. Afolabi, Omotade I. Oloyede, Oludare S. Agunbiade, Tajudeen O. Obafemi, B. Aline, A. Obajuluwa, K. Jaiyesimi, S. Anadozie
The α-glucosidase inhibitory activity of the extract was determined according to the method described by Ademiluyi and Oboh [25], with slight modifications. Briefly, 0–100 μL of free phenol/bound phenolic extract was incubated with 100 μL of 1.0 U mL−1 α-glucosidase solution in 100 mmol l−1 phosphate buffer (pH 6.8) at 37°C for 15 min. Thereafter, 50 μL of pNPG solution (5 mmol l−1) in 100 mmol l−1 phosphate buffer (pH 6.8) was added and the mixture was further incubated at 37°C for 20 min. The absorbance of the released p-nitrophenol was measured at 405 nm and the inhibitory activity was expressed as a percentage of a control sample without inhibitor.
Probing the role of an invariant active site His in family GH1 β-glycosidases
Published in Journal of Enzyme Inhibition and Medicinal Chemistry, 2019
Andrea Strazzulli, Giuseppe Perugino, Marialuisa Mazzone, Mosè Rossi, Stephen G. Withers, Marco Moracci
As previously reported, the catalytic efficiency of Ssβ-gly on disaccharides is lower than that on aryl glycosides, with the (kcat/KM)2 Np-Gal/(kcat/KM)lactose and the (kcat/KM)2 Np-Glc/(kcat/KM)cellobiose ratios of 60 and 137, respectively2, consistent with the better leaving group of the aryl glycosides. 2-Nitrophenol has a pKa of 7.22 and is assisted by the ortho effect produced by the presence in position 2 of the phenol ring of the NO2 group9,36. Instead, in disaccharides, the high pKa of 12.28 of glucose requires greater assistance by the acid/base in the glycosylation step of the reaction (Figure 1). Mutation affects more the (kcat/KM)2 Np-Glc/(kcat/KM)cellobiose ratio (1.5 × 103) while the ratio of the catalytic efficiencies on 2 Np-Gal and lactose is similar to the wild type (68 vs 60, for A150 and H150, respectively). The ratio of the catalytic efficiency of the mutant on 2 Np-Glc and -Gal is about 10-fold higher than that of the wild type with the (kcat/KM)2 Np-Glc/(kcat/KM)2 Np-Gal being 17.3 and 1.7 for A150 and H150, respectively.