Dataset Information

A Fluorescent Probe for the Specific Staining of Cysteine Containing Proteins and Thioredoxin Reductase in SDS-PAGE.

ABSTRACT: A naphthalimide-based fluorescent probe, Nap-I, with iodoacetamide as the alkylating group, has been synthesized, and its specific fluorescent staining of proteins containing cysteine (Cys) and selenocysteine (Sec) residues in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) has been evaluated. This molecule shows good fluorescence properties in the labeling of protein Cys/Sec residues, while reducing steric hindrance and minimizing changes in the water solubility of proteins. Reaction parameters, such as labeling time and pH, have been investigated, and the optimal labeling conditions for Cys-containing proteins have been determined. Thioredoxin reductase (TXNRD) is best stained at low pH. The probe Nap-I has been successfully used for the quantification of serum proteins and hemoglobin in Tan sheep serum, and TXNRD in Tan sheep liver and muscle has been labeled at low pH. Based on the probe Nap-I, we have also distinguished TXNRD1 and TXNRD2 by SDS-PAGE. The results showed that, compared with the normal microenvironment in which the protein resides, the lower the pH value, the greater the TXNRD activity.

SUBMITTER: Liu Y

PROVIDER: S-EPMC8146322 | biostudies-literature | 2021 Apr

REPOSITORIES: biostudies-literature

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A Fluorescent Probe for the Specific Staining of Cysteine Containing Proteins and Thioredoxin Reductase in SDS-PAGE.

Liu Yuning Y Yu Yanan Y Meng Qingshi Q Jia Xueting X Zhu Jiawei J Tang Chaohua C Zhao Qingyu Q Feng Xiaohui X Zhang Junmin J

Biosensors 20210423 5

A naphthalimide-based fluorescent probe, Nap-I, with iodoacetamide as the alkylating group, has been synthesized, and its specific fluorescent staining of proteins containing cysteine (Cys) and selenocysteine (Sec) residues in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) has been evaluated. This molecule shows good fluorescence properties in the labeling of protein Cys/Sec residues, while reducing steric hindrance and minimizing changes in the water solubility of proteins ...[more]

PMID: 33922498

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Project description:Thioredoxin reductase (TR) catalyzes the reduction of thioredoxin (TRX), which in turn reduces mammalian typical 2-Cys peroxiredoxins (PRXs 1-4), thiol peroxidases implicated in redox homeostasis and cell signaling. Typical 2-Cys PRXs are inactivated by hyperoxidation of the peroxidatic cysteine to cysteine-sulfinic acid, and regenerated in a two-step process involving retro-reduction by sulfiredoxin (SRX) and reduction by TRX. Here transient exposure to menadione and glucose oxidase was used to examine the dynamics of oxidative inactivation and reactivation of PRXs in mouse C10 cells expressing various isoforms of TR, including wild type cytoplasmic TR1 (Sec-TR1) and mitochondrial TR2 (Sec-TR2) that encode selenocysteine, as well as mutants of TR1 and TR2 in which the selenocysteine codon was changed to encode cysteine (Cys-TR1 or Cys-TR2). In C10 cells endogenous TR activity was insensitive to levels of hydrogen peroxide that hyperoxidize PRXs. Expression of Sec-TR1 increased TR activity, reduced the basal cytoplasmic redox state, and increased the rate of reduction of a redox-responsive cytoplasmic GFP probe (roGFP), but did not influence either the rate of inactivation or the rate of retro-reduction of PRXs. In comparison to roGFP, which was reduced within minutes once oxidants were removed reduction of 2-Cys PRXs occurred over many hours. Expression of wild type Sec-TR1 or Sec-TR2, but not Cys-TR1 or TR2, increased the rate of reduction of PRXs and improved cell survival after menadione exposure. These results indicate that expression levels of TR do not reduce the severity of initial oxidative insults, but rather govern the rate of reduction of cellular factors required for cell viability. Because Sec-TR is completely insensitive to cytotoxic levels of hydrogen peroxide, we suggest TR functions at the top of a redox pyramid that governs the oxidation state of peroxiredoxins and other protein factors, thereby dictating a hierarchy of phenotypic responses to oxidative insults.

Dataset Information

A Fluorescent Probe for the Specific Staining of Cysteine Containing Proteins and Thioredoxin Reductase in SDS-PAGE.

Publications

A Fluorescent Probe for the Specific Staining of Cysteine Containing Proteins and Thioredoxin Reductase in SDS-PAGE.

Similar Datasets

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