Next Article in Journal
The Influence of Vinification Methods and Cultivars on the Volatile and Phenolic Profiles of Fermented Alcoholic Beverages from Cranberry
Next Article in Special Issue
Transmission Electron Microscopy Study of Mitochondria in Aging Brain Synapses
Previous Article in Journal
A Simple and a Reliable Method to Quantify Antioxidant Activity In Vivo
Previous Article in Special Issue
Metabolomics Studies to Assess Biological Functions of Vitamin E Nicotinate
Open AccessArticle

Protein Redox State Monitoring Studies of Thiol Reactivity

1
Department of Pharmacology and Physiology, Georgetown University Medical Center, Washington, DC 20057, USA
2
Department of Biochemical Sciences “A. Rossi Fanelli”, Sapienza University of Rome, 00185 Rome, Italy
3
Dojindo Laboratories, 2025-5 Tabaru, Mashiki-machi, Kumamoto 861-2202, Japan
*
Authors to whom correspondence should be addressed.
Antioxidants 2019, 8(5), 143; https://doi.org/10.3390/antiox8050143
Received: 15 March 2019 / Revised: 8 May 2019 / Accepted: 20 May 2019 / Published: 22 May 2019
(This article belongs to the Special Issue Novel Aspects of Redox, Antioxidant and Mitochondrial Signaling)
Protein cysteine thiol status is a major determinant of oxidative stress and oxidant signaling. The -SulfoBiotics- Protein Redox State Monitoring Kit provides a unique opportunity to investigate protein thiol states. This system adds a 15-kDa Protein-SHifter to reduced cysteine residues, and this molecular mass shift can be detected by gel electrophoresis. Even in biological samples, Protein-SHifter Plus allows the thiol states of specific proteins to be studied using Western blotting. Peroxiredoxin 6 (Prx6) is a unique one-cysteine peroxiredoxin that scavenges peroxides by utilizing conserved Cysteine-47. Human Prx6 also contains an additional non-conserved cysteine residue, while rat Prx6 only has the catalytic cysteine. In cultured cells, cysteine residues of Prx6 were found to be predominantly fully reduced. The treatment of human cells with hydrogen peroxide (H2O2) formed Prx6 with one cysteine reduced. Since catalytic cysteine becomes oxidized in rat cells by the same H2O2 treatment and treating denatured human Prx6 with H2O2 results in the oxidation of both cysteines, non-conserved cysteine may not be accessible to H2O2 in human cells. We also found that untreated cells contained Prx6 multimers bound through disulfide bonds. Surprisingly, treating cells with H2O2 eliminated these Prx6 multimers. In contrast, treating cell lysates with H2O2 promoted the formation of Prx6 multimers. Similarly, treating purified preparations of the recombinant cyclic nucleotide-binding domain of the human hyperpolarization-activated cyclic nucleotide-modulated channels with H2O2 promoted the formation of multimers. These studies revealed that the cellular environment defines the susceptibility of protein cysteines to H2O2 and determines whether H2O2 acts as a facilitator or a disrupter of disulfide bonds. View Full-Text
Keywords: cysteine; HCN channel; hydrogen peroxide; peroxiredoxin; protein; redox state cysteine; HCN channel; hydrogen peroxide; peroxiredoxin; protein; redox state
Show Figures

Figure 1

MDPI and ACS Style

Suzuki, Y.J.; Marcocci, L.; Shimomura, T.; Tatenaka, Y.; Ohuchi, Y.; Brelidze, T.I. Protein Redox State Monitoring Studies of Thiol Reactivity. Antioxidants 2019, 8, 143.

Show more citation formats Show less citations formats
Note that from the first issue of 2016, MDPI journals use article numbers instead of page numbers. See further details here.

Article Access Map by Country/Region

1
Back to TopTop