Elsevier

Methods in Enzymology

Volume 474, 2010, Pages 165-179
Methods in Enzymology

Chapter 10 - Redox Clamp Model for Study of Extracellular Thiols and Disulfides in Redox Signaling

https://doi.org/10.1016/S0076-6879(10)74010-6Get rights and content

Abstract

Extracellular thiol/disulfide redox environments are highly regulated in healthy individuals and become oxidized in disease. This oxidation affects the function of cell surface receptors, ion channels, and structural proteins. Downstream signaling due to changes in extracellular redox potential can be studied using a redox clamp in which thiol and disulfide concentrations are varied to obtain a series of controlled redox potentials. Previous applications of this approach show that cell proliferation, apoptosis, and proinflammatory signaling respond to extracellular redox potential. Furthermore, gene expression and proteomic studies reveal the global nature of redox effects, and different cell types, for example, endothelial cells, fibroblasts, monocytes, and epithelial cells, show cell-specific redox responses. Application of the redox clamp to studies of different signaling pathways could enhance the understanding of redox transitions in many aspects of normal physiology and disease.

Introduction

Recognition of the highly regulated nature of extracellular thiol/disulfide couples, measured as GSH/GSSG and cysteine/cystine (Cys/CySS) redox potentials (Jones et al., 2000), prompted studies to determine whether variation in extracellular redox state changed with cell differentiation (Nkabyo et al., 2002) or affected cell growth and proliferation (Jonas et al., 2002, Jonas et al., 2003). The latter studies revealed that human cells in culture regulate the Cys/CySS redox potential (EhCySS) of the culture medium to the same value as found in plasma of young health adults (Fig. 10.1). Subsequent human research has revealed that EhCySS values are more oxidized in association with disease risk factors and specific diseases (Jones and Liang, 2009). In the present chapter, we describe the redox clamp approach which is useful to study mechanisms whereby variation in extracellular Eh value contributes to disease.

Section snippets

Key Concepts for Use

The GSH/GSSG and Cys/CySS couples represent distinct redox signaling nodes (Jones et al., 2004) which have not been fully delineated. The initial observations showed that the GSH/GSSG redox potential (EhGSSG) in human plasma is maintained at a more reducing steady-state value (approximately − 140 mV) compared to the EhCySS value (approximately − 80 mV). Importantly, the Cys/CySS pool size (> 90 μM in Cys equivalents) in human plasma is at least 10-fold greater than the GSH/GSSG pool (< 9 μM in GSH

Principles for Experimental Design

  • Desired Ehvalues are obtained by addition of selected concentrations of thiol and disulfide.

A redox clamp is obtained by using a relatively large pool of Cys plus CySS in which the concentrations of Cys and CySS are set to values which give desired Eh values according to the Nernst equation:EhCySS=Eo+(RT/nF)ln([CySS]/[Cys]2).In this equation, Eo is the standard half-cell potential (− 250 mV for pH 7.4) for the Cys/CySS couple relative to a standard hydrogen electrode, R is the gas constant, T is

Summary of Available Redox Clamp Studies

Hwang and Sinskey (1991) provided an important contribution to the understanding of redox potential and mammalian cell growth in a study designed to optimize in vitro conditions for production of biologic products using cultured mammalian cells. They noted that three parameters were critical to rapidly obtain maximum cell density, i.e., pO2, pH, and redox potential. They found that with pO2 and pH controlled, the redox potential (measured with a potentiometric electrode) could be maintained by

Perspectives and Conclusion

The redox clamp approach as described provides a straightforward and convenient means to test for redox signaling processes which are dependent upon extracellular EhCySS or EhGSSG. The cumulative evidence indicates that multiple extracellular and cell surface proteins are responsive to thiols and disulfides in the plasma and other extracellular fluids. Although information on Eh values for thiol/disulfide couples in biologic fluids is limited, the available data has been recently reviewed (Go

Acknowledgments

This work was supported by NIH grants ES011195 and ES009047.

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