Background

The redox status of proteins plays a key role in a number of cellular processes. Thiol-disulfide exchange reactions support the redox control of plant responses to fluctuating light under natural environments via a photochemical electron transfer cascade for posttranslational modification of proteins. Whereas thiols (R-SH) have relatively higher reactivity than other cellular components and can be directly detected by a variety of reagents and separation techniques (Fahey et al., 1980 and 1981; Newton et al., 1981; Riddles et al., 1983; Fenton and Fahey, 1986; Tyagarajan et al., 2003), disulfide bonds (R-S-S-R) have no such chemical characteristics. Therefore, the most common methodology for the detection of disulfide bonds consists of a three-step procedure: blocking of thiols, reduction of disulfide bonds, and subsequent detection of additionally exposed thiols. Dithiothreitol (DTT) is a well-known and frequently used reducing agent for the protection and exposure of thiol groups of proteins. However, the use of DTT in the laboratory is undesirable because of its harmful odor. Moreover, it can subsequently interfere with thiol detection reagents unless it is appropriately removed (Getz et al., 1999). Also, the reducing power of DTT is limited to pH > 7. Tris(2-carboxyethyl) phosphine-hydrochloride (TCEP) can be a promising alternative with advantages of being odorless and more effective in the reduction of disulfide bonds over a wide pH range from 1.5 to 8.5. Monobromobimane (mBB) has been used for fluorescent labeling of low molecular weight thiols by detecting fluorescent derivatives under a fluorescent microscope or with high-performance liquid chromatography (Fahey and Newton, 1987; Meyer et al., 2001). This protocol describes a simple method for discriminate labeling with mBB of free thiols, disulfide bonds, and total thiols in plant protein extracts. Here, we show an example of the procedure using Arabidopsis leaves. Free thiols are labeled without any preprocessing (Figure 1A), whereas labeling of disulfide bonds requires blocking of free thiols by iodoacetamide (IAA) and reduction of disulfide bonds by TCEP before the mBB treatment (Figure 1B). Total labeling of thiols just after TCEP treatment is helpful to estimate the redox state of total thiols in protein extracts (Figure 1C). This simple method allows the detection of modifications in samples on a gel and can be used for semi-quantitative analysis and provides an overview of the redox state of thiol-containing proteins without any special equipment other than conventional protein electrophoresis and imaging systems.


Figure 1. Flowchart for discriminate labeling of thiols in protein under different redox states. A. Labeling of free thiols in proteins. B. Specific labeling of thiols, forming disulfide bonds in protein. C. Total labeling of thiols in proteins. F in closed circle means mBB-derived fluorescence. IAA: Iodoacetamide, TCEP: Tris(2-carboxyethyl)phosphine Hydrochloride.