Quantifying Changes in the Cellular Thiol-Disulfide Status during Differentiation of B Cells into Antibody-Secreting Plasma Cells.

Plasma cells produce and secrete massive amounts of disulfide-containing antibodies. To accommodate this load on the secretory machinery, the differentiation of resting B cells into antibody-secreting plasma cells is accompanied by a preferential expansion of the secretory compartments of the cells and by an up-regulation of enzymes involved in redox regulation and protein folding. We have quantified the absolute levels of protein thiols, protein disulfides, and glutathionylated proteins in whole cells. The results show that while the global thiol-disulfide state is affected to some extent by the differentiation, steady-state levels of glutathionylated protein thiols are less than 0.3% of the total protein cysteines, even in fully differentiated cells, and the overall protein redox state is not affected until late in differentiation, when large-scale IgM production is ongoing. A general expansion of the ER does not affect global protein redox status until an extensive production of cargo proteins has started.

A fluorescent probe which allows highly specific thiol labeling at low pH.

Determination of the thiol-disulfide status in biological systems is challenging as redox pools are easily perturbed during sample preparation. This is particularly pertinent under neutral to mildly alkaline conditions typically required for alkylation of thiols. Here we describe the synthesis and properties of a thiol-specific reagent, fluorescent cyclic activated disulfide (FCAD), which includes the fluorescein moiety as fluorophore and utilizes a variation of thiol-disulfide exchange chemistry. The leaving-group character of FCAD makes it reactive at pH 3, allowing modification at low pH, limiting thiol-disulfide exchange. Different applications are demonstrated including picomolar thiol detection, determination of redox potentials, and in-gel detection of labeled proteins.

Quantifying the global cellular thiol-disulfide status.

It is widely accepted that the redox status of protein thiols is of central importance to protein structure and folding and that glutathione is an important low-molecular-mass redox regulator. However, the total cellular pools of thiols and disulfides and their relative abundance have never been determined. In this study, we have assembled a global picture of the cellular thiol-disulfide status in cultured mammalian cells. We have quantified the absolute levels of protein thiols, protein disulfides, and glutathionylated protein (PSSG) in all cellular protein, including membrane proteins. These data were combined with quantification of reduced and oxidized glutathione in the same cells. Of the total protein cysteines, 6% and 9.6% are engaged in disulfide bond formation in HEK and HeLa cells, respectively. Furthermore, the steady-state level of PSSG is <0.1% of the total protein cysteines in both cell types. However, when cells are exposed to a sublethal dose of the thiol-specific oxidant diamide, PSSG levels increase to >15% of all protein cysteine. Glutathione is typically characterized as the "cellular redox buffer"; nevertheless, our data show that protein thiols represent a larger active redox pool than glutathione. Accordingly, protein thiols are likely to be directly involved in the cellular defense against oxidative stress.

Kinetic and thermodynamic aspects of cellular thiol-disulfide redox regulation.

Regulation of intracellular thiol-disulfide redox status is an essential part of cellular homeostasis. This involves the regulation of both oxidative and reductive pathways, production of oxidant scavengers and, importantly, the ability of cells to respond to changes in the redox environment. In the cytosol, regulatory disulfide bonds are typically formed in spite of the prevailing reducing conditions and may thereby function as redox switches. Such disulfide bonds are protected from enzymatic reduction by kinetic barriers and are thus allowed to exist long enough to elicit the signal. Factors that affect the rate of thiol-disulfide exchange and stability of disulfide bonds are discussed within the framework of the underlying chemical foundations. This includes the effect of thiol acidity (pK(a)), the local electrostatic environment, molecular strain, and entropy. Even though a thiol-disulfide exchange reaction is thermodynamically favorable, it will only take place if the activation energy to form the transition state complex can be overcome. This is accomplished by enzymes, such as the oxidoreductases, that direct reactions in thermodynamically favorable directions by decreasing the activation energy barrier.

Quantification of protein thiols and dithiols in the picomolar range using sodium borohydride and 4,4'-dithiodipyridine.

Experimental determination of the number of thiols in a protein requires methodology that combines high sensitivity and reproducibility with low intrinsic thiol oxidation disposition. In detection of disulfide bonds, it is also necessary to efficiently reduce disulfides and to quantify the liberated thiols. Ellman's reagent (5,5'-dithiobis-[2-nitrobenzoic acid], DTNB) is the most widely used reagent for quantification of protein thiols, whereas dithiothreitol (DTT) is commonly used for disulfide reduction. DTNB suffers from a relatively low sensitivity, whereas DTT reduction is inconvenient because the reagent must be removed before thiol quantification. Furthermore, both reagents require a reaction pH > 7.0 where oxidation by ambient molecular oxygen is significant. Here we describe a quick and highly sensitive assay for protein thiol and dithiol quantification using the reducing agent sodium borohydride and the thiol reagent 4,4'-dithiodipyridine (4-DPS). Because borohydride is efficiently destroyed by the addition of acid, the complete reduction and quantification can be performed conveniently in one tube without desalting steps. Furthermore, the use of reverse-phase high-performance liquid chromatography for the thiol quantification by 4-DPS reduces the detection limit to the picomolar range (equivalent to 1 microg of a 50-kDa protein containing 1 thiol) while at the same time maintaining low pH throughout the procedure.

Increasing the reactivity of an artificial dithiol-disulfide pair through modification of the electrostatic milieu.

The thiol-disulfide exchange reaction plays a central role in the formation of disulfide bonds in newly synthesized proteins and is involved in many aspects of cellular metabolism. Because the thiolate form of the cysteine residue is the key reactive species, its electrostatic milieu is thought to play a key role in determining the rates of thiol disulfide exchange reactions. While modest reactivity effects have previously been seen in peptide model studies, here, we show that introduction of positive charges can have dramatic effects on disulfide bond formation on a structurally restricted surface. We have studied properties of vicinal cysteine residues in proteins using a model system based on redox-sensitive yellow fluorescent protein (rxYFP). In this system, the formation of a disulfide bond between two cysteines Cys149 and Cys202 is accompanied by a 2.2-fold decrease in fluorescence. Introduction of positively charged amino acids in the proximity of the two cysteines resulted in an up to 13-fold increase in reactivity toward glutathione disulfide. Determination of the individual pK(a) values of the cysteines showed that the observed increase in reactivity was caused by a decrease in the pK(a) value of Cys149, as well as favorable electrostatic interactions with the negatively charged reagents. The results presented here show that the electrostatic milieu of cysteine thiols in proteins can have substantial effects on the rates of the thiol-disulfide exchange reactions.