Studies have shown that modification of critical cysteine residues in proteins leads to the regulation of protein function. These modifications include disulfide bond formation, glutathionylation, sulfenic and sulfinic acid formation, and S-nitrosation. The biotin switch assay was developed to specifically detect protein S-nitrosation. In this assay, proteins are denatured with SDS in the presence of methyl methanethiosulfonate (MMTS) to block free thiols. After acetone precipitation or Sephadex G25 separation to remove excess MMTS, HPDP-biotin and 1 mM ascorbate are added to reduce the Snitrosothiol bonds and label the reduced thiols with biotin. The proteins are then separated on a nonreducing SDS PAGE and detected using either streptavidin-HRP or anti-biotin HRP conjugate. Our examination of this labeling scheme has revealed that the extent of labeling depends on the buffer composition and importantly, on the choice of metal ion chelator (DTPA vs. EDTA). Unexpectedly, using purified S-nitrosated albumin, we have found that "contaminating" copper is required for the ascorbate-dependent degradation of S-nitrosothiol; this is consistent with the fact that ascorbate itself does not rapidly reduce S-nitrosothiols. Removal of copper from buffers by DTPA and other copper chelators preserves approximately 90% of the S-nitrosothiol, while the inclusion of copper and ascorbate completely eliminates the S-nitrosothiol in the preparation and increases the specific biotin labeling. These biotin switch experiments were confirmed using triiodide-based and copper-based reductive chemiluminescence. Additional modifications of the assay using NEM (N-ethylmaleimide) for thiol blockade, ferricyanide pretreatment to stabilize S-nitrosated hemoglobin, and cyanine dye labeling instead of biotin, are presented for the measurement of cellular and blood S-nitrosothiols. These results indicate that degradation of S-nitrosothiol in the standard biotin switch assay is metal ion-dependent and that experimental variability in S-nitrosothiol yields using this assay occurs secondary to the inclusion of metal ion chelators in reagents and variable metal ion contamination of buffers and labware. The addition of copper to ascorbate allows for a simple assay modification that dramatically increases sensitivity while maintaining specificity.

It has become clear in recent decades that the post-translational modification of protein cysteine residues is a crucial regulatory event in biology. Evidence supports the reversible oxidation of cysteine thiol groups as a mechanism of redox-based signal transduction while the accumulation of proteins with irreversible thiol oxidations is a hallmark of stress-induced cellular damage. The initial formation of cysteine sulfenic acid (SOH) derivatives, along with the reactive properties of this functional group, serves as a crossroads whereby the local redox environment may dictate the progression of either regulatory or pathological outcomes. Protein-SOH are established as transient intermediates in the formation of more stable cysteine oxidation products both under basal conditions and in response to several redox-active extrinsic compounds. This review details both direct and multi-step chemical routes proposed to generate protein-SOH, the spectrum of secondary reactions that may follow their initial formation and the arsenal of experimental tools available for their detection. Both the pioneering studies that have provided a framework for our current understanding of protein-SOH as well as state-of-the-art proteomic strategies designed for global assessments of this post-translational modification are highlighted.

Pulmonary arterial endothelial cells possess transplasma membrane electron transport (TPMET) systems that transfer intracellular reducing equivalents to extracellular electron acceptors. As one aspect of determining cellular mechanisms involved in one such TPMET system in pulmonary arterial endothelial cells in culture, glycolysis was inhibited by treatment with iodoacetate (IOA) or by replacing the glucose in the cell medium with 2-deoxy-D-glucose (2-DG). TPMET activity was measured as the rate of reduction of the extracellular electron acceptor polymer toluidine blue O polyacrylamide. Intracellular concentrations of NADH, NAD(+), NADPH, and NADP(+) were determined by high-performance liquid chromatography of KOH cell extracts. IOA decreased TPMET activity to 47% of control activity concomitant with a decrease in the NADH/NAD(+) ratio to 34% of the control level, without a significant change in the NADPH/NADP(+) ratio. 2-DG decreased TPMET activity to 53% of control and decreased both NADH/NAD(+) and NADPH/NADP(+) ratios to 51% and 55%, respectively, of control levels. When lactate was included in the medium along with the inhibitors, the effects of IOA and 2-DG on both TPMET activity and the NADPH/NADP(+) ratios were prevented. The results suggest that cellular redox status is a determinant of pulmonary arterial endothelial cell TPMET activity, with TPMET activity more highly correlated with the poise of the NADH/NAD(+) redox pair.

In this study the mechanism by which S-nitrosocysteine (CysNO) activates soluble guanylyl cyclase has been investigated. CysNO is the S-nitrosated derivative of the amino acid cysteine, and has previously been shown to be transported into various cell types by amino acid transport system L (L-AT). Here we show using both neuroblastoma and pulmonary artery smooth muscle cells, that CysNO stimulates cGMP formation at low concentrations, but this effect is lost at higher concentrations. Stimulation of cGMP accumulation occurs only after its transport into the cell and subsequent flavoprotein reductase-mediated metabolism to form nitric oxide (NO). Consequently, CysNO can be regarded as a cell-targeted NO releasing agent. However, CysNO also functions as an NO-independent thiol-modifying agent and can compromise cellular antioxidant defenses in a concentration-dependent manner. The observed biphasic nature of CysNO-dependent cGMP accumulation appears to be due to these two competing mechanisms. At higher concentrations, CysNO likely inactivates guanylyl cyclase through modification of an essential thiol group on the enzyme, either directly or as a result of a more generalized oxidative stress. We show here that higher concentrations of CysNO can increase cellular S-nitrosothiol content to non-physiological levels, deplete cellular glutathione (GSH) and inhibit cGMP formation in parallel. Although the inhibition of sGC by S-nitrosation has been suggested as a mechanism of nitrovasodilator tolerance, in the case of CysNO, it appears to be more a reflection of a generalized oxidative stress placed upon the cell by the non-physiological levels of intracellular S-nitrosothiol generated upon CysNO exposure.

Gel-based detection of S-nitrosothiols has relied on the biotin-switch method developed by Jaffrey et al (Nat. Cell. Biol., 3, 193−197, 2001). This method attempts to replace the nitroso group with a biotin label to allow detection and isolation of S-nitrosated proteins, and has been extensively used in the literature. Here we describe a modification of this method that differs from the original in two major ways. First, it uses a combination of copper ions and ascorbate to achieve selective reduction of the S-nitrosothiol. Second, it replaces the biotin label with fluorescent cyanine dyes in order to directly observe the modified proteins in-gel and perform comparative studies using difference gel electrophorsesis (DIGE) analysis in two dimensions.