Copper dependence of the biotin switch assay: Modified assay for measuring cellular and blood nitrosated proteins

Abstract

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.

Identification and Quantification of S-Nitrosylation by Cysteine Reactive Tandem Mass Tag Switch Assay

“…A limiting factor in the sensitivity of the traditional biotin switch assays has been the use of ascorbate as the SNO-specific reducing agent (51). Ascorbate, although reasonably specific, has limited reducing power and, even in the presence of copper, has not achieved the levels of detection seen with other techniques (30,51). Recently, alternate chemistries including a solid phase and biotin based organomercury reagents have been utilized to target SNO-modified thiols.…”

“…This allowed for the labeling of all the endogenously available cysteine residues in the lysate. SNO-modified thiols were reduced and labeled by resuspending pellets in HENS (HEN containing 1.0% (w/v) SDS), 1 mM ascorbate, 1 mM CuSO 4 and 0.8 mM Biotin-HPDP, cysTMT 6 or, in the case of the positive controls, cysTMT 0 reagent and incubated at room temp for 2 h. CuSO 4 was included as it has been found to increase the sensitivity but not affect specificity of the ascorbate reduction of SNO-modifications (30). TMT samples were labeled with cysTMT 126 -131 in the order; untreated, GSH, GSSG, 2, 10 and 20 M GSNO for two of the replicates and labeled in reverse order for the third.…”

How widespread is stable protein S-nitrosylation as an end-effector of protein regulation?

“…Selective detection of S-nitrosothiols by the ascorbatedependent biotin-switch is further complicated by the frequent addition of copper. Copper I is thought to directly reduce the nitroso bond, resulting in copper II which is then reductively regenerated by ascorbate [136]. Whilst the addition of copper I dramatically increases the sensitivity of the method, what other oxidative modifications may be reduced is not well defined.…”

Reactive Nitrogen Species and Hydrogen Sulfide as Regulators of Protein Tyrosine Phosphatase Activity

“…Several studies addressed improvement of the biotin switch method outcomes by changing reaction buffer composition, ascorbate concentration, attempted to modify the content of catalytic metal ions present in the reaction mixture, or attempted to convert S-nitrosothiols to more stable or detectable conjugates by phosphine-mediated reactions (3,53,95,100). Among the particular problems addressed are inefficient S-nitrosothiol reduction (95), sunlight exposure-induced artifacts (28), and potential reduction of disulfides (51).…”