Conversion of S-phenylsulfonylcysteine residues to mixed disulfides at pH 4.0: utility in protein thiol blocking and in protein-S-nitrosothiol detection

Abstract

A three step protocol for protein S-nitrosothiol conversion to fluorescent mixed disulfides with purified proteins, referred to as the thiosulfonate switch, is explored which involves: (1) thiol blocking at pH 4.0 using S-phenylsulfonylcysteine (SPSC); (2) trapping of protein S-nitrosothiols as their S-phenylsulfonylcysteines employing sodium benzenesulfinate; and (3) tagging the protein thiosulfonate with a fluorescent rhodamine based probe bearing a reactive thiol (Rhod-SH), which forms a mixed disulfide between the probe and the formerly S-nitrosated cysteine residue. S-Nitrosated bovine serum albumin and the S-nitrosated C-terminally truncated form of AdhR-SH (alcohol dehydrogenase regulator) designated as AdhR*-SNO were selectively labelled by the thiosulfonate switch both individually and in protein mixtures containing free thiols. This protocol features the facile reaction of thiols with S-phenylsulfonylcysteines forming mixed disulfides at mild acidic pH (pH = 4.0) in both the initial blocking step as well as in the conversion of protein-S-sulfonylcysteines to form stable fluorescent disulfides. Labelling was monitored by TOF-MS and gel electrophoresis. Proteolysis and peptide analysis of the resulting digest identified the cysteine residues containing mixed disulfides bearing the fluorescent probe, Rhod-SH.

Fig. 1

% GSNO over time (min) taken from absorbance readings at 545 nm at different pH’s with and without equimolar DTT.

Fig. 2

% Conversion of SPSC to PhSO₂Na over 1 h as measured from absorbance detection at 265 nm from RP HPLC analysis.

Fig. 3

Deconvoluted mass spectra of either (red, top): BSA (100 μM) or (blue, middle): BSA-SNO (44% SNO content, 100 μM total protein) after addition of PhSO₂Na and Rhod-SH (1) for 30 min. (green, bottom): BSA-SNO (22% SNO content, 28% free thiol, 100 μM total protein) after sequential addition of SPSC (1 h), PhSO₂Na (30 min), and Rhod-SH (1) (10 min).

Fig. 4

Blue: Chromatogram for a tryptic digest of BSA-SS-Rhod (λ= 565 nm). Red: Chromatogram for a tryptic digest of BSA subjected to the same reaction and digestion conditions.

Fig. 5

Spot volume vs. μg BSA-SS-Rhod from 1D SDS-PAGE nonreducing gels. BSA-SNO (1.8, 1.2, 0.6, 0.3, 0.15, and 0.0 μg) was subjected to the sequential addition of SPSC (1 h), PhSO₂Na (30 min), Rhod-SH (1) (10 min), and MMTS (5 min). The error bars represent the standard deviation. The image above is the fluorescent image of the gel and coomassie stain.

Fig. 6

Deconvoluted mass spectra of (dotted line): AdhR*-SNO; (solid line): AdhR*-SNO+ ascorbate (20 mM), 1 h. Buffer= 100 mM HEPES, 1 mM EDTA, 0.1 mM neocuproine (HEN), pH 7.5. AdhR*-SNO = 53% SNO, 47% free thiol, 25 μM total protein.

Fig. 7

Deconvoluted mass spectra after labelling by the thiosulfonate switch of (red, top): AdhR*-SNO (53% SNO, 47% free thiol, 25 μM total protein); (blue, bottom): AdhR*-SH (25 μM). thiosulfonate switch = SPSC (1 h) + PhSO₂Na (30 min) + Rhod-SH (1) (10 min).

Fig. 8

Deconvoluted spectrum of thiosulfonate switch labelled (A) BSA-SNO^# and AdhR*-SH (50 μM each) and (B) Spot volume of BSA-SNO^# and AdhR*-SH (5 μM each) labelled by the thiosulfonate switch and run on a 1D SDS-PAGE nonreducing gel. The image shows the actual gel spots with their corresponding molecular weights. BSA-SNO^#= 52% SNO, 0% free thiol.

Fig. 9

(A) Deconvoluted spectrum of thiosulfonate switch labeled BSA and AdhR*-SNO (50 μM each) (B) Spot volume of BSA and AdhR*-SNO (5 μM each) labelled by TST and run on a 1D SDS-PAGE nonreducing gel. The image shows the actual gel spots with their corresponding molecular weights. Buffer = 100 mM ammonium formate, 1 mM EDTA, pH 4.0. AdhR*-SNO = 53% SNO, 47% free thiol.

Scheme 1

BSA-SNO is converted into BSA-SSO₂Ph and PhSO₂NHOH upon reaction with PhSO₂Na at pH 4.0.

Scheme 2

Reactions of PhSO₂Na and Rhod-SH (1) on GSNO, GSH and GSSG.

Scheme 3

Reactions of Rhod-SH (1) with GSSG, (GlyCys)₂, SPSC, MMTS, NEM, and IAM at pH 4.0.

Scheme 4

Reactions of SPSC with a series of thiols of varying pKa.

Scheme 5

Synthesis of S-phenylsulfonylcysteine (SPSC).

Scheme 6

Synthesis of compounds 2-4.

Scheme 7

Synthesis of cysteic acid side chain 6. i. Boc-Cys(Trt)-OSu, Na₂CO₃, aq. dioxane, rt, 2 d, 88%, ii. Et₃SiH, TFA, CH₂Cl₂, rt, 1 h, iii. HCO₂H, H₂O₂, °C, 1 h, 81%, 2 steps.

Scheme 8

Synthesis of Rhod-SH (1). i. 6, NMM, DMF, 23 h, 62%, ii. DCC, NHS, DMF, 17 h, 91%, iii. cysteamine hydrochloride, NMM, DMF, 22 h, 61%.