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Sulfur Assimilation

Sulfur Assimilation

Sulfur is an essential element in biological systems, contributing to synthesizing key biomolecules, including amino acids such as cysteine and methionine, and cofactors such as coenzyme A and biotin. Microorganisms primarily assimilate sulfur as sulfate (SO₄²⁻) from the environment, which must undergo a series of biochemical transformations before it can be incorporated into cellular components. As sulfate is highly oxidized, it must undergo assimilatory sulfate reduction to become...

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A Rapid and Specific Microplate Assay for the Determination of Intra- and Extracellular Ascorbate in Cultured Cells

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Published on: April 11, 2014

Sinapinic acid can replace ascorbate in the biotin switch assay.

Vasantha Madhuri Kallakunta¹, Andrea Staruch, Bulent Mutus

¹Department of Chemistry and Biochemistry, 275-1, EH, University of Windsor, 401 Sunset Avenue, Windsor, Ontario N9B3P4, Canada.

Biochimica Et Biophysica Acta

|October 20, 2009

Summary

Sinapinic acid offers a modification to the biotin switch assay, improving the detection of S-nitrosated proteins. This new method avoids disulfide reduction artifacts, leading to more accurate results in proteomics research.

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Published on: February 18, 2013

Area of Science:

Biochemistry
Proteomics
Post-translational modifications

Background:

Protein S-nitrosation is a critical post-translational modification impacting protein function.
Nitric oxide (NO) interactions with thiols are key to NO's biological roles.
The biotin switch assay is a standard method for identifying S-nitrosated proteins, but can yield false positives due to disulfide reduction.

Purpose of the Study:

To introduce a modified biotin switch assay using sinapinic acid.
To eliminate potential artifacts in S-nitrosated protein detection.
To improve the accuracy of identifying the S-nitrosoproteome.

Main Methods:

Assessed sinapinic acid's denitrosation ability via NO/NO2- release.
Compared disulfide reduction by sinapinic acid versus ascorbate using DTNB assay.
Evaluated sinapinic acid and ascorbate in biotin switch assays with RAW 264.7 cells.

Main Results:

Sinapinic acid denitrosates S-nitrosothiols at pH 7.0 and denitrates/denitrosates at pH 8-8.5.
Sinapinic acid degrades S-nitrosothiols without reducing disulfide bonds.
Ascorbate, in contrast, reduces disulfide bridges, potentially causing false positives.

Conclusions:

Sinapinic acid effectively denitrosates S-nitrosothiols and does not reduce disulfides.
Sinapinic acid is a suitable replacement for ascorbate in the biotin switch assay.
This modification enhances the reliability of detecting S-nitrosated proteins.