Office of Technology Development (OTD)

Balaraman Kalyanaraman, PhD

The reversible regulation of protein function by S-nitrosation has led to the proposal that S-nitrosothiols function as posttranslational modifications, analogous to those created by phosphorylation or acetylation. S-nitrosoproteins are identified or quantified using several different methods such as gas phase measurement of released NO, spectrophotometric measurement of released NO, fluorescence measurement of released NO, S-nitrosocysteine antibodies and ascorbate-based switch assays.

The labile properties of the nitrosothiol bond have hindered general identification of S-nitrosated proteins and the specific cysteinyl residues that are modified. The S-nitroso bond is subject to decomposition by light, transition metals, excess thiols, and antioxidants, including ascorbate. The methods listed in the State-of-the-Art all suffer drawbacks due to labile bonds or low specificity binding of anti-nitrosocysteine antibodies.

In the current invention a trifunctional molecule is presented for spin trapping the sulfhydryl radical from a disrupted nitrosothiol bond and form persistent, detectable spin adducts that can be directly assessed by a variety of known methods such as binding avidin through a biotin moiety on the trifunctional or by visualization by a fluorescent probe. In addition, the current invention is a spin trapping molecule that can not only trap free radicals in a Western blot, but also can be targeted to an organ, a cell, an organelle or a molecule of interest for other types of studies including EPR. The trifunctional molecule is especially applicable to S-nitrosoproteins.

This technology directly identifies and quantifies S-nitrosoproteins in Western blots or in situ.

Structure and production of the trifunctional molecule are complete and tested.