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System for detection of s-nitrosoproteins

System for detection of s-nitrosoproteins description/claims

This application is a continuation of U.S. patent application Ser. No. 10/319,457, filed on Dec. 12, 2002, now U.S. Pat. No. ______, which claims the benefit of U.S. Provisional Application No. 60/339,268, filed on Dec. 12, 2001. The entire contents of the foregoing are hereby incorporated by reference.

This invention was made with United States Government support under Grant Nos. 1RO1 HL59337, HL10026 and GM57601-01, awarded by the national Institute of Health. The United States Government has certain rights in the invention.

Nitric oxide (NO) has relatively recently been recognized as a biological messenger that reacts with a variety of sulfhydryl-containing molecules and enzymes to produce S-nitrosylated compounds. Since NO has a short half-life under physiological conditions, it generally exists in biological systems as adducts of amino acids, peptides, and proteins (“NO equivalents”). These NO equivalents are usually biologically active in that they behave as NO donors, and thereby possess unique pharmacological properties. The various targets for nitrosylation include serum albumin, tissue-type plasminogen activator, transcriptional activators, glyceraldehyde-3-phosphate dehydrogenase, human immunodeficiency virus protease, hemoglobin, and protein-phosphotyrosine phosphatase.

Nitrosylation can alter protein conformation, leading to the activation or inactivation of enzymes or receptor proteins. Like phosphorylation, the nitrosylation reaction behaves like a “chemical switch” that allows cells to transmit stimuli from the plasma membrane to the nucleus in a highly regulated manner. However, the functions and processes of nitrosylation are difficult to deconvolute, due to the high number of closely-related kinases, and due to the lack of currently available technology to easily and accurately measure the extent or presence of protein nitrosylation.

Sulfhydryl groups (—SH, also referred to as “thiol”) are among the most reactive groups found in protein molecules. S-nitrosoproteins, S-nitrosothiols, and protein S-nitrosylation reactions are terms that refer to compounds with linkages through the thiol (—SH) group. These types of compounds play important roles in cell signaling processes such as neurotransmission, anion channel regulation, host defense and gene regulation. The chemical modification of the —SH group in proteins thus has important regulatory implications and can be used as a tool in the discovery of novel therapeutics.

Chemical modification of thiol groups occurs physiologically via oxidation reactions yielding either mixed disulfides or S-nitrosylated compounds. Alternatively, modification can occur through persulfide and trisulfide bond formation. The “S-nitrosylation” of proteins refers to the transfer of nitric oxide (NO) to sulfhydryl groups on proteins.

By way of example, some cysteine proteases such as caspase-3 and cathepsin K have been demonstrated to be inhibited by NO donors. (See Wang, Peng et al., Inhibition of Papain by S-Nitrosothiols, J. of Biol. Chem., 275, 2000 pp 20467-20473). Cysteine proteases play important roles in various biological processes. Elevated proteolytic activity of cysteine proteases is associated with many disease conditions, such as muscular dystrophy, inflammation, and rheumatoid arthritis. The active sites of cysteine proteases contain a cysteine sulfhydryl group which is highly sensitive to oxidation.

Compounds such as S-nitrosoglutathione (GSNO) are relevant biological molecules involved in nitrosylation reactions under physiological conditions. These compounds are also known to fluoresce, which would theoretically make their detection facile in samples derived from biological systems. However, identification of S-nitrosoproteins and measurement of their concentration following certain cellular events has proven to be extraordinarily cumbersome, thus extremely limiting its potential utility.

In light of the significant physiological implications of NO levels, it would be useful to have a diagnostic technique that can readily detect levels of NO or NO equivalents, such as S-nitrosothiols and other nitrosylated NO equivalents, to determine whether levels are normal for normal physiological conditions, or whether a patient has an existing or predisposition towards a pathophysiological condition. There is a particular need for procedures that are affordable and manageable, yet sensitive enough to detect levels of NO, or NO-adducts such as S-nitrosothiols. (See Beckman, J. S. et al., Methods in Nitric Oxide Research, Feelisch and Stainler, Wiley, Chichester, U.K., 1996; U.S. Pat. No. 5,891,735 to Stamler).

Representative of prior art approaches to monitoring of nitrosylation, U.S. Pat. No. 5,459,076 to Stamler et al. (incorporated herein by reference) describes a detection method that requires pretreatment with mercurous ion and a protein-precipitating agent. The samples are then monitored by chemiluminescence. This method involves cumbersome pretreatment procedures with a toxic mercury compound and, thus, presents considerable difficulties in application. It would be useful to have a simple procedure with minimal manipulation and without the use of additional chemicals.

The present invention is directed to a practical electrophoresis-based separation and identification system for cellular nitrosoproteins. The detection system meets a recognized need in the art for monitoring of NO in normal states and in disease states, provides a method for identifying and quantifying NO in normal and in disease states, and would facilitate the understanding of these processes for the development of better therapeutic drug species.

The present invention is directed towards a method for detecting the presence of nitrosylated species in biological samples. In a preferred embodiment, the biological samples are comprised of amino acid-based species. Preferably, the nitrosylated species are adducts between NO and amino acids, peptides, or proteins. The atoms forming the adducts with NO include carbon, nitrogen, sulfur, and oxygen. Preferably, the adduct is between NO and sulfur groups. More preferably, the adduct is a nitrosylated protein. Still more preferably, the protein is a nitrosothiol, or an “S-nitrosoprotein.” Still more preferably, the nitrosylated protein is S-nitrosoalbumin.

In an embodiment of the invention, the method for detecting the presence of nitrosylated species in a biological sample comprises the steps of contacting the biological sample with developing reagents, exposing the sample to excitation radiation, and detecting the resultant emitted fluorescence.

In a preferred embodiment, the developing reagents comprise a fluorescence-developing agent and a molecular species bearing a reactive moiety capable of transnitrosylation. Preferably, the reactive moiety is a thiol bearing group. More preferably, the molecular species capable of transnitrosylation is cysteine.

In yet another embodiment, the developing reagents comprise a saturated solution of copper (I) chloride. In still another embodiment, the fluorescent agent is 4,5-diaminofluoroscein. In a preferred embodiment, the developing reagents are added to the biological sample simultaneously. Alternatively, one or more of the developing reagents are added to the biological sample sequentially. Alternatively or additionally, the method of detecting nitrosylated species involves heating or incubating the biological sample to which the fluorescent-developing agent has been added in the presence of ascorbate and carboxyPTIO.

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