| Analytical measurement of discrete hydrogen sulfide pools in biological specimens. | |
| | |
MedLine Citation:
|
PMID: 22561703 Owner: NLM Status: Publisher |
Abstract/OtherAbstract:
|
Hydrogen sulfide (H(2)S) is a ubiquitous gaseous signaling molecule that plays a vital role in numerous cellular functions and has become the focus of many research endeavors, including pharmacotherapeutic manipulation. Among the challenges facing the field is the accurate measurement of biologically active H(2)S. We have recently reported that the typically used methylene blue method and its associated results are invalid and do not measure bona fide H(2)S. The complexity of analytical H(2)S measurement reflects the fact that hydrogen sulfide is a volatile gas and exists in the body in various forms, including a free form, an acid-labile pool, and bound as sulfane sulfur. Here we describe a new protocol to discretely measure specific H(2)S pools using the monobromobimane method coupled with RP-HPLC. This new protocol involves selective liberation, trapping, and derivatization of H(2)S. Acid-labile H(2)S is released by incubating the sample in an acidic solution (pH 2.6) of 100mM phosphate buffer with 0.1mM diethylenetriaminepentaacetic acid (DTPA), in an enclosed system to contain volatilized H(2)S. Volatilized H(2)S is then trapped in 100mM Tris-HCl (pH 9.5, 0.1mM DTPA) and then reacted with excess monobromobimane. In a separate aliquot, the contribution of the bound sulfane sulfur pool was measured by incubating the sample with 1mM TCEP (tris(2-carboxyethyl)phosphine hydrochloride), a reducing agent, to reduce disulfide bonds, in 100mM phosphate buffer (pH 2.6, 0.1mM DTPA), and H(2)S measurement was performed in a manner analogous to the one described above. The acid-labile pool was determined by subtracting the free hydrogen sulfide value from the value obtained by the acid-liberation protocol. The bound sulfane sulfur pool was determined by subtracting the H(2)S measurement from the acid-liberation protocol alone compared to that of TCEP plus acidic conditions. In summary, our new method allows very sensitive and accurate measurement of the three primary biological pools of H(2)S, including free, acid-labile, and bound sulfane sulfur, in various biological specimens. |
| | |
Authors:
|
Xinggui Shen; Elvis A Peter; Shyamal Bir; Rui Wang; Christopher G Kevil |
Related Documents
:
|
6997283 - Identification and properties of the prosthetic group of choline oxidase from alcaligen... 442083 - Isolation and characterization of proteohyaluronic acid from human umbilical cord. 3782473 - Attachment of human c5a des arg to its cochemotaxin is required for maximum expression ... |
Publication Detail:
|
Type: JOURNAL ARTICLE Date: 2012-4-19 |
Journal Detail:
|
Title: Free radical biology & medicine Volume: - ISSN: 1873-4596 ISO Abbreviation: - Publication Date: 2012 Apr |
Date Detail:
|
Created Date: 2012-5-7 Completed Date: - Revised Date: - |
Medline Journal Info:
|
Nlm Unique ID: 8709159 Medline TA: Free Radic Biol Med Country: - |
Other Details:
|
Languages: ENG Pagination: - Citation Subset: - |
Copyright Information:
|
Copyright © 2012 Elsevier Inc. All rights reserved. |
Affiliation:
|
Department of Pathology, Louisiana State University Health Sciences Center, Shreveport, LA 71130, USA. |
Export Citation:
|
APA/MLA Format Download EndNote Download BibTex |
| MeSH Terms | |
Descriptor/Qualifier:
|
|
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine
Previous Document: Redox-sensitive YFP sensors monitor dynamic nuclear and cytosolic glutathione redox changes.
Next Document: Regulation of cell cycle and stress responses under nitrosative stress in Schizosaccharomyces pombe.