A nonradioactive assay for nitric oxide synthase activity in tissue extracts

Hazel Sutherland; Olga Zolle; Roba Khundkar; Alec W M Simpson; Jonathan C Jarvis; Stanley Salmons

doi:10.1385/1-59259-807-2:105

A nonradioactive assay for nitric oxide synthase activity in tissue extracts

Methods Mol Biol. 2004:279:105-11. doi: 10.1385/1-59259-807-2:105.

Authors

Hazel Sutherland¹, Olga Zolle, Roba Khundkar, Alec W M Simpson, Jonathan C Jarvis, Stanley Salmons

Affiliation

¹ Department of Human Anatomy and Cell Biology, University of Liverpool, UK.

PMID: 15199239
DOI: 10.1385/1-59259-807-2:105

Abstract

We describe a fluorescence assay for nitric oxide synthase activity based on a new indicator, 4,5-diaminofluorescein (DAF-2). The method offers the advantage of being safer and more convenient than the citrulline radioassay in common use. The rapid and irreversible binding of DAF-2 to oxidized nitric oxide (NO) enables NO production to be measured in real time. The protocol is applied to the measurement of nitric oxide synthase in crude extracts of skeletal muscle.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Animals
Citrulline / chemistry
Fluorescein / chemistry*
Muscle, Skeletal / enzymology*
Nitric Oxide / metabolism*
Nitric Oxide Synthase / metabolism*
Spectrometry, Fluorescence / methods
Tissue Extracts / metabolism*

Substances

4,5-diaminofluorescein
Tissue Extracts
Citrulline
Nitric Oxide
Nitric Oxide Synthase
Fluorescein