GE Analytical Instruments ©2006 14-3 DLM 14291 Rev. A
Preparation of S-Nitroso-Albumin
The procedure for preparing S-nitroso-albumin is described in this reference
and involves treatment of albumin with dithiothreitol and DTPA to fully reduce
the thiol groups, dialysis, then trans-nitrosylation using S-nitroso-cysteine
(prepared by reaction of equimolar amount of thiol and nitrite). Unreacted
thiols were alkylated with N-ethylmaleimide (1 mM) and the stock solution
stored at – 20 °C. S-nitroso-albumin concentration was determined by the
Saville reaction.
Treatment of Plasma Samples
Samples were collected in pre-chilled tubes containing EDTA (final
concentration 2 mM) and N-ethylmaleimide (final concentration 5 mM). After
centrifugation, the samples are treated with 0.5% sulfanilamide in 0.1 M HCl to
remove the nitrite.
A similar method has been developed by Gladwin and co-workers (Gladwin,
M.T., Shelhamer, J.H., Schechter, A.N., Pease-Fye, M.E., Waclasis, M.A.,
Panza, J.A., Ognibene, F.P.and Cannon III, R.O. Role of circulating nitrite and
S-nitrosohemoglobiun in the regulation of regional blood flow in humans Proc
Natl Acad Sci 2000; 97:11482-11487) for the measurement of S-
nitrosohemoglobin in red blood cells. The reagent is similar to the reducing
agent for nitrite (acetic acid/sodium iodide) but a crystal of I
2
is added to the
purge vessel to convert RSNOs.
To eliminate response from iron-bound NO, the samples are pretreated with
100-fold excess of 0.2 M KCN and 0.2 M K
3
Fe(CN)
6
in 0.5 mM EDTA. Nitrite was
removed through use of a Sephadex G25 column.
Measurement of Iron-bound NO
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