GE MOA 280i, Руководство по эксплуатации и обслуживанию

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GE Analytical Instruments ©2006
14-1
DLM 14291 Rev. A
14.
OTHER LIQUID MEASUREMENT TECHNIQUES
Measurement of Nitrosothiols
Several techniques have been developed to allow conversion of RSNO
compounds to NO for measurement by the NOA. Nitrosothiols are generally
present at low nanomolar concentrations and will require injection of larger
volume (50-1000 µL) of sample for detection. A brief description of some
reported techniques is given below, consult the original reference for more
information.
Cu(I)/Cysteine Reagent
Fang and coworkers (Fang, K., Ragsdale, N.V., Carey, R.M., MacDonald, T., and
Gaston, B. Reductive Assays for S-Nitrosothiols: Implications for Measurements
in Biological Systems. Biochem Biophys Res Comm 1999;252:535-540) have
reported the use of copper(I) ions in combination with cysteine for the
conversion of low molecular weight nitrosothiols to NO using the purge vessel.
While the efficiency of conversion is high for lower molecular weight
compounds such as S-nitroso-glutathione, lower efficiencies were reported for
high molecular weigh species such as S-nitroso-albumin. This regent was
specific for nitrosothiols, with no response reported for nitrite, nitrate or nitro-
containing compounds at pH > 6.
Preparation of Reducing Agent
The reducing agent is PBS, another buffer, or water that contains 1 mM
cysteine and is staturated with CuCl. Since CuCl has low water solubility, only a
few milligrams of the solid is sufficient to prepare a saturated solution. The
reagent can be used at room temperature, but more rapid conversions are
obtained at elevated temperatures (50 °C). The highest response for GSNO was