We are using 2,3-Diaminonaphthalene (Sigma Cat No. 2757) for assay of nitrosothiol. The chemical is stored at 4 degree Celsius in desiccator. Colour of the product is slightly brown. We are facing following problem with the product while performing fluorimetric assay: Fluorescence of 2,3-Diaminonaphthalene in acidic pH (in 0.62 N HCL) is 1346 and after adjusting the pH to 11, it becomes 1550. While performing fluorimetric assay for nitrosothiol, we are using mixture of DAN and HgCl2 (4:1) in 0.62 N HCL. for this mixture we are getting a very high fluorescence value of 5515 at pH 11 and 2342 at pH 3. So, as the mixture itself is giving a very high fluorescence, we are unable to perform the assay. What may be probable reason of such high fluorescence?
Fluorometric Detection of BiologicalS-Nitrosothiols
Juliet K.J. Park, Peter Kostka
A technique is presented for the quantitative detection of S-nitrosothiols formed by model biological thiols, cysteine, glutathione, and serum albumin. The technique is based on the detection of fluorescent compound 1-[H]-naphthotriazole formed between 2,3-diaminonaphthalene and nitrous acid released from S-nitrosothiols by treatment with mercuric chloride in an acidic environment. Concentration of S-nitrosothiols is determined from the difference in fluorescent signal (excitation/emission wavelengths of 363 nm/450 nm, respectively) observed in the presence and absence of 0.18 mM HgCl2. The yield of the reaction between 2,3-diaminonaphthalene and nitrous acid released from the S-NO bond by HgCl2 approaches 90-100% as documented by simultaneous assays of S-nitrosothiols by uv spectrophotometry and by Saville method. The assay can be applied to the analysis of mixtures containing excess of thiol and/or nitrite at neutral pH by pretreatment of samples with N-ethylmaleinimide and/or ammonium sulfamate, respectively. In analysis of S-nitrosothiols in protein-containing mixtures, HgCl2-mediated release of nitrous acid in the presence of 2,3-diaminonaphthalene is followed by neutralization of samples and precipitation of protein with 0.5 M 5-sulfosalicylic acid. The fluorometric assay is carried out at an excitation wavelength of 380 nm to eliminate the background fluorescence of 5-sulfosalicylic acid observed at lower wavelengths. The technique offers simple and rapid determination of S-nitrosothiols in complex reaction mixtures with the detection limit at low nanomolar concentrations.
Fluorescence intensity measurements are on an arbitrary scale. The actual numbers are meaningless except in terms of the dynamic range of the instrument. For example, if your instrument can read up to a value of 6000, then you have a problem, but if it can read up to 100,000 you don't.
The setup of the instrument can be changed to increase or decrease the reading. Adjustable instrument parameters on a spectrofluorometer include excitation and emission wavelengths, slit widths, and detector gain. If your blank is giving a reading that is near the top of the dynamic range of the instrument, one or more of these parameters can be adjusted to lower it. Plate readers usually have adjustable instrument parameters as well.
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