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Overview
This is the standard “Ellman’s Test” for the determination of free thiols. (Ref. 1) It works well for small peptides and proteins synthesized using standard solid phase synthetic methods. Peptides from these syntheses are usually in their reduced form, and are usually stable to oxidation in acidic solutions. Free thiol can be determined in solutions collected from chromatographic separations or from reconstituted lyophilized samples. This protocol has been used for peptides (3 to 26mer) with a single Cys residue present and lacking tryptophan. (Ref. 2, 3) The technique should be feasible for multiple Cys residues (Hint 5).
Procedure
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Turn on UV-Vis spectrophotometer and setup, in your notebook, the Table described below.
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In an appropriate cuvette (see Hint 1), add 50 µL of the DTNB solution, 100 µL Tris solution, and water up to (1000 µL - sample µL).
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Volume example: 50 DTNB
100 Tris
840 Water
990 µL initial volume (take blank)
+10 Sample (added via syringe!)
1000 µL final volume (take reading at 412 nm)
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Mix solution carefully using pipette. Place cuvette into UV-Vis spectrophotometer and take a background scan using the solution as background.
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Introduce sample solution into cuvette with a syringe (Hint 2), keeping the cuvette in the instrument (Hint 3).
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Carefully mix solution with a pipettemen without disturbing the cuvette.
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Scan sample and record Absorbance at 412 nm. (Hint 4)
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Calculate absorbance for each sample and then average the results, divide this by 13600 M-1 cm-1 (the extinction coefficient of the reagent) to get the molarity of the solution. (Hint 5)
Sample Table and Calculation (Volumes in µL):
|
DTNB |
Tris |
Water |
Sample |
Abs (412nm) |
Abs (sample) |
|
|
|
|
|
|
|
|
50 |
100 |
840 |
10 |
0.5010 |
50.1 |
|
50 |
100 |
835 |
15 |
0.7540 |
50.3 |
|
50 |
100 |
830 |
20 |
1.0080 |
50.4 |
Equation:
Abs(sample) = (Tot. Vol./sample vol.) x (Abs 412)
Sample Calculation: Ave. = 50.2/13600 = 3.7 mM thiol
Solutions
DTNB solution = 50 mM sodium acetate (NaOAc)
2 mM DTNB in H2O
(refrigerate)
TRIS solution = 1 M Tris / pH 8.0
Biochemicals
DTNB = Ellman’s reagent = 5,5’-Dithiobis(2-nitrobenzoic acid)
Protocol Hints
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1 mL disposable plastic cuvettes work well. (However, see Hint 3)
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Keep samples frozen until use and on ice. Since this is supposed to be quantitative, we recommend using a syringe, not a pipette, for the sample solution, especially for small volumes and dilute solutions.
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When using plastic cuvettes, our experience has been that removal and reintroduction into the instrument of the same or different cuvette leads to anomalous readings/data. Thus, careful technique is used to introduce the sample and mix it in the sample holder of the instrument. Quartz cuvettes have no such problem with this. (But you have to clean them up each use.)
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If you set up the cuvettes and sample on ice near the UV-Vis, you can minimize the time the determinations take. Also, you won’t be accused of monopolizing the instrument.
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The calculation is for total free thiol. Presumably you know beforehand the total number of thiols are in your peptide. Divide the determined concentration by the number of thiols, and this is the peptide concentration. (See Hint 6)
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If your peptide contains more than 4 cysteine residues, you may want to recalculate the excess of DTNB and use a greater amount (e.g. 75 or 100 µL of solution)
References
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Ellman, G. L. (1959) Arch. Biochem. Biophys. 82, 70-77. (Original determination)
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Bulaj, G.; Kortemme, T.; Goldenberg, D. P. (1998) Biochemistry 37, 8965-8972. (Recent usage)
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Van Horn, J. D.; Bulaj, G.; Burrows, C. J. (2001) Unpublished results. |
