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Quantitative Determination of Peptides by Sulfhydryl (-SH) Groups
| Quantitative Determination of Peptides by Sulfhydryl (-SH) Groups | ||||||||||||||||||||||||||||||
| Author: David Van Horn, Greg Bulaj | ||||||||||||||||||||||||||||||
| Source: Contributed by David Van Horn, Dept. of Chemistry, UC Berkeley Greg Bulaj, Dept. of Biology, University of Utah | ||||||||||||||||||||||||||||||
| Abstract: This is the standard �llman� Test?for the determination of free thiols. It works well for small peptides and proteins synthesized using standard solid phase synthetic methods. | ||||||||||||||||||||||||||||||
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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
Turn on UV-Vis spectrophotometer and setup, in your notebook, the Table described below. In an appropriate cuvette (see Hint 1), add 50 µ L of the DTNB solution, 100 µ L Tris solution, and water up to (1000 µ L - sample µ L). Volume example: 50 DTNB 100 Tris 840 Water 990 µ L initial volume (take blank) + 10 Sample (added via syringe!) 1000 µ L final volume (take reading at 412 nm) Mix solution carefully using pipette. Place cuvette into UV-Vis spectrophotometer and take a background scan using the solution as background. Introduce sample solution into cuvette with a syringe (Hint 2), keeping the cuvette in the instrument (Hint 3). Carefully mix solution with a pipettemen without disturbing the cuvette. Scan sample and record Absorbance at 412 nm. (Hint 4) 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 µ L):
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 H2 O
(refrigerate)
TRIS solution = 1 M Tris / pH 8.0
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