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 | |
Date Added: Tue May 14 2002 | |
Date Modified: Wed Apr 28 2004 | |
Abstract: This is the standard 揈llman抯 Test?for the determination of free thiols. It works well for small peptides and proteins synthesized using standard solid phase synthetic methods. | |
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
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
References