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Thiols and disulfides: Difference between revisions
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= Expression of proteins containing disulfides = | == Expression of proteins containing disulfides == | ||
Expression of proteins containing disulfides in the native state can make a lot of trouble when expressed using standard vectors and strains for cytoplasmic expression. | Expression of proteins containing disulfides in the native state can make a lot of trouble when expressed using standard vectors and strains for cytoplasmic expression. | ||
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= Determination of thiols and disulfides in proteins = | == Determination of thiols and disulfides in proteins == | ||
= Method according to Riddles: determination of thiols with DTNB = | === Method according to Riddles: determination of thiols with DTNB === | ||
Dilute protein in 1 ml of 6 M Guanidine-HCl, 50mM Tris-HCl, pH 8.3, 1 mM EDTA to a final concentration of 10-40 µM thiols. Add 10 µl of 10 mM DTNB (Dithionitrobenzoic acid; Ellman’s reagent) in 100 mM Tris-Cl, pH 7.6. Read absorption at 412 nm. Extinction coefficient is 13,600 M-1 cm-1 per thiol. | Dilute protein in 1 ml of 6 M Guanidine-HCl, 50mM Tris-HCl, pH 8.3, 1 mM EDTA to a final concentration of 10-40 µM thiols. Add 10 µl of 10 mM DTNB (Dithionitrobenzoic acid; Ellman’s reagent) in 100 mM Tris-Cl, pH 7.6. Read absorption at 412 nm. Extinction coefficient is 13,600 M-1 cm-1 per thiol. | ||
If the thiols in the protein are oxidizing very fast keep the protein at low pH, which will keep the thiol protonated. Only the thiolate is oxidizing very fast. At low pH the assay with DTNB does not work. | If the thiols in the protein are oxidizing very fast keep the protein at low pH, which will keep the thiol protonated. Only the thiolate is oxidizing very fast. At low pH the assay with DTNB does not work. | ||
Method according to Pedersen: determination of thiols with DTP: | === Method according to Pedersen: determination of thiols with DTP: === | ||
Dilute protein in 950 µl of 6 M Guanidine-HCl, 100 mM acetate, pH 4.0, 1 mM EDTA to a final concentration of 10-40 µM thiols. Add 50 µl of 3.2 mM DTP (2,2-Dithiopyridin) in 0.2 M acetate, pH 4.0. Read absorption at 343 nm. Extinction coefficient is 7,600 M-1 cm-1 per thiol. | Dilute protein in 950 µl of 6 M Guanidine-HCl, 100 mM acetate, pH 4.0, 1 mM EDTA to a final concentration of 10-40 µM thiols. Add 50 µl of 3.2 mM DTP (2,2-Dithiopyridin) in 0.2 M acetate, pH 4.0. Read absorption at 343 nm. Extinction coefficient is 7,600 M-1 cm-1 per thiol. | ||
Method according to Thannhauser: determination of disulfides and thiols | === Method according to Thannhauser: determination of disulfides and thiols === | ||
The amount of disulfides in a protein is assessed by determination of thiols generated through cleavage of disulfides by sulfite. For the measurements a derivative of DTNB has to be synthesized: | The amount of disulfides in a protein is assessed by determination of thiols generated through cleavage of disulfides by sulfite. For the measurements a derivative of DTNB has to be synthesized: | ||
NTSB synthesis: | NTSB synthesis: | ||
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* 6. Subtraction of number of determined thiols yields number of disulfides | * 6. Subtraction of number of determined thiols yields number of disulfides | ||
References | |||
=== References === | |||
* Pedersen, A. O., and Jacobsen, J. (1980) Reactivity of the thiol group in human and bovine albumin at pH 3-9, as measured by exchange with 2,2'-dithiodipyridine. Eur. J. Biochem. 106, 291-5. | * Pedersen, A. O., and Jacobsen, J. (1980) Reactivity of the thiol group in human and bovine albumin at pH 3-9, as measured by exchange with 2,2'-dithiodipyridine. Eur. J. Biochem. 106, 291-5. | ||