Cysteine detection using Ellman

Figure 19.20 Cysteine also may be used in an Ellman s assay to determine the maleimide activation level of SMCC-derivatized proteins. Reaction of the activated carrier with different amounts of cysteine results in various levels of sulfhydryls remaining after the reaction. The coupling must be done in the presence of EDTA to prevent metal-catalyzed oxidation of sulfhydryls. Detection of the remaining thiols using an Ellman s assay indirectly indicates the amount of sulfhydryl uptake into the activated carrier. Comparison of the Ellman s response to the same quantity of cysteine plus an unactivated carrier indicates the absolute amount of sulfhydryl that reacted. Calculation of the maleimide activation level then can be done.

It should be noted that Ellman s assay does not require the use of external standards, or the generation of a standard curve, which is very convenient. However, it is limited in its sensitivity. These assay limitations are directly because of the inherent limitations in spectrophotometric detection. For instance, if an Ellman s assay sample shows an absorbance of 0.05 at 412 nm (approaching the lower limit of detection), the thiol concentration is calculated to be 3.7 /zM. This may be suitable when assaying proteins in the mg/mL concentration range (1 mg/ mL bovine serum albumin =15 uM), but not for lower concentrations. In some cases, the presence of unmodified cysteine thiols is aberrant or undesirable a more sensitive assay to detect low levels of such species would be useful. 

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