Ellman’s assay

The degree of —SH modification may be determined using the Ellman s assay (Section 4.1, this chapter). 

The deacetylated protein should be used immediately to prevent loss of sulfhydryl content through disulfide formation. The degree of —SH modification may be determined by performing an Ellman s assay (see Ellman s Assay for the Determination of Sulfhydryls, this chapter). 

Monitor the elution of reduced peptide from the column by measuring the absorbance at 280 nm (if peptide absorbs at this wavelength) as well as by performing an Ellman s assay (Section 4.1, this chapter) for sulfhydryl groups using a small aliquot (10-20 pi) of each collected fraction. 

Depending on the conditions, an Ellman s assay can detect as little as lOnM cysteine concentration. The linearity can extend into the mM range, making the test extremely flexible for different sample situations. 

Add iodoacetate to a concentration of 50mM in the reaction solution. Alternatively, add a quantity of iodoacetate representing a 10-fold molar excess relative to the number of —SH groups present. An estimation of the sulfhydryl content in the protein to be modified can be accomplished by performing an Ellman s assay (Chapter 1, Section 4.1). Readjust the pH if necessary. To aid in adding a small quantity of iodoacetic acid to the reaction, a concentrated stock solution may be made in the reaction buffer, the pH re-adjusted, and an aliquot added to the protein solution to give the desired concentration. 

An Ellman s assay comparing the unmodified protein to the iodoacetylated protein may be done to assess the degree of modification. 

Figure 19.19 An Ellman s assay may be done to determine the maleimide activation level of SMCC-derivatized proteins. Reaction of the activated carrier with different amounts of 2-mercaptoethanol results in various levels of sulfhydryls remaining after the reaction. 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 2-mercaptoethanol plus an unactivated carrier indicates the absolute amount of sulfhydryl that reacted. Calculation of the maleimide activation level then can be done.

Figure 19.21 The rate of reaction of cysteine with maleimide-activated BSA was determined using an Ellman s assay for remaining sulfhydryl groups after the reaction, according to Figure 19.20. Nearly all of the available maleimides are coupled with sulfhydryls within 2 hour.

Purify the thiolated toxin from unreacted Traut s reagent by gel filtration using 0.1M sodium phosphate, 0.15M NaCl, pH 7.5, containing lOmM EDTA. The presence of EDTA in this buffer helps to prevent oxidation of the sulfhydryl groups with resultant disulfide formation. The degree of —SH modification in the purified protein may be determined using the Ellman s assay (Chapter 1, Section 4.1). 

BMS, DMH and DTA are solids at room temperature. We recommend that their stock solutions ( 10 mM) in phosphate buffer (50 mM sodium phosphate, pH 7, 1 mM in EDTA) be prepared fresh by brief sonication to ensure complete solubilization. These solutions can be assayed for thiol groups by Ellman s assay (8). 

Ricin A-chain (Inland Labs) at 3 mg/mL in PBS was reduced for 30 min with 30 mM DTT at 30°C. Excess DTT was removed by Sephadex G-25 gel filtration in 5 mM sodium acetate buffer, pH 4.7, containing 50 mM NaCl and 0.5 mM EDTA. The thiol content was assessed by Ellman s assay (Ellman, 1959). MIANS (Molecular Probes) was added at a ratio of 0.9 mole MIANS 1.0 mole thiol. The reaction mixture was incubated at ambient temperature for 15 min after the pH was raised to 7.0 using 1 M Tris.HCl buffer, pH 7.4. The MIANS was quenched by addition of 1 mole equivalent of freshly prepared cysteine.HCl/mole MIANS and incubation for an additional 15 min. Any remaining thiol groups were alkylated by adding a 5-fold molar excess of iodoaceteimide over total thiol and after an additional 30 min, the protein (MIANS-ricin A-chain) was dialyzed against 0.1 M Tris.HCl buffer, pH 8.5. 

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