Sulfhydryl oxidation metal catalyzed

Purify the reduced IgG from excess 2-mercaptoethylamine and reaction by-products by dialysis or gel filtration using a desalting resin. All buffers should contain 1-10 mM EDTA to preserve the free sulfhydryls from metal-catalyzed oxidation. The sulfhydryl-containing half antibody now may be used in conjugation protocols that use —SH-reactive heterobifunctional crosslinkers (Chapter 5, Section 1). 

Proteins modified with 2-iminothiolane are subject to disulfide formation upon sulfhydryl oxidation. This can cause unwanted conjugation, potentially precipitating the protein. The addition of a metal-chelating agent such as EDTA (0.01-0.1M) will prevent metal-catalyzed oxidation and maintain sulfhydryl stability. In the presence of some serum proteins (i.e., BSA) a 0.1M concentration of EDTA may be necessary to prevent metal-catalyzed oxidation, presumably due to the high contamination of iron from hemolyzed blood. 

Including EDTA in the final preparation inhibits metal-catalyzed oxidation of the sulfhydryl groups to disulfides. The modified peptide or protein should be used immediately to assure full sulfhydryl reactivity. 

Dissolve the sulfhydryl-containing protein or macromolecule to be modified at a concentration of l-10mg/ml in 50mM Tris, 0.15M NaCl, 5mM EDTA, pH 8.5. EDTA is present to prevent metal-catalyzed oxidation of sulfhydryl groups. The presence of Tris, an amine-containing buffer, should not affect the efficiency of sulfhydryl modification. Not only do amines generally react slower than sulfhydryls, the amine in Tris buffer is of particularly low reactivity. If Tris does pose a problem, however, use 0.1M sodium phosphate, 0.15M NaCl, 5mM EDTA, pH 8.0. 

The following protocol is a suggested method for labeling a protein with AMCA-HPDP. It is assumed that the presence of a sulfhydryl on the protein has been documented or created. The reaction conditions can be carried out in a variety of buffers between pH 6 and 9. Avoid the presence of extraneous sulfhydryl-containing compounds (such as disulfide reductants) that will compete in the reaction. The inclusion of EDTA in the modification buffer prevents metal-catalyzed sulfhydryl oxidation. Optimization for a particular labeling experiment should be done to obtain the best level of fluorophore incorporation. 

Dissolve the thiol-containing proteins to be crosslinked in 50 mM sodium phosphate, pH 6.5-7.5, containing 10 mM EDTA to prevent metal-catalyzed sulfhydryl oxidation. 

Figure 19.19 shows a plot of the results of such an assay done to determine the maleimide content of activated BSA. This particular assay used 2-mercaptoethanol which is relatively unaffected by metal-catalyzed oxidation. For the use of cysteine or cysteine-containing peptides in the assay, however, the addition of EDTA is required to prevent disulfide formation. Without the presence of EDTA at 0.1 M, the metal contamination of some proteins (especially serum proteins such as BSA) is so great that disulfide formation proceeds preferential to maleimide coupling. Figure 19.20 shows a similar assay for maleimide-activated BSA using the more innocuous cysteine as the sulfhydryl-containing compound. 

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.

Add at least 5 mg/ml of a sulfhydryl-containing protein or other molecule to the SPDP-modified vesicles to effect the conjugation reaction. Molecules lacking available sulf-hydryl groups may be modified to contain them by a number of methods (Chapter 1, Section 4.1). The conjugation reaction should be done in the presence of at least lOmM EDTA to prevent metal-catalyzed sulfhydryl oxidation. 

Dissolve the antibody to be modified at a concentration of 1—10 mg/ml in 0.1 M sodium phosphate, 0.15 M NaCl, pH 7.2, containing 10 mMEDTA. High levels of EDTA often are required to completely stop metal-catalyzed oxidation of sulfhydryl groups when working with serum proteins—especially polyclonal antibodies purified from antisera. Presumably, carryover of iron from partially hemolyzed blood is the contaminating culprit. 

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