Proteins maleimide activated

React the protein with activated dendrimer for 2 hours at room temperature with mixing. At the completion of the reaction, cysteine may be added at 50 mM to block excess maleimide-reactive sites, which are not coupled with protein. 

After a carrier protein has been activated with sulfo-SMCC, it is often useful to measure the degree of maleimide incorporation prior to coupling an expensive hapten. Ellman s reagent may be used in an indirect method to assess the level of maleimide activity of sulfo-SMCC-activated proteins and other carriers. First, a sulfhydryl-containing compound such as 2-mercaptoethanol or cysteine is reacted in excess with the activated protein. The amount of unreacted sulfhydryls remaining in solution is then determined using the Ellman s reaction (Chapter 1, Section 4.1). Comparison of the response of the sample to a blank reaction using... 

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. 

Using this type of cysteine-uptake assay, it is possible to determine the percentage of maleimides that reacted over time. Thus, an indication of the reaction efficiency of a sulfhydryl-containing compound coupling with a maleimide-activated protein may be determined. Figure 19.21 shows the reaction rate for the coupling of cysteine to maleimide-activated BSA. Note that maximal coupling is obtained in less than 2 hours, and over 80 percent yield is achieved in less than 30 minutes. 

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.

Immediately purify the maleimide-activated protein by applying the reaction mixture to a desalting column. Do not dialyze the solution, since the maleimide activity will be lost over... 

Conjugate MCA-Gly peptides or maleimid-activated proteins to the iminothiolane-acivated carriers. 

However, due to the aromatic ring adjacent to its maleimide functional group, MBS displays less stability toward maleimide ring opening than SMCC (see this chapter Section 1.3). Unlike SMCC, MBS is therefore not recommended for preparing freeze-dried, maleimide-activated proteins, since during the processing necessary to purify and stabilize the derivative much activity can be lost by hydrolysis. 

Figure 287 Carrier proteins may be activated with sulfo-SMCC to produce maleimide derivatives reactive with sulfhydryl-containing molecules. The graphs show the gel filtration separation on Sephadex G-2S of maleimide-activated BSA (A) and OVA (B) after reaction with sulfo-SMCC. The first peak is the protein and the second peak is excess cross-linker. The maleimide groups create increased absorbance at 280 nm in the activated proteins.

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