SMCC

SMCC frequently is used to prepare hapten-carrier or antibody-enzyme conjugates. In both applications, one of the molecules is activated (usually the carrier or the enzyme) with the... 

Figure 5.4 SMCC reacts with amine-containing molecules to form stable amide bonds. Its maleimide end then may be conjugated to a sulfhydryl-containing compound to create a thioether linkage.

Since SMCC is a water-insoluble crosslinker, it must be dissolved first in organic solvent (DMSO or DMF) before adding it to a protein to be modified. In some cases, addition of even a small amount of organic solvent to a protein solution may be detrimental to activity. To be safe, no more than 10-20 percent solvent should be present in the aqueous reaction medium. 

The following is a generalized protocol for the activation of a protein with sulfo-SMCC with subsequent conjugation to a sulfhydryl-containing second molecule or protein. Specific examples of the use of this crosslinker to make antibody-enzyme or hapten-carrier conjugates may be found in Chapter 20, Section 1.1 and Chapter 19, Section 5, respectively. 

Dissolve 10 mg of a protein or other macromolecule to be activated with sulfo-SMCC in 1 ml of 0.1 M sodium phosphate, 0.15 M NaCl, pH 7.2. 

Weigh out 2 mg of sulfo-SMCC and add it to the above solution. Mix gently to dissolve. To aid in measuring the exact quantity of crosslinker, a concentrated stock solution may be made in water (or DMSO) and an aliquot equal to 2 mg transferred to the reaction solution. If a stock solution is made, it should be dissolved rapidly and used immediately to prevent extensive hydrolysis of the active ester. As a general guideline of addition for a particular protein activation, the use of a 40- to 80-fold molar excess of crosslinker over the amount of protein present usually results in good activation levels. 

The generalized protocol for performing a multi-step conjugation reaction with MBS or sulfo-MBS is similar to that described for SMCC (Section 1.3, this chapter). Specific examples may be found in the cited references. 

Use of sulfo-NHS-LC-SPDP or other heterobifunctional crosslinkers to modify PAMAM dendrimers may be done along with the use of a secondary conjugation reaction to couple a detectable label or another protein to the dendrimer surface. Patri et al. (2004) used the SPDP activation method along with amine-reactive fluorescent labels (FITC or 6-carboxytetramethylrhodamine succinimidyl ester) to create an antibody conjugate, which also was detectable by fluorescent imaging. Thomas et al. (2004) used a similar procedure and the same crosslinker to thiolate dendrimers for conjugation with sulfo-SMCC-activated antibodies. In this case, the dendrimers were labeled with FITC at a level of 5 fluorescent molecules per G-5 PAMAM molecule. 

A common choice of crosslinker for this type of reaction is sulfo-SMCC, which has been used extensively for antibody conjugation (Chapter 20, Section 1.1). A better option for dendrimer conjugation is to use a similar crosslinker design, but one that contains a hydrophilic PEG spacer arm to promote dendrimer hydrophilicity after modification. Derivatization of an amine-dendrimer with a NHS-PEG-maleimide can create an intermediate that is coated with water-soluble PEG spacers. This modification helps to mask any potential for nonspecific interactions that the PAMAM surface may have, while providing terminal thiol-reactive maleimides for coupling ligands (Figure 7.10). 

If a NHS-PEG-maleimide compound is used for this type of activation and coupling, the intermediate maleimide-activated dendrimer should be quickly purified of excess crosslinker and reaction by-products and immediately used to couple ligand. This is due to the fact that the maleimide hydrolyzes in aqueous solution at a higher rate than an SMCC-type crosslinker, because of the extreme hydrophilicity of the PEG spacer arm compared to the cyclohexane spacer of SMCC. 

Figure 9.62 Sulfo-SMCC can be used to conjugate amine-containing QDs with thiol-containing proteins or other molecules using a two-step coupling procedure.

Add 1 mg of sulfo-SMCC to the QD solution with mixing to dissolve the crosslinker. 

Figure 19.16 A common way of conjugating sulfhydryl-containing haptens to carrier proteins is to activate the carrier with sulfo-SMCC to create an intermediate maleimide derivative. The maleimide groups then can be coupled to thiols to form thioether bonds.

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 19.18 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-25 of male-imide-activated BSA (A) and OVA (B) after reaction with sulfo-SMCC. The first peak is the protein and the second peak is excess crosslinker. The maleimide groups create increased absorbance at 280 nm in the activated proteins.

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.

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