Enzyme maleimide activation

An affinity label is a molecule that contains a functionality that is chemically reactive and will therefore form a covalent bond with other molecules containing a complementary functionality. Generally, affinity labels contain electrophilic functionalities that form covalent bonds with protein nucleophiles, leading to protein alkylation or protein acylation. In some cases affinity labels interact selectively with specific amino acid side chains, and this feature of the molecule can make them useful reagents for defining the importance of certain amino acid types in enzyme function. For example, iodoacetate and A-ethyl maleimide are two compounds that selectively modify the sulfur atom of cysteine side chains. These compounds can therefore be used to test the functional importance of cysteine residues for an enzyme s activity. This topic is covered in more detail below in Section 8.4. 

Figure 20.3 The reaction of SMCC with the amine groups on enzyme molecules yields a maleimide-activated derivative capable of coupling with sulfhydryl-containing antibody molecules.

Figure 20.4 Reduction of the disulfide bonds within the hinge region of an IgG molecule produces half-anti-body molecules containing thiol groups. Reaction of these reduced antibodies with a maleimide-activated enzyme creates a conjugate through thioether bond formation.

A final consideration is to provide adequate desalting of the reduced antibody molecule from excess reducing agent. If even a small amount of a thiol-containing reductant remains, subsequent conjugation with a maleimide-activated enzyme will be inhibited. 

Pool the fractions containing antibody and immediately mix with an amount of maleimide-activated enzyme to obtain the desired molar ratio of antibody-to-enzyme in the conjugate. Use of a 4 1 (enzymerantibody) molar ratio in the conjugation reaction usually results in high-activity conjugates suitable for use in many enzyme-linked immunoassay procedures. Higher molar ratios also have been used with success. 

The number of sulfhydryls created on the immunoglobulin using thiolation procedures such as this one is more critical to the yield of conjugated enzyme molecules than the molar excess of maleimide-activated enzyme used in the conjugation reaction. Therefore, it is important to use a sufficient excess of Traut s reagent to obtain a sufficient number of available sulfhydryls. 

Figure 20.6 Available amine groups on an antibody molecule may be modified with the NHS ester end of SATA to produce amide bond derivatives containing terminal protected sulfhydryls. The acetylated thiols may be deprotected by treatment with hydroxylamine at alkaline pH. Reaction of the thiolated antibody with a maleimide-activated enzyme results in thioether crosslinks.

Add 10—40 ul of the SATA stock solution per ml of lmg/ml antibody solution. This will result in a molar excess of approximately 12- to 50-fold of SATA over the antibody concentration (for an initial antibody concentration of lmg/ml). A 12-fold molar excess works well, but higher levels of SATA incorporation will potentially result in more maleimide-activated enzyme molecules able to couple to each thiolated antibody molecule. For higher concentrations of antibody in the reaction medium, proportionally increase the amount of SATA addition however do not exceed 10 percent DMF in the aqueous reaction medium. 

The amine groups on these fragments also may be modified with thiolating agents, such as SATA or 2-iminothiolane, to create sulfhydryl residues suitable for coupling to maleimide-activated enzymes (Section 1.1, this chapter) (Figure 20.13). Amine groups further may be utilized... 

Figure 20.13 The thiolation reagent SATA can be used to create sulfhydryl groups on Fab fragments. After deprotection of the acetylated thiol of SATA with hydroxylamine, conjugation with a maleimide-activated enzyme can take place, producing thioether linkages.

Figure 23.4 Avidin may be modified with 2-iminothiolane to produce sulfhydryl groups. Subsequent reaction with a maleimide-activated enzyme produces a thioether-linked conjugate.

Reduction of the pyridyl disulfide end after SPDP modification releases the pyridine-2-thione leaving group and generates a terminal—SH group. This procedure allows sulfhydryl-reactive derivatives such as maleimide-activated enzymes (Chapter 26, Section 2.3) to be conjugated with DNA probes for use in hybridization assays (Malcolm and Nicolas, 1984). 

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