Heterobifunctional reagents

A most promising group of such reagents contains maleimide derivatives (Ishikawa et al., 1978). They produce results far superior to GA (Deelder and De Water, 1981). 

A-Hydroxysuccinimide ester of p-formylbenzoic acid, on the other hand, is satisfactory for the labeling of MPOase to antibody (Krae-henbuhl et al., 1974 Tijssen and Kurstak, 1974). 

N-Hydroxysuccinimide (NHS) esters of maleimide derivatives Maleimide groups react rapidly under mild conditions 

MBS contains a reactive NHS ester and a reactive maleimide residue (Fig. 11.12) both of which react under mild conditions. Acylation of the free amino groups of one protein (e.g., IgG) occurs 

The conjugates are purified by chromatography on a Sephadex G-200 column (70 ml sample 1.5 ml), equilibrated with 10 mM Tris-HCl buffer, pH 7.0, containing 10 mM MgC and 50 mM NaCl. The conjugate elutes with the void volume and can be stored in the presence of 0.1% BSA and 0,02% NaNs for at least a year at 4°C. This method will separate free IgG from free enzyme and the conjugates. SpA-Sepharose may, in some cases, be used to separate enzyme from conjugate (Section 11.2.4). 

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The greatest degree of control in crosslinking procedures is afforded using heterobifunctional reagents (Chapter 5). Since a heterobifunctional crosslinker has different reactive groups on either end of the molecule, each side can be directed specifically toward different functional... 

SIAX, succinimidyl-6-((iodoacetyl)amino)hexanoate, is a heterobifunctional reagent containing an NHS ester on one end and an iodoacetyl group on the other end (Brinkley, 1992)... 

Figure 5.11 NPIA is one of the shortest heterobifunctional reagents. It reacts with amine-containing molecules through its p-nitrophenyl ester end to produce amide bonds. The iodoacetyl group then can be used to couple with thiol compounds to give stable thioether linkages.

Sulfo-SAMCA, sulfosuccinimidyl-7-azido-4-methylcoumarin-3-acetate, is a heterobifunctional reagent similar in design to SAED (Section 3.9, this chapter) (Thermo Fisher). One end of the crosslinker contains an amine-reactive sulfo-NHS ester, while the other end is an AMCA derivative (Chapter 9, Section 3) that contains a photosensitive phenyl azide group. Unlike... 

The most common type of heterobifunctional reagent used for the activation of lipid components includes the amine- and sulfhydryl-reactive crosslinkers containing an N-hydroxysuccinim-ide (NHS) ester group on one end and either a maleimide, iodoacetyl, or pyridyl disulfide group on the other end (Chapter 5, Section 1). Principle reagents used to effect this activation process include SMCC (Chapter 5, Section 1.3), MBS (Chapter 5, Section 1.4), SMPB (Chapter 5, Section 1.6), SIAB (Chapter 5, Section 1.5), and SPDP (Chapter 5, Section 1.1). Other... 

Carlsson, J., Drevin, H., and Axen, R. (1978) Protein thiolation and reversible protein-protein conjugation. N-Succinimidyl 3(2-pyridyldithio)propionate, a new heterobifunctional reagent. Biochem. J. 173, 723-737. 

Ji, T.H., and Ji, I. (1982) Macromolecular photoaffinity labeling with radioactive photoactivatable heterobifunctional reagents. Anal. Biocbem. 121, 286-289. 

Kumar, P., Agarwal, S.K., and Gupta, K.C. (2004) N-(3-Trifluoroethanesulfonyloxypropyl)-anthraqui-none-2-carboxamide a new heterobifunctional reagent for immobilization of biomolecules on a variety of polymer surfaces. Bioconjugate Chem. 15, 7-11. 

Politz, S.M., Noller, H.F., and McWhirter, P.D. (1981) Ribonucleic acid-protein cross-linking in Escherichia coli ribosomes (4-azidophenyl) glyoxal, a novel heterobifunctional reagent. Biochemistry 20, 372-378. 

The non-radioactive labeling utilizes fluorescence, chemiluminescence, or biotin/avidin interactions. Capillary electrophoresis with laser-induced fluorescence was first employed in PAL by Miller et al. [51]. Gilbert and Rando recently reported several biotin-containing heterobifunctional reagents and used them successfully [18] (Fig. 5). 

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