Thiol-functionalised microspheres allow covalent coupling of numerous molecules that can be derivatized into an iodoacetyl or maleimide compounds. Commercially available heterobifunctional cross-linkers are routinely used for chemical modifications of proteins at one end, and covalent attachment to one microspheres at the other end.  

 

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Thiol-Reactive Chemical Reaction

 

Reactive groups able to couple with thiol contained in molecules (or coated onto the surface of the microspheres) are frequently used on cross-linking, especially in the design of heterobifunctional cross-linkers where sulfhydryl-reactive groups are present on one of two ends. The other end of such cross-linkers is often an amine-reactive functional group.

 

The primary coupling chemical reactions for modification of Thiol-microspheres proceed by alkylation or disulfide interchange. Many of the reactive groups involved in these reactions are stable in aqueous environment and allow a two-step conjugation strategy.

 

Moreover, these reactions present the advantage to be rapid and to give stable thioether or disulfide bonds. Chemical reactives able to effect a coupling with a thiol functional group are numerous: Haloacetyl and Alkyl halide derivatives; Maleimides; Aziridines; Acryloyl derivatives;
Arylating Agents; Thiol-Disulfide Exchange reagents.

 

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Main Chemical reactions

 

Main functional groups involved in reactions with Thiol to create sulfhydryl-reactive compounds are Haloacetyls and Maleimides.

 

Haloacetyl and alkyl Halide Derivatives 

 

Three forms of activated halogen derivatives can be used to create sulfhydryl-reactive compounds: haloacetyl and benzyl halides that react through a resonance activation process with the neighboring benzene ring; alkyl halides that possess the halogen β to a nitrogen or sulfur atom. In each of these compounds the halogen is easily displaced by an attacking nucleophilic substance to form an alkylated derivative with loss of HX. The iodoacetyl derivative has the highest reactivity toward sulfhydryl and may be directed specifically for SH modification. The specificity of this modification has been used in the design of heterobifunctionnal cross-linking reagents, where one end of the cross-linker is an iodoacetamide derivative and the other end contains a different group directed at another chemical target.

 

Maleimides
 

The double bond of maleimide may undergo an alkylation reaction with sulfhydrylgroups to form stable thioether bonds. Maleimide reactions are specific for sulfhydryl groups in the pH range 6.5-7.5. At pH 7, the reaction of the maleimide with sulfhydryl proceeds at a rate 1000 times greater than its reaction with amines. At higher pH values, some cross-reactivity with amino groups takes place.

 

Some examples of heterobifunctionnal cross-linkers with an sulfhydryl reactive group

 

• N-succinimidyl (4-iodoacetyl) aminobenzoate (SIAB) with amine-reactive and sulfhydryl-reactive ends.The NHS ester of SIAB can couple to primary amine-containing molecules, forming stable amide linkages. The iodoacetyl group creates stable thioether bonds. SIAB is water-insoluble; it must be first dissolved in organic solvent (DMSO and DMF) prior to addition to an aqueous reaction medium.
• Succinimidyl-4-(N-maleimidomethyl) cyclohexana-1-carboxylate (SMCC) with NHS ester andmaleimide ends. The SMCC is a heterobifunctional cross-linker with significant utility in cross-linking proteins (antibody, enzyme) and hapten-carrier conjugates. The maleimide end of SMCC is specific when the reaction pH is in the range of 6.5-7.5. At pH 7 the reaction of the maleimides with sulfhydryls proceeds at a rate 1000 times greater than its reaction with amines. SMCC is a water-insoluble cross-linker; it must be dissolved first in organic solvent (DMF) before it is added to a protein to be modified.
• m-Maleimidobenzoyl-N-hydroxysuccimide ester (MBS) with NHS ester and maleimide ends. The NHS ester can react with primary amines in proteins and other molecules to form stable amide bonds, while the maleimide end nearly exclusively reacts with sulfhydryl groups to create stable thioether linkages. These characteristics allow highly controlled conjugation to be done with MBS using two- or  three-step processes. In this sense, the NHS ester end of the reagent typically is reacted with the first protein to be cross-linked, forming an maleimide-activated intermediate. The maleimide group is more stable to breakdown by hydrolysis than the NHS ester, so the activated intermediaries can be quickly purified from excess cross-linker and reaction by-products before it is added to the sulfhydryl present onto the surface of microspheres. MBS is a water-insoluble reagent so it must be first dissolved in organic solvent (DMF or DMSO).
• N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP) may be the most popular heterobifunctional cross-linking agent available. The activated NHS ester end reacts with amine groups in proteins and other molecules to form an amide linkage. The 2- pyridylthiol group at the other end reacts with sulfhydryl residues to form a disulfide linkage with sulfhydryl present onto the surface of microspheres.

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Coupling IgG on SH-Microspheres with SMCC

 

Particle Characteristics

 

• Reference: Estapor R00-07
• Polymer : Polystyrene
• Mean size : 1.46 µm
• Number of SH : 18 µeq/g (Ellman method) or 290 µeq/g (Sulphate assay)

 

Protocol

 

1°) Dialyse the protein against 50 volumes of PBS.
2°) Incubate SMCC (1 mg/ml in DMF/PBS) and protein (1-10 mg/ml in PBS) for 75 min at +4°C.
3°) Eliminate free SMCC and unmodified proteins on a Sephadex G15 column Elute with HEPES
0.1 M pH 7.5. The elution of the protein-SMCC can be monitored at 206 nm with a
spectrophotometer.
4°) Prepare 100 µl of Estapor® microspheres at 10% solid, previously washed twice with PBS.
5°) Incubate the Estapor® microspheres with 1 ml of DTT (dithiothreitol 3 mg/ml) for 15 min at room
temperature, and wash the Estapor® microspheres 3 times with PBS.
6°) Mix 100 µl of the reduced Estapor® microspheres with the protein-SMCC conjugate, and
incubate for 12hrs at +4°C.
7°) Wash the Estapor® microspheres and resuspended in the desired buffer.

 

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Literature

 

Hashida, S., and Ishikawa, E. (1985) Use of normal IgG and its fragments to lower the non-specific binding of Fab’-enzyme conjugates in sandwich enzyme immunoassay.
Anal.Lett.18 (B9), 1143-1155

 

Dewey, R.E. et al. (1987) A mitochondrial protein associated with cytoplasmic male sterility in the T cytoplasm of maize.
Proc.Natl.Acad.Sci.USA 84, 5374-5378

 

Smyth, D.G. et al (1964) Reaction of N-ethylmaleimide.
J.Am.Chem.Soc.82, 4600

 

Hermanson, G.T., (1996) in "Bioconjugates Techniques.
Academic Press, San Diego