Crosslinkers disulfide cleavable

Control experiments showed that all the crosslinks were cleavable. No crosslinks were formed if the reagent was omitted, if photolysis was omitted, or if the reagent was first inactivated by treatment with ammonium acetate. These experiments proved that neither monofunctional cross-linking, nor disulfide interchange to give crosslinking in the dark occurred. If the membranes were solubilized in SDS before irradiation no cross-... 

Reported structures for homobifunctional aryl azides include a biphenyl derivative and a naphthalene derivative (Mikkelsen and Wallach, 1976), a biphenyl derivative containing a central, cleavable disulfide group (Guire, 1976), and a compound containing a central l,3-diamino-2-propanol bridge between phenyl azide rings that are nitrated (Guire, 1976). The only commercially available homobifunctional photoreactive crosslinker is BASED. 

Figure 4.21 BASED can react with molecules after photoactivation to form crosslinks with nucleophilic groups, primarily amines. Exposure of its phenyl azide groups to UV light causes nitrene formation and ring expansion to the dehydroazepine intermediate. This group is highly reactive with amines. The cross-bridge of BASED is cleavable using a disulfide reducing agent.

SADP, N-succinimidyl-(4-azidophenyl)l,3 -dithiopropionate, is a photoreactive heterobifunctional crosslinker that is cleavable by treatment with a disulfide reducing agent (Thermo Fisher). The crosslinker contains an amine-reactive NHS ester and a photoactivatable phenyl azide group, providing specific, directed coupling at one end and nonselective insertion capability at the other end. 

Figure 6.2 The trifunctional reagent sulfo-SBED reacts with amine-containing bait proteins via its NHS ester side chain. Subsequent interaction with a protein sample and exposure to UV light can cause crosslink formation with a second interacting protein. The biotin portion provides purification or labeling capability using avidin or streptavidin reagents. The disulfide bond on the NHS ester arm provides cleavability using disulfide reductants, which effectively transfers the biotin label to an unknown interacting protein.

The use of periodate as a cleavage agent does have advantages, however. Unlike the use of cleavable crosslinkers that contain disulfide bonds which require a reductant to break the conjugate, cleavage of diol-containing crosslinks with periodate typically preserves the indigenous disulfide bonds and tertiary structure of proteins and other molecules. As a result, with most proteins bioactivity usually remains unaffected after mild periodate treatment. 

However, since SMCC forms nonreversible thioether linkages with sulfhydryl groups, it only can be used in the preparation of immunotoxins if intact A-B toxins are employed in the conjugate. In such conjugates, the A chain still have the potential for reductive release from the B-chain subunit after cellular docking and internalization. Immunotoxins prepared with A-chain or single-subunit toxins will not display cytotoxicity if crosslinked with SMCC, since the crosslinker does not create cleavable disulfide bonds upon conjugation. 

In another case in which a number of exemplary control experiments were done, Markwell and Fox (1980) crosslinked the outer membranes of enveloped viruses with methyl 3-(p-azidophenyl)dithio]propionimidate. Virus (4 mg protein/ml) was reacted with the imidate (0.1 to 0.5 mM) at 0°C for 30 min at pH 8.5. The reaction was quenched with 50 mM ammonium acetate, 50 mM NEM (30 min, 25 °C), and the vims recovered by centrifugation. After irradiation the crosslinked polypeptides were examined in a two-dimensional SDS-polyacrylamide gel. One complication was that the crosslinking pattern had to be compared with a native pattern of disulfide linkages, and a reagent with a different cleavable crosslink may have been a better choice. As mentioned above, the analysis was simplified by the use of surface labeling. 

SPDP is a heterobifunctional cleavable crosslinker containing a /V-hydroxysuccinimdc residue and a pyridyl disulfide residue to react with primary amines and sulfhydryls, respectively (8). SPDP is stable as a solution in ethanol at room temperature as long as it is kept free of moisture thus a 20-mM solution may be prepared and used for several days. The powder form of SPDP should be stored surrounded by silica gel (or another drying agent) because it is very unstable in water. 

Lee, H. and T. G. Park 1998. Reduction/oxidation induced cleavable/crosslinkable temperatme-sensitive hydrogel network containing disulfide linkages. Polymer Journal. 30 976-80. 

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