Disulfide bridge formation

Disulfide bridges formation ChEs contain 8-10 cysteines six of these form three internal disulfide bridges. The cysteine that is located four amino acids upstream the carboxyl terminus forms a disulfide bridge with a cysteine of an identical subunit, creating an interchain disulfide bridge, which stabilizes the dimeric structure. 

Disulfide-Bridge Formation in Homodimeric Two-Stranded Coded Coils... 

Remarkably, the mutation of the type 1 copper site Cys-310 to Ser in the Al domain resulted in an inactive enzyme that was partially defective for secretion from the cell (Tagliavacca et al., 1997). The occurrence of intact type 1 copper sites in F8 is also supported by the fact that their putative cysteine ligand residues were shown not to be involved in disulfide bridge formation (McMullen et al., 1995). Thus the F8 has hve halfcysteines in each A domain. Analysis of the disulhde bond pattern in F8 showed that four half-cysteines are involved in the formation of two disulfide bonds, while the fifth remains free. The free Cys residues have been identified as Cys-310 in domain Al, Cys-692 in domain A2, and Cys-2000 in domain A3. All these Cys residues match analogous residues involved in type 1 copper binding in ceruloplasmin. However, as mentioned above, only two of the domains, Al and A3, contain the complete set of ligands for the type 1 copper. 

Stir vigorously the solution at room temperature for 12 h to incorporate atmospheric oxygen into the solution. Ideally, disulfide bridge formation should be monitored by reverse phase HPLC. Purification (step 4) should be performed immediately after the end of the reaction. 

Five oxidation procedures were compared for the disulfide bridge formation. 

Additional factors that could influence the delivery efficiency and possible side effects of PNA-vector chimeras include the physical linkage between the PNA sequence and vector. Below we provide an overview of different conjugation strategies and provide a step-by-step protocol for disulfide bridge formation between PNA and carrier peptide. 

Organic solvents in disulfide bridge formation protocols increase PNA solubility. If the PNA solubility in buffer is known to be high, their amount can be reduced. If PNA is insoluble in organic solvents, 20 )uL of TFA may be added to the pellet. 

The protected peptide is isolated, before disulfide bridge formation, by precipitation from its N-methylpyrrolidone solution (95.5 % yield) with water. 

Different protecting groups (S-Trityl, S-Acetamidomethyl...) have been introduced on the cysteine side chains to optimise the reaction conditions of disulfide bridge formation. The conditions developed by Kamber and his group have been used to make the disulfide bridge (ref. 5). The best results have been obtained when we use S trityl on the L-cysteine and S-acetamidomethyl on the 7 cysteine in N,N-dimethylformamide in the presence of an excess of iodine (4 equivalents). The excess of iodine is eliminated with ascorbic acid. The final peptide is isolated by precipitation with water. 

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