Sulfitolysis

Proinsulin fusion E. coli cells sulfitolysis SEC Affix UV 61 B p... 

J. S. Pati ick and A. L. Lagu, Determination of recombinant human proinsulin fusion protein produced in Escherichia coli using oxidative sulfitolysis and two-dimensional HPLC, Chem. 64 507-511 (1992). 

Sulfitolysis occurs simultaneously with Keratin denatured through direct reduction... 

In some processes sulfite is added and undoubtedly forms derivatives of sulfhydryls by sulfitolysis of disulfide bonds. Little is known about these products. 

Photometric determinations with p-chloromercuribenzoate (3) were carried out in phosphate at pH 8 27 1SH groups were found per mole of unmodified and also of acetyl glutamate-activated carbamyl-P synthetase. With cold-inactivated enzyme, however, only 19 to 20 groups could be detected. The same results were obtained by amperometric titration in aqueous solution (2) and in 8M urea (5) after sulfitolysis on the cold-inactivated enzyme, an average of 23 SH groups per mole of enzyme was determined. The cases thus far examined, of substrate-induced enzyme inactivation, show also a change in SH group content (1, 14). 

The basis utilized by Petrides et al.15 is 1500 kg of purified BHI per year. They indicate that this represents 10-15% of the world demand.17 In essence, the following downstream steps are involved in sequence during synthetic BHI production. The fermentation step (not a downstream step) is also included to provide some continuity. The steps are fermentation, cell harvesting, cell disruption, inclusion body recovery, inclusion body solubilization, enzymatic conversion, refolding, sulfitolysis, CNBr cleavage, final purification steps, and crystallization. In the flow chart provided by Petrides et al.15, a surge tank separates the upstream from the downstream processes. This tank is in between the fermentor and the downstream processing steps. 

The chimeric protein is then cleaved with CNBr into Trp-LE -Met(which contains 121 amino acids) and the denatured proinsulin (82 amino acids). This denatured proinsulin must be converted to the active insulin form. Sulfitolysis is carried out under alkaline conditions wherein the proinsulin... 

By oxidative sulfitolysis. Originally this method was conceived as involving (i) fission of disulfide by sulfite to produce thiol and S-sulfo-cysteine, and (ii) oxidation of this thiol to disulfide which again reacts with sulfite as in (i). The over-all reaction is the conversion of disulfide to >S-sulfocysteine. On this basis the reactions can be formulated as in reaction (II). 

Sulfonate derivatives of cysteine peptides, also referred to as Bunte salts, are prepared by oxidative sulfitolysis of cysteine or cystine peptides (Scheme These derivatives... 

The S-sulfo derivative is stable at neutral and acidic pH. Sulfitolysis has been employed as a mild procedure for the cleavage of disulfide bonds in multi-chain proteins, as a preliminary to polypeptide fractionation, and also to provide a protective blocking group for the cysteinyl residue. The S-sulfo derivative is readily reduced back to the thiol by the addition of sulfhydryl compounds, e.g. -mercaptoethanol. 

The following sulfitolysis procedure is based upon the work of Chan (1968). 

The purified inclusion body fraction is en solubilized and the residual insoluble material is pelleted and discarded. Solubilization of inclusion bodies is accomplished by using a combination of denaturants to disrupt non-covalent interactions and thiol reagents or sulfitolysis to break disulfide bonds. 

Oxidative decarboxylations. See Decarboxylation Oxidative -elimination. See ( Elimination Oxidative sulfitolysis, 239-240. V-, P- and 5-Oxides deoxygenation, 115 Oximes Beckmann rearr., 136-137 heterocycles from, 307-308 reduction of, 98, 112 Oxirane (ethylene oxide) ... 

---