Insulin disulfide bonds, reduction

Insulin reductase activity is assayed according to Holmgren with a few modifications by measuring the catalytic reduction of insulin disulfide bonds at 30°. Pf PDO (approximately 20 pg) is added in 1 ml of 100 mM sodium phosphate buffer, pH 7.0, containing 2 mM EDTA and 1 mg of bovine insulin. A control cuvette contains only buffer and insulin. The reaction is started by addition of 1 mM dithiothreitol (DTT) to both cuvettes. Increasing turbidity from precipitation of insulin B chain is recorded at 650 nm. The stock solution of insulin (10 mg/ml) is prepared according to the Holmgren protocol. 

Insulin possesses two polypeptide chains, A and B, linked by disulfide bonds. Upon denaturation and reduction of insulin, followed by reoxidation, only 7% recovery of activity was obtained. This is the level of activity expected for random pairing of disulfide bridges. How can these data be reconciled with the hypothesis that the amino acid sequence directs protein folding ... 

Insulin is inactivated in vivo by (a) an immunochemical system in the blood of insulin-treated patients, ib) reduction of disulfide bonds (probably by glutathione), and (c) insu-linase (a proteolytic enzyme) that occurs in liver. Pepsin and chymotrypsin hydrolyze some peptide bonds that lead to... 

Markus, G. (1964) Electrolytic reduction of disulfide bonds of insulin. Journal of Biological Chemistry, 239, 4163-4170. 

Protein folding fthioredoKin promotes correct disulfide bond formation) pj>ssible control of insulin le els> through C(>ntrol of insulin reduction Control of melanin formation people with high levels of thioredmin reductase tan easdy) Regulation of photosyntlietic carl n fixation see Chapter 17)... 

The existence of polarographically reducible disulfide bonds in proteins appears to be intimately related to many Brdicka reactions. The reduction of disulfides to sulfhydryls at mercury electrodes, and the reverse process, have also been studied extensively. Insulin and other disulfide-containing proteins give rise to polarographic currents characteristic of this reaction. 

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