Human gene 2 relaxin chain combination and folding.

Relaxin is a small 6 kD two-chain peptide member of the insulin superfamily that is principally produced in the corpus luteum of the ovary and which plays a key role in connective tissue remodeling during parturition. Like insulin, it is produced on the ribosome as preprohormone that undergoes oxidative folding and subsequent proteolytic processing to yield the mature insulin-like peptide. In contrast to the now considerable insight into insulin chain folding and oxidation, comparatively little is known about the folding pathway of relaxin. A series of synthetic pairwise serine substituted relaxin A-chain cysteine analogues was prepared, and their oxidation behavior was studied both on their own and in the presence of native relaxin B-chain. It was observed that native S-reduced A-chain oxidized rapidly to a bicyclic product, whereas individual formation of each of the intramolecular disulfide bonds between Cys11 and Cys24 and the native Cys10 and Cys15 was considerably slower. Curiously, the non-native, isomeric Cys11-Cys15 disulfide bond formed most rapidly, although circular dichroism spectroscopy analysis showed this product to be devoid of secondary structure. This suggested that it may in fact be an intermediate in the subsequent formation of the native Cys10-Cys15 intramolecular disulfide. Combination of the native A-chain with the B-chain proceeded rapidly as compared with the A-chain analogue that lacked the intramolecular disulfide bond suggesting that this latter element is required as a first step in the folding process. It is therefore probable that relaxin is generated from its constituent A- and B-chains in a stepwise organization manner similar to that of insulin chain combination and folding. Further studies showed that the efficiency of combination of A-chain to B-chain was not markedly influenced by reaction temperature and that a reasonable yield of relaxin could be obtained on combination of the preoxidized A-chain with the S-reduced B-chain.