Intramolecular cross-linking of insulin. Preparation and properties of oxalyl- and malonyl-bis(methionyl) insulin.
The bifunctional reagents, oxalyl-(Met-ONp)2 and malonyl-(Met-ONp)2 have been prepared and investigated as reversible cross-linking reagents for insulin and model compounds. The removal of the cross-linking residues was demonstrated by the cyanogen bromide cleavage of oxalyl-(Met-Phe-OMe)2 and malonyl-(Met-Phe-OMe)2. Zinc-insulin reacted with a molar equivalent of oxalyl-(Met-ONp)2 or malonyl-(Met-ONp)2 in presence of excess triethylamine to yield oxalyl-(Met)2-insulin and malonyl-(Met)2-insulin, respectively. In these derivatives the N-terminal phenylalanine (B1 residue) was free. Thus the cross-link was between A1 and B29 residues in insulin. All three disulfide bonds of these insulin derivatives undergo reduction with tributylphosphine to give six sulfhydryls. Air-oxidation of reduced oxalyl-(Met)2-insulin and malonyl-(Met)2-insulin in 0.05 M disodium phosphate, pH 9.5, yielded products which were indistinguishable from oxalyl-(Met)2-insulin and malonyl-(Met)2-insulin respectively, as measured by physicochemical and biological methods. Cyanogen bromide cleavage of reduced and reoxidized malonyl-(Met)2-insulin in 70% formic acid regenerated insulin quantitatively, but only 40% of insulin was determined from similar treatment of oxalyl-(Met)2-insulin. The regenerated insulins exhibited the biological activity of native insulin. These studies strongly suggest that disulfide bonds formed during oxidation of reduced oxalyl-(Met)2-insulin and malonyl-(Met)2-insulin are identical to those found in insulin.
D000273:Adipose Tissue / Q000187:drug effects; D000818:Animals; D003432:Cross-Linking Reagents / Q000138:chemical synthesis; D003488:Cyanogen Bromide; D005947:Glucose / Q000378:metabolism; D006868:Hydrolysis; D066298:In Vitro Techniques; D007328:Insulin / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000494:pharmacology; D007700:Kinetics; D008297:Male; D008314:Malonates / Q000138:chemical synthesis; D010070:Oxalates / Q000138:chemical synthesis; D010084:Oxidation-Reduction; D051381:Rats; D011919:Rats, Inbred Strains
D003432:Cross-Linking Reagents; D007328:Insulin; D008314:Malonates; D010070:Oxalates; C039042:oxalylbis(methionyl-4-nitrophenyl ester); C039039:malonylbis(methionyl-4-nitrophenyl ester); D005947:Glucose; D003488:Cyanogen Bromide