Preparation and activity of nitrated insulin dimer.
Nitration of insulin using tetranitromethane causes polymerisation involving cross-linked tyrosyl residues. By performing this reaction with insulin crystals, in which it is known that B16 tyrosine of one monomer is closely associated with B26 of the neighbouring monomer within the dimer, it has been possible to isolate a covalent dimer of insulin cross-linked between these two tyrosines. It was, however, first necessary to block the reactive A14 tyrosine. Both rhombohedral (hexameric) and cubic (dimeric) pig insulin crystals were used, the latter proving successful in yielding a pure dimeric product as shown by oxidative sulphitolysis and HPLC. The purified nitrated dimer was biologically active (ca. 10% potency compared to monomeric insulin in a lipogenesis assay) suggesting that the residues responsible for insulin's action are present on the surface of the dimer and not buried in the interface.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001704:Biopolymers; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D003460:Crystallization; D007328:Insulin / Q000302:isolation & purification* / Q000502:physiology; D046911:Macromolecular Substances; D009566:Nitrates / Q000302:isolation & purification / Q000502:physiology; D013329:Structure-Activity Relationship; D013552:Swine; D013774:Tetranitromethane / Q000494:pharmacology; D014443:Tyrosine / Q000378:metabolism / Q000502:physiology; D014961:X-Ray Diffraction / Q000379:methods
D000596:Amino Acids; D001704:Biopolymers; D007328:Insulin; D046911:Macromolecular Substances; D009566:Nitrates; D014443:Tyrosine; D013774:Tetranitromethane