A sedimentation equilibrium study of platypus insulin: the HB10D mutant does not associate beyond dimer.
An extensive study of the self-association patterns of zinc-free synthetic native and mutant (HB10D) platypus insulin in solution (pH = 7.0; I = 0.1 M; 25 degrees C) has been undertaken using the method of sedimentation equilibrium. The data was fitted to a mathematical equation describing the indefinite duoisodesmic (IDI) model of self-association [A.E. Mark, P.D. Jeffrey, Biol. Chem. Hoppe-Slayer, 371 (1990) 1165]. From this the relevant association constants, KA and KB, describing the polymerising system were calculated. This information allows the calculation of the complex distribution of odd and even numbered polymeric species within the insulin system in solution. In the studies on the self-association of the synthetic native and mutant platypus insulin, each was compared with bovine insulin as well as with each other. It is concluded that there is some reduction in the extent of the self-association of native platypus insulin compared to bovine insulin. A reduction, in specifically the dimer-dimer interaction, is indicated by the higher KA and lower KB values. HB10D platypus insulin shows a dramatic reduction in self-association compared to native platypus and to bovine insulin. Analysis of the self-association pattern yielding a KB value of effectively zero suggests that the substitution of an aspartic acid residue for a histidine at B10 virtually abolishes its dimer-dimer interaction. Platypus insulin has essentially the same biological activity as that of porcine (submitted for publication) but a somewhat lower self-association, while the introduction of one amino acid in a critical region increases the activity twofold while abolishing self-association beyond dimer.
Nourse
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Division of Biochemistry and Molecular Biology, John Curtin School of Medical Research, Australian National University, Canberra, Australia.
Jeffrey
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D000818:Animals; D002417:Cattle; D007328:Insulin / Q000737:chemistry* / Q000235:genetics*; D008956:Models, Chemical; D008970:Molecular Weight; D009154:Mutation / Q000502:physiology*; D010987:Platypus / Q000097:blood*; D013816:Thermodynamics; D014461:Ultracentrifugation
D007328:Insulin