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 synth etic 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 sedi mentation 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, K-A and K-B, describing the polymerising system were calculated. This information allows the calculation of the complex distribution of odd and even numbered polym eric species within the insulin system in solution. In the studies on the self-association of the synthetic native and mutant platypus insul ins, 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 sel f-association of native platypus insulin compared to bovine insulin. A reduction, in specifically the dimer-dimer interaction, is indicated by the higher K-A and lower K-B values. KB10D platypus insulin shows a dramatic reduction in self-association compared to native platypus an d to bovine insulin. Analysis of the self-association pattern yielding a K-B value of effectively zero suggests that the substitution of an aspartic acid residue for a histidine at B10 virtually abolishes its d imer-dimer interaction. Platypus insulin has essentially the same biol ogical activity as that of porcine (submitted for publication) but a s omewhat lower self-association, while the introduction of one amino ac id in a critical region increases the activity twofold while abolishin g self-association beyond dimer. (C) 1998 Elsevier Science B.V.