The use of insulin as an injected therapeutic agent for the treatment of diabetes has been one of the outstanding successes of modern medicine. The therapy has, however, had its associated problems, not least because injection of insulin does not lead to normal diurnal concentrations of insulin in the blood. This is especially true at meal times when absorption from subcutaneous tissue is too slow to mimic the normal rapid increments of insulin in the blood. In the neutral solutions used for therapy, insulin is mostly assembled as zinc-containing hexamers and this self-association, which under normal physiological circumstances functions to facilitate proinsulin transport, conversion and intracellular storage, may limit the rate of absorption. We now report that it is possible, by single amino-acid substitutions, to make insulins which are essentially monomeric at pharmaceutical concentrations (0.6 mM) and which have largely preserved their biological activity. These monomeric insulins are absorbed two to three times faster after subcutaneous injection than the present rapid-acting insulins. They are therefore capable of giving diabetic patients a more physiological plasma insulin profile at the time of meal consumption.

ABSTRACT

␤ 2 -Microglobulin, a small protein localized in serum and on cell surfaces, can adopt specific aggregating conformations that generate amyloid in tissues and joints as a complication to long-term hemodialysis. We characterize a proteolytic variant of ␤ 2 -microglobulin (cleaved after Lys 58 ) that as a trimmed form (Lys 58 is removed) can be demonstrated in the circulation in patients with chronic disease. An unexpected electrophoretic heterogeneity of these two cleaved variants was demonstrated by capillary electrophoresis under physiological conditions. Each separated into a fast and a slow component while appearing homogeneous, except for a fraction of oxidized species detected by other techniques. The two components had different binding affinities for heparin and for the amyloid-specific dye Congo red, and the equilibrium between the two forms was dependent on solvent conditions. Together with analysis of the differences in circular dichroism, the results suggest that ␤ 2 -microglobulin cleaved after Lys 58 readily adopts two equilibrium conformations under native conditions. In the cleaved and trimmed ␤ 2 -microglobulin that appears in vivo, the less populated conformation is characterized by an increased affinity for Congo red. These observations may help elucidate why ␤ 2 -microglobulin polymerizes as amyloid in chronic hemodialysis and facilitate the search for means to inhibit this process.1 is a small protein (M r 11,729) localized in the circulation and on cell surfaces, where it constitutes the nonpolymorphic light chain of the class I major histocompatibility complex (1). ␤ 2 m can be cleaved C-terminally to Lys 58 (Lys 58 -␤ 2 m) by activated complement component C1s. In serum, the exposed Lys 58 residue of this cleaved molecule is subsequently removed by a carboxypeptidase B-like activity. This generates a cleaved and trimmed ␤ 2 m variant called desLys 58 -␤ 2 m (␤ 2 -microglobulin cleaved C-terminally to Ser 57 and lacking Lys 58 ) (2). Wild-type ␤ 2 m is a one-chain protein belonging to the immunoglobulin superfamily characterized by two antiparallel ␤-sheets connected by an internal disulfide bridge in a ␤-sandwich topology without helical structures (3, 4). The Lys 58 -␤ 2 m and des-Lys 58 -␤ 2 m variants are disulfide-linked two-chain heterodimers (Fig. 1). des-Lys 58 -␤ 2 m has been demonstrated in sera from patients in hemodialysis treatment (5) and in patients with malignancies and autoimmune and immunodeficiency diseases (2, 6, 7), but, to our knowledge, analyses of the conformational structure of the cleaved variants have not previously been published. In chronic renal failure, plasma concentrations of ␤ 2 m may increase 10 -70 times despite dialysis, and within 2 years after the onset of dialysis, about 20% of patients suffer from complications due to the formation of insoluble deposits of ␤ 2 m (␤ 2 m amyloid) in tissues and joints (8). The events leading to the formation of amyloid in this and other chronic diseases such as Alzheimer's disease are still largely unknown (9). There a...

Vacuum UV circular dichroism spectra measured down to 178 nm for hexameric 2-zinc human insulin, zinc-free human insulin, and the two engineered and biologically active monomeric mutants, [B/S9D] and [B/S9D,T27E] human insulin, show significant differences. The secondary structure analysis of the 2-zinc human insulin (T6) in neutral solution was determined: 57% helix, 1% beta-strand, 18% turn, and 24% random coil. This is very close to the corresponding crystal structure showing that the solution and solid structures are similar. The secondary structure of the monomer shows a 10-15% increase in antiparallel beta-structure and a corresponding reduction in random coil structure. These structural changes are consistent with an independent analysis of the corresponding difference spectra. The advantage of secondary structure analyses of difference spectra is that the contribution of odd spectral features stemming mainly from side chain chromophores is minimized and the sensitivity of the analyses improved. Analysis of the CD spectra of T6 2-zinc, zinc-free human insulin and monomeric mutant insulin by singular value decomposition indicates that the secondary structure changes following the dissociation of hexamers into dimers and monomers are two-state processes.