FORMATION OF AN ACTIVE DIMER DURING STORAGE OF INTERLEUKIN-1 RECEPTORANTAGONIST IN AQUEOUS-SOLUTION

The degradation products of recombinant human interleukin-1 receptor a ntagonist (rhlL-1ra) formed during storage at 30 degrees C in aqueous solution were characterized. Cationic exchange chromatography of the s tored sample showed two major, new peaks eluting before (P1) and after (L2) the native protein, which were interconvertible. Size-exclusion chromatography and electrophoresis documented that both the P1 and L2 fractions were irreversible dimers, formed by noncovalent interactions . A competition assay with interleukin-1 indicated that on a per monom er basis the P1 and L2 dimers retained about two-thirds of the activit y of the native monomer. Infrared and far-UV circular dichroism spectr oscopies showed that only minor alterations in secondary structure aro se upon the formation of the P1 dimer. However, alteration in the near -UV circular dichroism spectrum suggested the presence of disulfide bo nds in the P1 dimer, which are absent in the native protein. Mass spec troscopy and tryptic mapping, before and after carboxymethylation, dem onstrated that the P1 dimer contained an intramolecular disulfide bond between Cys-66 and Cys-69. Although conversion of native protein to t he P1 dimer was irreversible in buffer alone, the native monomer could be regained by denaturing the P1 dimer with guanidine hydrochloride a nd renaturing it by dialysis, suggesting that the intramolecular disul fide bond does not interfere with refolding. Analysis of the time cour se of P1 formation during storage at 30 degrees C indicated that the p rocess followed first-order, and not second-order, kinetics, suggestin g that the rate-limiting step was not dimerization. It is proposed tha t a conformational change in the monomer is the rate-limiting step in the formation of the P1 dimer degradation product. Sucrose stabilized the native monomer against this process. This result can be explained by the general stabilization mechanism for this additive, which is due to its preferential exclusion from the protein surface.