Oxidation of methionine residues in recombinant human interleukin-1 receptor antagonist: implications of conformational stability on protein oxidation kinetics

Oxidation of methionine residues is involved in several biochemical processes and in degradation of therapeutic proteins. The relationship between conformational stability and methionine oxidation in recombinant human interleukin-1 receptor antagonist (rhIL-1ra) was investigated to document how thermodynamics of unfolding affect methionine oxidation in proteins. Conformational stability of rhIL-1ra was monitored by equilibrium urea denaturation, and thermodynamic parameters of unfolding (DeltaGH2O, m, and Cm) were estimated at different temperatures. Methionine oxidation induced by hydrogen peroxide at varying temperatures was monitored during "coincubation" of rhIL-1ra with peptides mimicking specific regions of the reactive methionine residues in the protein. The coincubation study allowed estimation of oxidation rates in protein and peptide at each temperature from which normalized oxidation rate constants and activation energies were calculated. The rate constants for buried Met-11 in the protein were lower than for methionine in the peptide with an associated increase in activation energy. The rate constants and activation energy of solvent exposed methionines in protein and peptide were similar. The results showed that conformational stability, monitored using the Cm value, has an effect on oxidation rates of buried methionines. The rate constant of buried Met-11 correlated well with the Cm value but not DeltaGH2O. No correlation was observed for the oxidation rates of solvent-exposed methionines with any thermodynamic parameters of unfolding. The findings presented have implications in protein engineering, in design of accelerated stability studies for protein formulation development, and in understanding disease conditions involving protein oxidation.     

The conformational stability and flexibility of insulin with an additional intramolecular cross-link

The conformational stability and flexibility of insulin containing a cross-link between the alpha-amino group of the A-chain to the epsilon-amino group of Lys29 of the B-chain was examined. The cross-link varied in length from 2 to 12 carbon atoms. The conformational stability was determined by guanidine hydrochloride-induced equilibrium denaturation and flexibility was assessed by H2O/D2O amide exchange. The cross-link has substantial effects on both conformational stability and flexibility which depend on its length. In general, the addition of a cross-link enhances conformational stability and decreases flexibility. The optimal length for enhanced stability and decreased flexibility was the 6-carbon link. For the 6-carbon link the Gibbs free energy of unfolding was 8.0 kcal/mol compared to 4.5 kcal/mol for insulin, and the amide exchange rate decreased by at least 3-fold. A very short cross-link (i.e. the 2-carbon link) caused conformational strain that was detectable by a lack of stabilization in the Gibbs free energy of unfolding and enhancement in the amide exchange rate compared to insulin. The effect of the cross-link length on insulin hydrodynamic properties is discussed relative to previously obtained receptor binding results.     

Unequal meiotic crossover: a frequent cause of NF1 microdeletions

Neurofibromatosis type 1 is a common autosomal dominant disorder caused by mutations of the NF1 gene on chromosome 17. In only 5%-10% of cases, a microdeletion including the NF1 gene is found. We analyzed a set of polymorphic dinucleotide-repeat markers flanking the microdeletion on chromosome 17 in a group of seven unrelated families with a de novo NF1 microdeletion. Six of seven microdeletions were of maternal origin. The breakpoints of the microdeletions of maternal origin were localized in flanking paralogous sequences, called "NF1-REPs." The single deletion of paternal origin was shorter, and no crossover occurred on the paternal chromosome 17 during transmission. Five of the six cases of maternal origin were informative, and all five showed a crossover, between the flanking markers, after maternal transmission. The observed crossovers flanking the NF1 region suggest that these NF1 microdeletions result from an unequal crossover in maternal meiosis I, mediated by a misalignment of the flanking NF1-REPs.     

Comparative oxidation studies of methionine residues reflect a structural effect on chemical kinetics in rhG-CSF

The effect of protein conformation on the rate of chemical degradation is poorly understood. To address the role of structure on chemical degradation kinetics, comparative oxidation studies of methionine residues in recombinant human granulocyte colony-stimulating factor (rhG-CSF) were performed. The kinetics of oxidation of methionine residues by hydrogen peroxide (H2O2) in rhG-CSF and corresponding chemically synthesized peptides thereof was measured at different temperatures. To assess structural effects, equilibrium denaturation experiments also were conducted on rhG-CSF, yielding the free energy of unfolding as a function of temperature. A comparison of the relative rates of oxidation of methionine residues in short peptides with those of corresponding methionine residues in rhG-CSF yields an understanding of how protein tertiary structure affects oxidation reactions. For the temperature range that was studied, 4-45 degrees C, the oxidation rate constants followed an Arrhenius equation quite well, suggesting the lack of temperature-induced local structural perturbations that affect chemical degradation rates. One of the four methionine residues, Met 122, exhibited an activation energy significantly different from that of the corresponding peptide. Extrapolation of kinetic data predicts non-Arrhenius behavior around the melting temperature. Three phenomenological models based on different mechanisms are discussed, and an application to shelf life prediction of pharmaceuticals is presented.     

Second polar body inclusion results in diploid/triploid mixoploidy

We report three cases with a typical diploid/triploid mixoploidy. Cytogenetic analysis showed a normal diploid karyotype in peripheral blood lymphocytes and a mixture of diploid and triploid cells in skin fibroblasts. We analysed microsatellite markers in patients blood lymphocytes and skin fibroblasts and compared the results with the microsatellite markers in the parents. The extra haploid set was in all three cases of maternal origin. In one case the markers were not very informative but in two cases pericentromeric markers showed a single dose of one paternal allele and a double dose of one maternal allele, more telomeric markers showed one paternal allele and two different maternal alleles. These observations can only be explained by the inclusion of the second polar body in one of the blastomeres at the cleavage stage.