Capillary isoelectric focusing of a difficult-to-denature tetrameric enzyme using alkylurea–urea mixtures

David Gervais, Darryl King
2014 Analytical Biochemistry  
Alkylurea Capillary isoelectric focusing Capillary electrophoresis a b s t r a c t Capillary isoelectric focusing (cIEF) is normally run under denaturing conditions using urea to expose any buried protein residues that may contribute to the overall charge. However, urea does not completely denature some proteins, such as the tetrameric enzyme Erwinia chrysanthemi L-asparaginase (ErA), in which case electrophoresis-compatible alternative denaturants are required. Here, we show that alkylureas
more » ... h as N-ethylurea provide increased denaturation during cIEF. The cIEF analysis of ErA in 8 M urea alone resulted in a cluster of ill-resolved peaks with isoelectric points (pI values) in the range 7.4 to 8.5. A combination of 2.0 to 2.2 M N-ethylurea and 8 M urea provided sufficient denaturation of ErA, resulting in a main peak with a pI of 7.35 and an acidic species minor peak at 7.0, both comparing well with predicted pI values based on the sum of protein residue pK a values. Recombinant deamidated ErA mutants were also demonstrated to migrate to pI values consistent with predictions (pI 7.0 for one deamidation). The quantitation of ErA acidic species in samples from full-scale manufacturing (1.0-3.5% of total peak area) was found to be reproducible and linear. Use of alkylureas as denaturing agents in capillary electrophoresis and cIEF should be considered during biopharmaceutical assay development. Crown
doi:10.1016/j.ab.2014.08.004 pmid:25132564 fatcat:m3dnholvg5fjbdwakl5rry66ii