Modification of acidic residues normalizes sodium dodecyl sulfate-polyacrylamide gel electrophoresis of caldesmon and other proteins that migrate anomalously.

Caldesmon migrates as a 140-kDa protein during polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS), although its true molecular mass is close to 90 kDa. Since caldesmon's high acidic residue content may be responsible for this anomaly, it was reasoned that modification of these residues, with a loss of negative charge, might restore normal electrophoretic migration. Therefore caldesmon was reacted with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide in the presence of excess ethanolamine, which results in negatively charged carboxylates being converted to neutral amides without protein cross-linking. The absence of cross-linking was shown by rotary shadow electron microscopy. In accord with expectations, modified caldesmon migrated as a 94-kDa protein when compared to standards, which were much less affected by modification. The anomalous migration of caldesmon might be due to the repulsion of negatively charged SDS by caldesmon's acidic residues. Low binding of SDS to caldesmon is consistent with the fact that SDS, up to 1%, had little or no effect on the secondary structure of caldesmon, as monitored by circular dichroism. However, other mechanisms can also explain these observations. The abnormal migration of tropomyosin and calsequestrin, both of which have a high percentage of acidic amino acids, was also "normalized" by this treatment. Thus this method might have general application for the electrophoresis of proteins which have a high acidic residue content and migrate anomalously.