Divalent metal cation chelators enhance chromatographic separation of structurally similar macromolecules: separation of human growth hormone isoforms

Human GH isoforms were separated by anion-exchange chromatography using a linear NaCl gradient in the presence and absence of EDTA and EGTA. SDS–PAGE showed that glycosylated 24-kDa hGH did not appreciably separate from other hGH variants in the absence of metal chelators. However, in the presence of metal chelators, glycosylated 24-kDa hGH separated from the bulk of the hGH isoforms. Human GH isoforms were also separated by size-exclusion chromatography in the presence and absence of metal chelators. Glycosylated 24-kDa hGH eluted with the bulk of the hGH isoforms in both separations. The inclusion of metal chelators in chromatographic buffers to alter the charge and/or size of proteins by stripping their metals may be a generally useful strategy in their fractionation.