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.
Haro
L S
LS
Division of Life Sciences, The University of Texas at San Antonio, 78249-0609, USA. lharo@lonestar.utsa.edu
Cubriel
A
A
Bustamante
J
J
Flores
R
R
Martinez
A O
AO
D002413:Cations, Divalent; D002614:Chelating Agents / Q000737:chemistry*; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D019382:Human Growth Hormone / Q000737:chemistry / Q000302:isolation & purification*; D006801:Humans; D008670:Metals / Q000737:chemistry*; D020033:Protein Isoforms / Q000737:chemistry / Q000302:isolation & purification
D002413:Cations, Divalent; D002614:Chelating Agents; D008670:Metals; D020033:Protein Isoforms; D019382:Human Growth Hormone