Preparative alkaline urea gradient PAGE: application to purification of extraordinarily-stable disulfide-linked homodimer of human growth hormone.
The 40-60 pituitary human growth hormone (hGH) isoforms are so similar in their physico-chemical properties (charge, size, hydrophobicity) that the limited resolutions of chromatographic separation methodologies have not permitted most of them to be isolated. However, application of high-resolution preparative alkaline urea gradient PAGE has facilitated isolation of a disulfide-linked mercaptoethanol-resistant (MER) 45 kDa hGH dimer. Human pituitary extracts were separated by Sephadex G-100 chromatography under alkaline conditions. Pooled fractions containing MER-45 kDa hGH, as determined by SDS-PAGE, were then separated by Sephadex G-100 chromatography under acidic conditions followed by diethylaminoethyl (DEAE) anion-exchange chromatography. Pooled DEAE fractions containing MER-45 kDa hGH and other hGH isoforms were then separated by preparative electrophoresis in an alkaline polyacrylamide gradient (5-20%) slab gel containing 8 M urea into five distinct protein zones. One electroeluted zone contained pure MER-45 kDa hGH. The dimeric hGH isoform was immunoreactive at low concentrations (effective dose to produce 50% response (ED(50)) +/- S.E. = 58 +/- 5.00 pM) in a hGH radioimmunoassay, similar to that of standard monomeric hGH (ED(50) +/- S.E. = 22.93 +/- 3.90 pM), indicating that is was conformationally intact. Alkaline urea gradient PAGE is a valuable tool for preparative separation of structurally similar proteins such as isoforms of the hGH family.
Grigorian
Alexei L
AL
Department of Biochemistry and Molecular Genetics, University of Alabama School of Medicine, Birmingham, AL, USA.
Bustamante
Juan J
JJ
Muñoz
Jesus
J
Aguilar
Roberto M
RM
Martinez
Andrew O
AO
Haro
Luis S
LS
D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D019281:Dimerization; D004220:Disulfides / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D019382:Human Growth Hormone / Q000737:chemistry* / Q000302:isolation & purification*; D006801:Humans; D008623:Mercaptoethanol / Q000737:chemistry; D008970:Molecular Weight; D010902:Pituitary Gland / Q000737:chemistry; D017434:Protein Structure, Tertiary; D014508:Urea / Q000737:chemistry*
D004220:Disulfides; D019382:Human Growth Hormone; D008623:Mercaptoethanol; D014508:Urea