Efficient isolation of the subunits of recombinant and pituitary glycoprotein hormones.
Complete dissociation into subunits was attained by incubating Chinese hamster ovary (CHO)-derived or native human thyrotropin, follitropin and lutropin overnight at 37 degrees C in acetic acid. The alpha-and beta-subunits of the pituitary glycoprotein hormones were rapidly and quantitatively isolated by reversed-phase high-performance liquid chromatography (RP-HPLC). A dissociation efficiency of > 98% was obtained on the basis of mass determinations of the heterodimers and subunits carried out via mass spectrometry. CHO-derived or native subunits were isolated on a C4 column (80-90% total recovery) and characterized comparatively for purity, hydrophobicity, molecular mass and charge distribution by HPLC, mass spectrometry, sodium dodecylsulfate-polyacrylamide gel electrophoresis and isoelectric focusing. Thyrotropin was used as a model for showing that, after subunit reassociation, the in vivo bioactivity of the hormone was completely restored. The method described is mild, practical, flexible, and can be adapted to dissociate microgram amounts of native or recombinant glycoprotein hormones, allowing characterization of each subunit.
Carvalho
C M
CM
Biotechnology Department, IPEN-CNEN, Av. Prof. Lineu Prestes 2242, Cidade Universitária, 05508-900 São Paulo, Brazil.
Oliveira
J E
JE
Almeida
B E
BE
Ueda
E K M
EK
Torjesen
P A
PA
Bartolini
P
P
Ribela
M T C P
MT
D000818:Animals; D016466:CHO Cells; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D006224:Cricetinae; D003412:Cricetulus; D015292:Glycoprotein Hormones, alpha Subunit / Q000302:isolation & purification* / Q000378:metabolism; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D007525:Isoelectric Focusing; D010908:Pituitary Hormones, Anterior / Q000302:isolation & purification* / Q000378:metabolism; D021122:Protein Subunits / Q000302:isolation & purification* / Q000378:metabolism; D011994:Recombinant Proteins / Q000302:isolation & purification* / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
D015292:Glycoprotein Hormones, alpha Subunit; D010908:Pituitary Hormones, Anterior; D021122:Protein Subunits; D011994:Recombinant Proteins