Partial purification from human serum of a specific binding protein for human growth hormone.
Following the recent identification and characterization of a highly specific binding protein(s) for human growth hormone (hGH) in human sera, we now report our initial studies on its purification. Outdated blood blank serum was first fractionated by ammonium sulphate precipitation. The majority of the binding activity appeared in the 30-45% saturation fraction, which was then applied in sequence to an hGH-affinity column, to strong anion exchange (SAX) chromatography (a Mono Q column 0.01-0.2 M phosphate gradient), to a TSK phenyl hydrophobic interaction column (HIC) (0.75-0 M sulphate gradient in 0.1 M phosphate) and finally to a second SAX step. By Scatchard analysis the final product was purified approximately 16,500-fold compared to serum with an overall recovery of 5%. The binding affinity (Ka) of the purified material was approximately 0.4 X 10(9) M-1, very similar to that of whole serum (approximately 0.5 X 10(9) M-1). On silver-stained, sodium dodecyl sulphate-polyacrylamide gels, the final SAX product yielded a major doublet, under reduced conditions, of molecular weights (MW) 55,000 and 59,000. Although it is not known whether one or both of these bands might have hGH binding activity, the MW 59,000 band is very similar in size to one (MW 60,000) of two specific binding proteins observed by covalent crosslinking techniques. These data indicate that the specific hGH binding activity of human serum can be substantially purified and that it appears to exist in at least two forms. Further studies are required to determine the biochemical and physiological relationship, if any, between the two forms.
Herington
A C
AC
Medical Research Centre, Prince Henry's Hospital, Melbourne, Australia.
Smith
A I
AI
Wallace
C
C
Stevenson
J L
JL
D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D008970:Molecular Weight; D011986:Receptors, Somatotropin / Q000097:blood / Q000302:isolation & purification*
D011986:Receptors, Somatotropin