Hydrophobic interaction chromatography (HIC) for the separation of protein-bound and free steroids. Application to binding protein and receptor assays.
Protein-bound steroids can be separated from free steroids using microcolumns of silica gel coated with an hydrophobic (octadecyl) solid phase. The bound fraction is eluted in the assay buffer, whereas the free fraction is retained quantitatively on the column in the first step and can be recovered in methanol. Both fractions can be quantitated directly (e.g. by liquid scintillation spectrometry when using radioactive ligands) or kept for further analysis (e.g. by TLC, HPLC etc.). Separation of the bound and free fractions is rapid, accurate and reproducible; intra- and inter-assay coefficients of variation are lower than 5 and 10%, respectively. Recovery of radioactive steroids is high (usually over 85%) and can be estimated separately for each sample. Since assay blanks are very low (typically less than 0.1% of input), this new method, which could be termed "hydrophobic interaction chromatography" (HIC), should prove especially useful for the development of sensitive binding assays, particularly in the field of steroid receptors. The HIC method compared well with three methods currently used for steroid binding assays, namely adsorption of unbound steroids on dextran-coated charcoal, gel filtration on Sephadex LH-20 and adsorption of steroid-protein complexes on DEAE-cellulose filters. Examples of application described here include studies on human plasma sex hormone binding globulin (SHBG) and SP2 placental protein (saturation analysis, binding specificity etc.), the separation of antibody-bound steroids in a radioimmunoassay and the estimation of androgen binding to rat epididymal androgen binding protein (rABP). Receptor assays are illustrated by saturation analysis of the mouse uterine oestrogen receptor and of the androgen receptor in the human genital skin.
D000727:Androgen-Binding Protein / Q000032:analysis; D000818:Animals; D002352:Carrier Proteins / Q000032:analysis*; D002845:Chromatography / Q000379:methods; D004822:Epididymis / Q000378:metabolism; D005260:Female; D006801:Humans; D008297:Male; D051379:Mice; D010920:Placenta / Q000378:metabolism; D011257:Pregnancy Proteins / Q000032:analysis; D011485:Protein Binding; D051381:Rats; D011944:Receptors, Androgen / Q000032:analysis; D011960:Receptors, Estrogen / Q000032:analysis / Q000302:isolation & purification; D011987:Receptors, Steroid / Q000032:analysis*; D012738:Sex Hormone-Binding Globulin / Q000032:analysis; D013256:Steroids / Q000302:isolation & purification*; D014599:Uterus / Q000378:metabolism
D000727:Androgen-Binding Protein; D002352:Carrier Proteins; D011257:Pregnancy Proteins; D011944:Receptors, Androgen; D011960:Receptors, Estrogen; D011987:Receptors, Steroid; D012738:Sex Hormone-Binding Globulin; D013256:Steroids