HPLC analysis of estrogen receptor by a multidimensional approach

Previously we demonstrated the polymorphism of estrogen receptors (ER) in cytosol of various tissues based upon properties of size, shape and surface charge. This study describes the application of a multidimensional approach utilizing HPLC for characterization of ER. Cytosols from human uterus and endometrial carcinomas were characterized sequentially by high performance size exclusion chromatography (HPSEC) on Spherogel TSK-3000 SW, and high performance ion-exchange chromatography (HPIEC) using SynChropak AX-1000 anion exchange columns. Using HPSEC, specific estrogen binding was exhibited by a 30 Å isoform and by one appearing after the V 0 (~60Å) in human uterus. However, in endometrial carcinoma other smaller binding components with Stoke's radii of < 20 Å were observed also. In buffers containing 400 mM KCl, predominantly a 28–30 Å species was observed by HPSEC. Further characterization of the 28–30 Å isoform from low and high salt elution from HPSEC was accomplished with an AX-1000 column. With either condition, 2 forms were eluted on HPIEC, 1 in the column wash (retention time 8–9 min), and the other at 50–70 mM phosphate. The elution profile of the larger species (~60Å by HPSEC) on the ion-exchange column was time dependent. Immediate analysis (within 15 min) showed a profile similar to that of the original cytosol which contained minor components eluting in wash buffer and at 50–70 mM phosphate and a major isoform at 180mM phosphate. However delayed analysis (after 2 h) of the 60 Å isoform showed a similar profile (components in buffer wash and at 50–70 mM phosphate) obtained with the 30 Å species. This time dependent change was not observed for the 30 Å species or for the original cytosol. Estrogen receptors in cytosol sedimented at 10S and 4S in low ionic strength gradients and at 4S in sucrose gradients containing 400 mM KCl. The 28–30 Å and 60 Å species recovered from HPSEC sedimented at 3.5S. This multidimensional approach indicates that native estrogen receptors dissociated into a number of smaller molecular isoforms, which were distinguishable by different surface charge properties.