Purification of PSA-ACT complex: characterization of PSA-ACT complex by various chromatographic procedures.
We have explored various chromatographic procedures with the intention of establishing an isolation procedure that would allow us to isolate a large quantity of PSA-ACT (prostate specific antigen-alpha 1-antichymotrypsin) complex either from patients' sera or from incubation mixtures of free PSA and protease inhibitors. We found that at pH 7.2, both free PSA and PSA-ACT molecules are negatively charged and bind to the DEAE-Sepharose column. However, they could be separated from each other using a linear gradient of NaCl at pH 7.2. Both free PSA and PSA-ACT molecules were also found to be retained by the Con A Sepharose column because of the carbohydrate moiety of the PSA molecule. These two molecules were not separable by Con A chromatography. These two molecules apparently differ in their isoelectric points and were well separated by chromatofocusing using a pH gradient from pH 9 to 6. It appears that chromatofocusing can also be used to identify the isoforms of free PSA because of its high resolving power. The large difference in molecular size between free PSA and PSA-ACT complex allowed their separation by gel filtration chromatography on a column containing either S-100, S-200, or S-300 gel. S-200 gel appeared to be the best for the separation of free PSA from PSA-ACT and for the removal of other contaminating serum proteins.(ABSTRACT TRUNCATED AT 250 WORDS)
Wu
J T
JT
Department of Pathology, University of Utah, School of Medicine, Salt Lake City, USA.
Zhang
P
P
Bandhauer
M E
ME
Wilson
L
L
Astill
M E
ME
Colemere
J T
JT
D002845:Chromatography; D002850:Chromatography, Gel; D003208:Concanavalin A; D003911:Dextrans; D004983:Ethanolamines; D006801:Humans; D017430:Prostate-Specific Antigen / Q000097:blood / Q000302:isolation & purification*; D012685:Sepharose; D000514:alpha 1-Antichymotrypsin / Q000097:blood / Q000302:isolation & purification*
D003911:Dextrans; D004983:Ethanolamines; D000514:alpha 1-Antichymotrypsin; D003208:Concanavalin A; D012685:Sepharose; C025614:sephadex; D017430:Prostate-Specific Antigen; C007369:2-diethylaminoethanol