Open AccessPurification of psa‐act complex: Characterization of psa‐act complex by various chromatographic procedures
Author(s) -
Wu James T.,
Zhang Ping,
Bandhauer Michael E.,
Wilson Lorraine,
Astill Mark E.,
Colemere Jeffrey T.
Publication year - 1995
Publication title -
journal of clinical laboratory analysis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.536
H-Index - 50
eISSN - 1098-2825
pISSN - 0887-8013
DOI - 10.1002/jcla.1860090104
Subject(s) - chromatofocusing , chemistry , chromatography , isoelectric focusing , sepharose , size exclusion chromatography , prostate specific antigen , biochemistry , prostate , enzyme , medicine , cancer
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‐α 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. We believe that the combined use of these chromatographic procedures would permit the isolation of a large quantity of pure PSA‐ACT complex that should facilitate the preparation of more specific antibodies as well as a calibrator for the establishment of a new generation of PSA assay.©1995 Wiley‐Liss, Inc.