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BACKGROUNDProstate-specific antigen (PSA), a marker for prostate cancer (CaP), forms a covalent complex with alpha(1)-antichymotrypsin (ACT) in human blood. Structural analysis of the PSA-ACT complex is difficult, and complexation may be a reason for biased immunological assays when compared with the analysis of free PSA. We developed a method to cleave the PSA-ACT complex chemically. The liberated PSA was thus available for analysis as free PSA (F-PSA).METHODSPSA was released from the PSA-ACT complex by cleaving the interprotein ester bond with ethanolamine under alkaline conditions. The release was followed by reversed-phase HPLC and an immunoassay for F-PSA. Released PSA obtained from human blood was further immunopurified and analyzed by matrix-assisted laser desorption-induced time of flight (MALDI-TOF) mass spectrometry.RESULTSIn vitro-prepared PSA-ACT complex was completely cleaved by treatment with nucleophilic compounds such as ethanolamine at pH 9-10. The released PSA was stable under these conditions and could be measured by reversed-phase HPLC as well as the ENZYMUN immunoassay for F-PSA. When plasma from a CaP patient [containing 190 microg/L F-PSA and 1890 microg/L total PSA (T-PSA)] was treated under similar conditions, a concentration of approximately 1600 microg/L F-PSA was measured at the end of the incubation, indicating that the PSA-ACT complex was completely cleaved. Two benign prostatic hyperplasia and CaP sera panels (12 and 13 sera, respectively) containing 4-45 microg/L T-PSA were similarly treated. The concentrations of F-PSA measured after incubation were, on average, 85% of the T-PSA values of the untreated sera. Finally, the PSA released from the complex of the CaP plasma was isolated by immunosorption, analyzed by MALDI-TOF mass spectrometry, and compared to PSA obtained from semen. The intact PSA as well as the peptides observed after digestion with endoproteinase Lys C did not reveal any structural difference between the PSA from these two sources.CONCLUSIONSPSA complexed to ACT in plasma of a CaP patient seems to be structurally very similar to the PSA reference material from semen. The release of PSA from the PSA-ACT complex allows F-PSA and T-PSA to be measured by the same immunological assay, thus eliminating any possible bias between two different assays.

Analysis of Free Prostate-specific Antigen (PSA) after Chemical Release from the Complex with α1-Antichymotrypsin (PSA-ACT)