PURIFICATION AND CHARACTERIZATION OF DIFFERENT MOLECULAR-FORMS OF PROSTATE-SPECIFIC ANTIGEN IN HUMAN SEMINAL FLUID

We have developed a new procedure for purifying prostate-specific anti gen (PSA) from human seminal fluid. The method is based on ammonium su lfate precipitation, hydrophobic interaction chromatography, gel filtr ation, and anion-exchange chromatography. It can be completed within 2 days with a recovery of intact PSA of 30%, By anion-exchange chromato graphy, five isoforms of PSA (A, B, C, D, and E) can be separated. The major form (PSA-B) consists of the intact enzyme, as shown by the occ urrence of only one band of 33 kDa in sodium dodecyl sulfate-polyacryl amide gel electrophoresis under reducing or nonreducing conditions, ac id by amino acid sequencing, which reveals only one amino-terminal seq uence corresponding to the reported amino-terminal sequence of intact PSA. The specific absorbance of 1 g/L PSA-B at 280 nm was 1.61, and 80 % of the PSA-B formed a complex with alpha(1)-antichymotrypsin, indica ting that it is enzymatically active. Three cleaved forms of PSA with different nicking sites and low enzymatic activity were separated from intact PSA by ion-exchange chromatography. In addition, we isolated a glycosylation variant, PSA-A, which showed a higher isoelectric point (pI = 7.2) than PSA-B (pI = 6.9) but similar enzymatic activity; this form accounts for 5-10% of total PSA. After treatment with sialidase, PSA-A and B had the same isoelectric point value (pI = 7.7).