Lyophilized PSA-ACT complex is stable.

Pettersson et a!. recently reported that prostate-specific antigen (PSA) corn-plexed to a1-antichymotrypsin (PSA-ACT) is unstable in aqueous solutions [1]. Recognizing the usefulness of PSA-ACT calibrators, given that most of the PSA in male serum is in the complexed form, they pointed out that establishment of optimal conditions for preparation and storage of PSA-AGT complexes to prevent dissociation is of crucial importance [1]. They reported three major mechanisms for minimizing the dissoci-ation of PSA-AGT complex in aqueous solutions during storage: (a) slightly acidic pH (i.e., pH 6.8-7.4), (b) 100-1000-fold molar excess of native ACT, and (c) bow temperatures. However, we have recently found that purified PSA and AGT complexed in vitro is highly stable in the byophilized form even at warm temperatures. The suggestion of Pettersson et al. to store aqueous solutions of PSA-AGT complex at a slightly acidic pH is theoretically correct because the lower pH stops the enzymatic activity of PSA, a serine proteinase that cleaves substrates at an optimal pH of 7.8 [2]. We used a bow pH (sodium acetate buffer, pH 5.6) in our in vitro complexation procedures to avoid unnecessary enzymatic activity of PSA during purification of the PSA-ACT complex [3]. Our final products, 90% PSA-ACT complex and 10% free PSA, were prepared in a matrix of pH 7.4 immediately before lyophilization [3]. Addition of 100-1000-fold molar excess of ACT to the preparation obviously stabilizes the PSA-AGT complex. However , because our final product contains 10% enzymatically active PSA, we cannot add excess AGT to our preparation. We used 10 g/L bovine serum albumin (BSA) in phosphate-buffered saline, pH 7.4, as a matrix, which theoretically could protect the PSA-ACT complex by offering an alternative substrate to any latent enzymaticably active PSA in the PSA-AGT preparations. The amount of protein in 10 g/L BSA is i0 to 106 times more than that of dissociated, enzymati-calby active PSA and is much less expensive. The PSA-AGT complex should be much more stable at low temperatures than at high temperatures, because the framework of most protein molecules is stable in the absence of inputs of thermal energy. In fact, all of our procedures for PSA-AGT preparation, except for the initial complexation of PSA to AGT at 37 #{176}G, were performed at 4 #{176}G [3]. We stored the final product, PSA-AGT, at-20 #{176}G. The most important mechanism for stabilizing PSA-AGT complex is byophi-lization. Our ongoing stability studies of PSA-AGT complex demonstrate …