Lyophilized PSA-ACT complex is stable.

Abstract

Pettersson et a!. recently reported that prostate-specific antigen (PSA) cornplexed to a1-antichymotrypsin (PSAACT) is unstable in aqueous solutions [1]. Recognizing the usefulness of PSAACT 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 dissociation of PSA-AGT complex in aqueous solutions during storage: (a) slightly acidic pH (i.e., pH 6.8-7.4), (b) 1001000-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 PSAACT 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 PSAAGT preparations. The amount of protein in 10 g/L BSA is i0 to 106 times more than that of dissociated, enzymaticalby 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 byophilization. Our ongoing stability studies of PSA-AGT complex demonstrate that lyophilized PSA-AGT is stable for at least 3 months at room temperature (Table 1). We were surprised to find that even at a temperature of 54 #{176}G the immunoreactivity (both free and total PSA) of lyophibized PSA-AGT remained unchanged for several days (data not shown); at 45 #{176}G in an aqueous solution (20 mmol/L phosphate-buffered saline with 10 g/L BSA, pH 7.4) PSA-AGT was stable for only a few hours (data not shown). Therefore, we conclude that lyophibized PSA-ACT is an ideal form for a long-term storageatbow temperature. To understand why lyophibization or freeze-drying, a commonly used method to prepare proteins for long-term storage, did not affect the stability of the PSA-ACT bimolecular protein complex, we studied the binding mechanism of serine proteinase inhibitors (serpins) to serine proteinases. Serpins are single pobypeptide chains containing a conserved domain structure of -390 residues usually flanked by Nor C-terminal extensions [4]. All serpins are proposed to have the same basic conformation, featuring five antiparablel stranded $3-sheets (sheet A), and a reactive site loop (RSL) protruding from the globular inhibitor molecule. At or near the center of the RSL is an amino acid residue termed P1, which is specifically recognized by the primary substrate-binding site of the target proteinase [5]. Amino acids flanking the P1 residue are labeled P1 through PN in the N-terminal direction and P1’ through PN’ in the G-terminal direction. RSL is composed of an exposed loop (P8 to P5’) and a hinge region (P9 to P15) immediately proximal to the exposed loop [6]. Most serpins combine with the catalyticand substrate-binding site of the target proteinase to form a tight and stable complex. Having found that the complex of proteinase (E) and serpins (I) did not dissociate in the presence of a denaturing reagent such as sodium dodecyb sulfate (SDS), but was sensitive to nucleophiles [7], earlier investigators [5, 8, 9] were led to the hypothesis that a covalent, acyl intermediate between E and an inactivated (or modified) inhibitor (1*) was formed during complex formation. However, more recent data showing the interaction of catalytically inactive anhydroproteinases to serpins [10, ii] and the reversibility of certain serpin-proteinase complexes [12, 13] argue against the above hypothesis. Previous studies by nuclear magnetic resonance [14] demonstrate that serpin and serine proteinase form a stable tetrahedral intermediate (EI) during complex formation. The following scheme describes this hypothesis [15]:

1 Figure or Table

Cite this paper

@article{Zhi1996LyophilizedPC, title={Lyophilized PSA-ACT complex is stable.}, author={Chen Zhi and Angelo Prestigiacomo and Thomas A. Stamey}, journal={Clinical chemistry}, year={1996}, volume={42 8 Pt 1}, pages={1297-8} }