The BPTI decamer observed in acidic pH crystal forms pre-exists as a stable species in solution

bovine pancreatic trypsin inhibitor (BPTI) crystallizes under acidic pH con ditions in the presence of thiocyanate, chloride and sulfate ions, yielding three different polymorphs in P2(1), P6(4)22 and P6(3)22 space groups, res pectively. In all three crystal forms, the same decamer is found in the pac king (ten BPTI molecules organized through two perpendicular 2-fold and 5-f old axes as a well-defined and compact object) in contrast to the monomeric crystal forms observed at basic pH conditions. The crystallization of BPTI under acidic conditions (pH 4.5) was investigated by small angle X-ray sca ttering with both under- and supersaturated BPTI solutions. Data showed the oligomerization of BPTI molecules under all investigated conditions. Accor dingly. Various mixtures of discrete oligomers (n = 1 to 10) were considere d. Calculated scattering curves were obtained using models based on the cry stallographic structures, and the experimental patterns were analyzed as a linear combination of the model curves using a non-linear curve fitting pro cedure. The results, confirmed by gel filtration experiments, unambiguously demonstrate the coexistence of two different BPTI particles in solution: a monomer and a decamer, with no evidence of any other intermediates. Moreov er, using both approaches, the fraction of decamers was found to increase w ith increasing salt concentration, even beyond the solubility curve. We the refore propose that at acidic pH, BPTI crystallizes following a two step pr ocess: decamers are first built in under- and supersaturated solutions, upo n which crystal growth proceeds by decamer stacking. Indeed, those BPTI cry stals should best be described as "BPTI decamer" crystals. (C) 2000 Academi c Press.