Comparison of solubility and interactions of aprotinin (BPTI) solutions inH2O and D2O

Small-angle neutron scattering experiments are often performed with protein s solubilized in heavy water because of the large difference in neutron sca ttering properties of protons and deuterons. In order to characterize the e ffect of D2O on physico-chemical properties of protein solutions, we invest igated the effect of D2O on the phase diagram and the interactions of bovin e pancreatic trypsin inhibitor (BPTI) in solution. We measured the solubili ty in D2O of BPTI solutions in the presence of NaCl (reverse solubility) an d KSCN (direct solubility) and compared with the values measured by Lafont et al. in H2O under the same conditions [Lafont et al., J. Crystal Growth 1 73 (1997) 132]. In the two salts, we found that BPTI solubility in D2O is s ignificantly lower than in H2O. The curves representing the solubility of B PTI in KSCN are shifted by 7.2 degrees C between light and heavy water, a s hift obtained previously with lysozyme and representing the difference in t he temperature of maximum density of both types of water [Gripon et al., J. Crystal Growth 177 (1997) 238; 178 (1997) 575]. In the case of BPTI in NaC l, we did not find this relationship between the solubility in H2O and D2O. We found, by dynamic light scattering, that BPTI attractive intermolecular interactions in the presence of NaCl in D2O are significantly stronger tha n in H2O. We investigated the association of BPTI molecules in crystallizat ion conditions in the presence of NaCl in H2O and D2O by small-angle X-ray and neutrons scattering, respectively. In the presence of heavy water, the transition monomer-multimer is observed at about 2 mg/ml of BPTI in 1 M NaC l whereas in light water and in 1.4 M NaCl solution this transition is obse rved at about 15 mg/ml. These results dearly showed that BPTI in crystalliz ation conditions is a multimer and confirm the importance of the isotopic n ature of water in the crystallization of proteins. The replacement of H2O b y D2O decreases the solubility and increases the attractive intermolecular interactions. (C) 2000 Elsevier Science B.V. All rights reserved.