The growth of a large single crystal of cubic porcine insulin for characterization of hydrogen and hydration in cubic insulin crystals by neutron diffraction analysis is reported. Growth in D2O was investigated based on the phase diagram for cubic insulin to determine appropriate growth conditions, and a large single crystal was then successfully grown by a dialysis method to a size of 4.0 x 4.0 x 1.3 mm3. Neutron diffraction analysis of the cubic insulin crystals was carried out using a single-crystal diffractometer at the JRR-3M reactor of the Japan Atomic Energy Research Institute. In preliminary analysis, Npi appears to be protonated and Ntau deprotonated in His5 in the B-chain, whereas both Npi and Ntau are protonated in His10.
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Maeda et al. (2003) studied this question.