A Neutron crystallographic analysis of a cubic porcine insulin at pD 6.6

Ishikawa, Takuya*; Chatake, Toshiyuki*; Onishi, Yuki*; Tanaka, Ichiro*; Kurihara, Kazuo; Kuroki, Ryota; Niimura, Nobuo*

A neutron diffraction study has been carried out at 2.7 resolution on cubic porcine insulin at pD 6.6 using the BIX-4 at the JRR-3 reactor of JAEA. The ionization states of several amino acids in porcine insulin have been obtained at pD 6.6 and they are compared with those at pD 9 obtained by neutron diffraction as well as those at pH 6.50 and 6.98 obtained by X-ray diffraction. For the present work, a large single crystal of 2.7 mm was obtained by dialysis. Refinement of the structure was carried out and the -factor was 21.4% with the free being 29.4%. Protonation and deprotonation of various ionizable amino acid residues were observed and discussed on the basis of the charged states estimated by the values. In the case of His5, both N and N of an imidazole ring are protonated at pD 6.6, but at pD 9 only N is protonated. In contrast, for His10, both N and N are protonated at pD 6.6 as well as at pD 9.