Optical and thermodynamical properties of proteolytic fragments of cytochrome b5.

Haem containing fragments of cytochrome b5 prepared from rabbit liver microsomes by trypsin and chymotrypsin treatment, and checked by isoelectrofocussing, were identified as sequences 1--90 (tryptic fragment) and 12--97 (chymotryptic fragment) of the protein. The two fragments exhibit the same helix content as judged from circular dichroism spectra. Thermal unfolding measured by scanning microcalorimetry proceeds as a two-state process at transition temperatures Ttrs equals 70 degrees C (fragment 1--90) and Ttrs equals 73 degrees C (fragment 12--97) at neutral pH. The Gibbs energy change at unfolding of the fragments calculated from the calorimetric results amounts to delta G25 degrees equals 26 + or minus 2 kJ mol-1. The results indicate that the region ranging at least from residues 90--97 does not essntially contribute to the secondary structure and stability of the haem containing domain of cytochrome b5. The findings confirm the existence of a junction region between the membrane binding moiety and the haem containing domain of cytochrome b5 which enables lateral movement of the functionally important part of the molecule to certain extent.