J. Biochem, 1974, Vol. 76, No. 6 1303-1317
© 1974 Japanese Biochemical Society
research-article |
Dilatometric Studies on the Conformational Changes of Hemoglobin
I. Volume Changes on the Formation of Azide-methemoglobin from Deoxyhemoglobin
Department of Biophysics and Biochemistry, Faculty of Science, University of Tokyo Bunkyo-ku, Tokyo 113
The volume changes, 
V, associated with the reactions: Hb (or Mb) + Fe3+(CN)6+N3– 
Hb+N3–)+Fe2+ (CN)6 were measured on whale myoglobin, horse hemoglobin, and horse hemoglobin digested by carboxypeptidase A [EC 3.4.12.2
[EC]
], by carboxypeptidase B [EC 3.4.12.3
[EC]
], and by both enzymes. The measurements were carried out with a new type of dilatometer convenient for dilatometric titration. The values of V observed were corrected for the volume change due to the reaction of buffers with protons released as a result of the Bohr effect. The Bohr effect, as the number of protons, H+, released in the conversion of deoxyhemoglobin into its azide-met derivative was obtained by summing the values of H+ in the binding of oxygen and in the conversion of oxyhemoglobin into the azide-met derivative. The so-called residual Bohr effect was absent in the conversion of deoxyhemoglobin into azide-methemoglobin and that of deoxymyoglobin into azide-metmyoglobin. The volume changes were proportional to the amount of ferricyanide added for all the derivatives at all pH values studied. The magnitude of V varied with pH and was affected by the removal of the C-terminal residues. All the data indicated that the volume changes less that due to the reaction of the heme groups reflect mainly the number of salt bridges which are expected to dissociate with the conformational change of the hemoglobins. The average of V for the formation of salt bridges was about 9 ml per mole of salt bridge and no significant difference was observed between the types of salt bridge.