Abstract

The structure of bovine deoxyhaemoglobin has been determined at 2·2 Å resolution and refined to an R-factor of 0·193 for all 32,583 reflections, and a free R-factor of 0·249 for 1527 reflections excluded from the refinement. The structure shows no significant differences between the α-carbon positions of bovine and human haemoglobin, except at the N-terminal segment and the first helix (A) which are closer to the dyad symmetry axis and pushed more tightly against the rest of the β-subunits in the bovine form. In a search for the predicted chloride-binding sites, three-dimensional data were collected from crystals suspended in 50% polyethylene glycol buffered either with 50 mM Na phosphate (pH 7·3) ± 0·1 M NaCl or with 0·1 M Hepes (pH 7·3) ± 0·1 M NaBr. Difference electron density maps with and without NaCl or NaBr showed no evidence of specific halide ion-binding sites. Oxygen equilibria were measured in 10 mM Hepes buffer without added NaCl, with 0·1 mM NaCl, 0·1 M NaCl + 1 mM 2,3-diphosphoglycerate, and 0·1 M NaCl + 1 mM inositol hexaphosphate. Without added chloride, P 50 of stripped bovine haemoglobin was similar to that of human haemoglobin with 0·1 M NaCl. With 0·1 M NaCl it was similar to that of human haemoglobin saturated with 2,3-diphosphoglycerate. In 0·1 M NaCl neither organic phosphate significantly affected the oxygen affinity. Titration of P 50 with NaCl showed Δ log P 50 /Δ log[Cl -] of bovine and human haemoglobin to be identical. Analysis of the oxygen equilibrium curves showed the low intrinsic oxygen affinity of bovine haemoglobin to be due to a larger oxygen dissociation constant from the T-structure. The influence of chloride on P 50 and on the alkaline Bohr effect is the same in bovine and human haemoglobins. It is proposed that this is due to the excess positive charges in the central cavity and its widening in the transition from the R to the T-structure. The widening would allow more chloride ions to enter and neutralize the positive charges, but these ions would remain mobile and therefore do not show up as peaks of high electron density. Repulsion between excess positive charges in the central cavity raises the free energy of the T-structure relative to the R-structure, thereby raising the oxygen affinity. Conversely, entry of chloride ions on widening of the cavity reduces the free energy of the T-structure and therefore lowers the oxygen affinity. Such an action of an allosteric effector in the absence of specific effector binding sites has not hitherto been found in the study of cooperative effects in proteins.

Full Text
Paper version not known

Talk to us

Join us for a 30 min session where you can share your feedback and ask us any queries you have

Schedule a call

Disclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.