ALLOSTERIC MODULATION BY TERTIARY STRUCTURE IN MAMMALIAN HEMOGLOBINS - INTRODUCTION OF THE FUNCTIONAL-CHARACTERISTICS OF BOVINE HEMOGLOBIN INTO HUMAN HEMOGLOBIN BY 5 AMINO-ACID SUBSTITUTIONS

Bovine erythrocytes do not contain 2,3-diphosphoglycerate, the princip al allosteric effector of human hemoglobin. Bovine hemoglobin has a lo wer oxygen affinity than human hemoglobin and is regulated by physiolo gical concentrations of chloride (Fronticelli, C., Bucci, E., and Razy nska, A. (1988) J. Mol. Biol. 202, 343-348). It has been proposed that the chloride regulation in bovine hemoglobin is introduced by particu lar amino acid residues located in the amino-terminal region of the A helix and in the E helix of the beta subunits (Fronticelli, C. (1990) Biophys. Chem. 37, 141-146). In accordance with this proposal we have constructed two mutant human hemoglobins, beta(V1M+H2deleted+T4I+P5A) and beta(V1M+H2deleted+T4I+P5A+A76K). These are the residues present a t the proposed locations in bovine hemoglobin except for isoleucine at position 4. Oxygen binding studies demonstrate that these mutations h ave introduced into human hemoglobin the low oxygen affinity and chlor ide sensitivity of bovine hemoglobin and reveal the presence of a prev iously unrecognized allosteric mechanism of oxygen affinity regulation where all the interactions responsible for the lowered affinity and c hloride binding appear to be confined to individual beta subunits.