ALLOSTERIC MODIFIERS OF HEMOGLOBIN - 2-[4-[[(3,5-DISUBSTITUTED ANILINO)CARBONYL]METHYL]PHENOXY]-2-METHYLPROPIONIC ACID-DERIVATIVES THAT LOWER THE OXYGEN-AFFINITY OF HEMOGLOBIN IN RED-CELL SUSPENSIONS, IN WHOLE-BLOOD, AND INVIVO IN RATS

Two new potent allosteric effectors of hemoglobin, RSR-4 [2-[4-[[(3,5-dichloroanilino)carbonyl]methyl]phenoxy]-2-methylpropionic acid] and RSR-13 [2-[4-[[(3,5-dimethylanilino)carbonyl]methyl]phenoxy]-2-methylpropionic acid], are compared to the previously reported compounds L3,5 and L3,4,5 [Lalezari, I., Lalezari, P., Poyart, C., Marden, M., Kister, J., Bohn, B., Fermi, G., & Perutz, M. F. (1990) Biochemistry 29,1515]. Unlike L3,5 and L3,4,5, RSR4 and RSR-13 are less impeded by physiological concentrations of serum albumin. RSR-4 has also been shown to be more effective than L3,5 in shifting the allosteric equilibrium of bovine Hb toward the low-affinity T-state. X-my crystal studies show that both RSR-4 and RSR-13 bind to only one pair of symmetry-related sites in the Hb central water cavity whereas previous studies on L3,5 and L3,4,5 demonstrated a second pair of symmetry-related binding sites near Arg 104beta. Three major interactions between these allosteric effectors and Hb include the acid group with the guanidinium group of C-terminal Arg 141alpha, the effector's amide oxygen with the ammonium ion of Lys 99alpha, and the pi electrons of the halogenated or methylated aromatic ring and Asn 108beta. No explanation has been found for the difference in number of binding sites observed for RSR-4 and RSR-13 (two sites) compared to L3,5 and L3,4,5 (four sites); also no correlation has been made between the number of binding sites and degree of allosteric shift in the oxygen equilibrium curve. We also show that the new allosteric effectors (1) readily cross the red cell membrane in the presence of serum albumin solutions, (2) restore to normal the oxygen equilibrium curves of outdated blood, (3) are not inhibited from entering erythrocytes in the presence of an anion-channel blocking agent (DIDS), and (4) shift the oxygen dissociation curve to the right in vivo in rats. The possible use of these effectors as therapeutic agents is discussed.