The enhancement of the alkaline Bohr effect of some fish hemoglobins with adenosine triphosphate

Abstract

The oxygen equilibria of the hemoglobins of one holostean fish, the spotted gar ( Lepisosteus osculatus), and of four teleost fish, the carpsucker ( Carpiodes carpio), the small mouth buffalo fish ( Ictiobus bubalus), the Rio Grande cichlid ( Cichlasoma cyanoguttatum), and the redear sunfish ( Lepomis microlophus), have been measured as a function of pH in the presence and absence of ATP. The oxygen equilibria of the teleost hemoglobins in the presence of 200 μ m ATP can be superimposed within experimental error upon the data obtained in the absence of ATP by a simple downward shift of the pH scale by 0.5 unit. Thus the effects of proton and ATP binding appear equivalent: Both can be interpreted in terms of a two-state allosteric model in which binding occurs preferentially to the low-affinity T-state. The oxygen affinities of each of the teleost hemoglobins approach asymptotically a maximal value at high pH. Although these high affinities are associated with decreased cooperativity of oxygen binding, as reflected by the Hill coefficient n, the asymptotic value of n never appears lower than 1.2 to 1.4. This indicates that the data cannot be completely described in terms of a single high-affinity R-state in alkaline solution: At least two different conformations are required. The oxygen affinity of the spotted gar hemoglobin, like that of each of the teleost hemoglobins, reaches a maximal value (minimal value of log PO 2 for half-saturation) above pH 8, but unlike teleost hemoglobins, the Hill coefficient reaches maximal values of 2.6 to 2.7 at high pH. The data in the absence of ATP are superimposable on the data in its presence by a downward shift of the pH scale by 0.25 unit. The measurement of the Bohr effect ( Δlog P 30 ΔpH ) in the presence and absence of ATP shows that the Bohr effect in each of the hemoglobins is substantially enhanced by organic phosphates, as it is in mammalian hemoglobins. The extent of the enhancement of the Bohr effect by 200 μ m ATP for each of the hemoglobins is approximately the same in the range pH 6.7 to 7.3 (increase in Δlog P 50 ΔpH ~ 0.3 ). This is a direct consequence of the equivalence of the linked-function relationship to the effects of ATP and proton binding on oxygenation.