The effects of temperature and pH on the binding of ATP to carp (Cyprinus carpio) deoxyhemoglobin.

The linkage relationships between the binding of protons, organic phosphate, and oxygen to hemoglobin are well documented phenomena. These interactions provide a model for the investigation of linkage equilibria. Since ATP is the major organic phosphate in the erythrocytes of many fish, the binding of ATP to a representative fish hemoglobin (Carp I) was investigated. In these studies, both temperature and pH were varied so that their effects on ATP binding could be elucidated. The experimental binding constants were fitted to an equation that included the two major ionizable forms of ATP present within the experimental pH range. The resultant fitted parameters indicate three protons are taken up as deoxyhemoglobin binds the fully ionized form to ATP while only two protons are bound for the partially ionized form. The enthalpy of binding (delta H), provides insight concerning the possible physiological and ecological significance of this phenomenon.

Effects of anions on the activation thermodynamics and fluorescence emission spectrum of alkaline phosphatase: evidence for enzyme hydration changes during catalysis.

The effect of anions on the thermodynamic activation functions for a model enzyme, calf intestinal alkaline phosphatase (EC 3.1.3.1), have been studied in order to examine the role of protein hydration changes in establishing the energetics of enzyme catalysis. The influences of these anions on the activation volume (delta V) and activation free energy (delta G) reflected clear Hofmeister (lyotropic) series effects, in the order F- greater than Cl- greater than Br- greater than I- (order of increasing salting-out potential). A pronounced covariation was observed between the influences of these anions on Vmax, which is proportional to delta G, and on the negative activation volume of the reaction. Fluoride was able to counteract the influences of Br- and I- on both Vmax and delta V when combinations of these anions were employed. The effects of Br- and I- on Vmax and delta V were more pronounced at lower temperatures. The control delta V was increasingly negative at reduced temperatures. The effects of the neutral salts and propanol on delta V and delta G, as well as the effects of salting-in anions on the activation enthalpy and the negative activation entropy of the reaction, are consistent with a model which proposes that peptide groups or polar side chains on the native enzyme exergonically increase their exposure to solvent during the catalytic activation event. These conclusions are in accord with the known free energy, enthalpy, entropy, and volume changes which occur when model peptide groups are transferred between water and concentrated salt solutions. Consistent with the kinetic results, the fluorescence emission wavelength maximum of alkaline phosphatase increased in the presence of anions in the order F- greater than Cl- greater than Br- greater than I-. The salting-out ion (F-) and the salting-in ions (Br- and I-) shifted lambda max in different directions, and these lambda max shifts could be counterbalanced by using equimolar combinations of salting-in and salting-out anions. Control experiments with a model compound, N-acetyltryptophanamide, showed that the spectra shifts caused by the salts did not result solely from differential quenching by the anions of the solvent-exposed tryptophan(s) on the enzyme. Hofmeister additivity phenomena indicated that the solvent is at the basis of these salt-induced enzyme structural changes. It is concluded that changes in protein solvation during enzymic reactions contribute significantly to the thermodynamic activation parameters in both the native and the salt-perturbed enzyme.

Allosteric modifiers of fish hemoglobins: in vitro and in vivo studies of the effect of ambient oxygen and pH on erythrocyte ATP concentrations.

Fundulus heteroclitus decreases erythrocyte adenosine triphosphate and increases blood hematocrit when acclimated to hypoxic conditions. A defined medium has been developed which allows isolated F. heteroclitus erythrocytes to be maintained for several hours without an appreciable loss of cellular ATP. The effect of oxygen tension, pH and metabolic inhibitors on the cellular concentration of ATP of fish red cells has been investigated as an in vitro model to explain in vivo responses to environmental changes. The isolated red cells significantly decrease their ATP/Hb molar ratio when exposed either to anaerobiosis or metabolic inhibitors. It is concluded that the in vivo response is mediated at the red cell level via decreased oxidative phosphorylation in the presence of low environmental oxygen. The length of time necessary to elicit the responce both in vivo and in vitro is also discussed.