Effects of pH, CO2 and organic phosphates on oxygen affinity of sea turtle hemoglobins

The affinities of adult-type hemoglobins of green (Chelonia mydas) and loggerhead (Caretta caretta) sea turtles were determined at 25.7 degrees C as a function of pH, PCO2 and each of the organic phosphates found in erythrocytes of these species (ATP, IHP (substitute for IPP) and DPG). Each species showed a single hemoglobin band in cellulose acetate electrophoresis which isoelectric focusing resolved into one major and three minor components having isoelectric points near pH 7.2. Oxygen binding curves were recorded using a modified 'Hemoscan' (Instrument Division, Travenol Laboratories, Savage, MD). Hemolysates were kept saturated with CO during preparation to prevent methemoglobin formation. CO was driven off immediately before performing each oxygen binding determination, using the photodissociative effect of the Hemoscan light beam. In both species, phosphate-free hemoglobin has higher affinity than whole blood, and had lower pH sensitivity. CO2 (37 Torr) reduces affinity, particularly in the green sea turtle, and eliminates pH sensitivity. Each organic phosphate tested reduces affinity, with IHP being most effective at a given concentration and DPG least effective. Organic phosphates are most effective at low pH, and restore both pH sensitivity and oxygen affinity to whole-blood levels taking into account the difference between plasma and intracellular pH. Reasons are suggested why the organic phosphate effect was not detected in a previous study. Hemolysates of both species are distinctive in giving upward-curving Hill plots, with n = 1 asymptotes at low saturation that are insensitive to pH, CO2 and organic phosphates. The oxygen affinities corresponding to these asymptotes are about 100 Torr in both species. Results are compared to oxygen dissociation curves obtained from whole blood.