Comp. Biochem. Physiol. Vol. 78B, No. 2, pp. 443-446, 1984 0305-0491/84 $3.00+0.00 Printed in Great Britain (~ 1984 Pergamon Press Ltd OXYGEN BINDING AND ACID BASE STATUS OF THE BLOOD FROM THE FRESHWATER TURTLE, PHRYNOPS HILARH E. REISCHL,* W. JELKMANN, K. H. GOTZ, C. ALBERS and C. BAUER lnstitut fiir Physiologie, Universit~t Regensburg, D-8400 Regensburg, FRG. (Received 7 October 1983) Abstract--1. Oxygenation studies with the whole blood of Phrynops hilarii show a Ps0 of 38 torr at extracellular pH (pile) of 7.4 which corresponds to an intracellular pH (pHi) of 7.05 at 25°C. 2. The blood CO 2 Bohr effect was -0.56 when related to pHi. 3. pHi is related to pile by the following equation: pHi = 0.75.pile + 1.54 (r = 0.99); pHi = 0.72. pile + 1.72 (r = 0.96) at 10 and 25°C respectively. Blood pH, pile, for 25°C, was 7.519 + 0.254 (n = 6). 4. Blood gas partial pressures were: pCO 2 = 25.8 + 3.8 torr (n = 6); pO 2 = 61.7 + 21.2 torr (n = 6). 5. The major red cell phosphates, in mmole/1 erythrocytes, n = 6, were: ATP (3.66___0.86); GTP (0.53 _+0.28); 2.3-DPG (0.32_ 0.12) and inorganic phosphates (2.00 +_0.35). 6. The plasma inorganic ion composition, n = 6, was, in mEq/l: K ÷ (3.04 + 0.40); Na ÷ (148.4 + 12.6); Ca 2÷ (4.75 _ 1.32); C1 (106.6 _ 5.0). 7. Additional blood parameters of interest (n = 6) were: lactate (2.07 + 1.72mM in plasma); erythrocytes/mm 3 (416 x 103+ 4.6 x 103); leucocytes/mm 3 (4636 + 2618); haematocrit (~o) (14.5 + 3.6); haemoglobin, g/dl (3.2 + 0.5); plasma protein g/dl (4.4 + 0.4); osmolarity (293 + 10 mOsm/1). 8. The non-bicarbonate buffer value was -22.6 mmol/kg H20/pH. 9. For a constant CO, content, ApHe/At = 0.0141 +0.002 (n = 18) and ApHi/At =0.0157 +0.003 (n = 18). INTRODUCTION Studies on the two isolated haemoglobins and on the unfractionated haemolysate of the turtle, Phrynops hilarii (Reischl et al., 1984) have shown, in terms of oxygen binding, the stripped isolated components (CI, isoelectric point=7.6 and CII, isoelectric point = 6.3) to be different functionally, and that the Ps0s of the unfractionated haemolysate, at pH around 7.0, are a mean of the ones of the isolated components, but at higher pH values depart progressively from the mean; an indication that more alkaline pHs favour molecular interaction among the CI and CII and that these two components no longer function independently as seen in the more acid pH range. In the present paper we have studied oxygen binding in the whole blood, and blood parameters known to influence 02 binding to haemoglobins, like organic phosphate concentration and intracellular pH (pHi). Blood oxygenation properties of turtles and other ectotherms may be more complex than mammalian ones because haemoglobin cooperativity changes with saturation (Maginnis et al., 1980; Lapennas and Lutz, 1982). Our results indicate that the oxygenation proper- ties of the whole blood can be derived from those of the unfractionated stripped haemolysate added with ATP (10 mM) and referred to pHi. *Current address: Departmento de Fisiologia, Farmaco- logia e Biofisica UFRGS, 90.000 Porto Alegre, RS- Brasil. MATERIALS AND METHODS Phrynops hilarii is a South-American freshwater turtle (Freiberg, 1970), from the family Chelidae. The animals were kept in captivity in a large aquarium with running water at a temperature of around 20°C. They were fed with bovine heart meat ad libitum. Blood was collected by cardiac puncture, with sodium heparin (Parmonta, Hamburg) as anticoagulant. Blood osmolarity was determined by means of the lowering of the freezing point (Halb-Mikro Osmometer, Knauer, Oberursel/Taunus, FRG). Plasma Na ÷, K ÷ and Ca 2÷ were determined by flame photometry, CI by means of the chlor-o-counter (Marius, Utrecht/ Netherlands). Lactate was analysed enzymatically (Boeh- ringer, Mannheim, FRG). The concentration of plasma protein was determined by the biuret method. Haematocrit, haemoglobin concentration and blood cell counts were determined by routine methods. Measurements of the actual blood gases and pH were done with the electrodes equi- librated at a temperature close to the animals body temperature. (For details see Albers, 1972.) pH-logpCO 2 buffer lines were established with 3 different concentrations of CO2, mixed with 02 and N2, respectively, at 25cC. lntraerythrocytic pH was determined in haemolysates of freeze-thawed erythrocytes at 10 and 25"C. Red cell phosphates were determined as cited in Jelkmann et al. (1981). Oxygen equilibrium curves were obtained by measuring the O2-content of blood samples (Lex-O2-Con , Lexington) which were equilibrated with water vapour saturated mixtures of 02, CO2 and N 2 as described (Jelk- mann et al., 1981). pH was varied by changing the pCO 2. Unless otherwise stated results are given as a mean + SD. RESULTS Figure 1 shows the CO: Bohr effect of whole blood, related to pHi. The regression line of the fixed acid 443