Camp. Biochem. Physiol. Vol. zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA 93A, No. 2, pp. 319-325, 1989 Printed in Great Britain 0300-9629189 $3.00 + 0.00 C 1989 Pergamon Pressplc zyxwvut THE FUNCTIONAL PROPERTIES OF AMPHIBIAN HEMOGLOBIN: THE CASE OF ~A~A~AN~~R ~A~A~AN~~R AND ~Y~R~~AN~~~ Goner SAVERIO G. CONDO*, ANDREA BELLELLI?, MAURIZIO BRUNORI,‘~ MARCELLA CORDA,~ MARIA G. E%LLEGRINI,~ ELISABETTA M. CLEMENTI* and BRUNO GIARDINA* *Department of Experimental Medicine and Biochemical Sciences, II University of Rome, Rome, Italy; TDepartment of Biochemical Sciences and CNR, Center of Molecular Biology, University of Rome “La Sapienza”, Rome, Italy; and IDepartment of Biological Chemistry, University of Cagliari, Cagliari, Italy zyxwvutsrqpon (Received 30 September 1988) zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQP Abstract-l. The ligand-binding properties of hemoglobins from the urodelan amphibians Salamander saiamander and Hydromantes genei genei have been studied and compared with those of hemoglobins from the urodelan, Trifurus cristabs (Condcj et al., 1980; Cond6 et al., 1983). 2. Depending on the experimental conditions, the Bohr effect is absent or reversed in S. salamander hemoglobin, similarly to almost all the known hemoglobins from the Urodela (and tadpoles of both Anurans and the Urodela). 3. H. genei shares, with anuran amphibia, a normal Bohr effect (reduced oxygen affinity of hemoglobin at acid pH), although smaller than that of mammals. 4. The difference in the Bohr coefficient of hemoglobin from members of the two major subclasses of amphibians (Anura and Urodela: -0.3 and +O.l, respectively, averaged from data of Table I), almost vanishes in the whole blood (-0.2 to -0.4 in the two groups), due to the presence of intracellular effecters. 5. Biochemical and physiological implications of the (apparent) reverse Bohr effect are reviewed. INTRODUCTION The pH sensitivity of hemoglobin oxygen affinity is responsible for the enhanced oxygen retease to acidic tissues. The presence of a Bohr effect has been described for almost all hemoglobins from verte- brates and apparently represents a highly conserved physiological property of this respiratory protein. In fishes the specific need of high gas pressures in some organs (i.e. swimbladder and choroid rete at level of the eye) has been met by a strongly increased Bohr effect, called the Root effect (after Root, 1931), sometimes observed also in amphibian hemoglobins (Condd et al., 1981; Perutz and Brunori, 1982). Moreover, it should be recalled that both Bohr and Root effects are apparently enhanced in the presence of organic phosphates, due to the effect of pH on the affinity of hemoglobin for these effecters (Antonini et al., 1982). In this respect it is therefore surprising that some amphibians display a “reverse” Bohr effect (oxygen affinity of hemoglobin decreases as pH is increased) where, contrary to “normal” Bohr effect, oxygen release is accompanied by proton release. This reversed pH dependence of p,j2 although evident beyond doubt (Morpurgo et al., 1970, Condi, et al., 198 1) is usually less marked than any normal alkaline Bohr effect (the Bohr coefficient, A logp,,,/A pH, ~.__ *All correspondence should be addressed to: Prof. Saverio G. Cond6, II Universiti di Roma, Dipartimento di Medicina Sperimentale e Scienze Biochimiche, via Orazio Raimondo, 00173 Roma, Italia. being -0.6 in the case of HbA and f0.2 for Triturus cristatus hemolysate). Even more surprisingly, some amphibian hemo- globins with a reversed Bohr effect, in the absence of organic phosphates, display a normal Bohr effect when these effecters are added @ mph&ma means; Bonaventura et al., 1977); thus, we often observe a “normal” Bohr effect in red blood cells and a “reverse” one in purified hemoglobin stripped from organic phosphates. Within the line of research on comparative mol- ecular physiology of blood from amphibians, func- tional characterization of hemoglobins from many urodela and anurans is available; however, com- parison of the results is hindered since many of the available data are older than, or immediately sub- sequent to, the discovery of the effect of organic phosphates on hemo~obin (Benesch and Benesch, 1967) and are obtained on preparations con- taminated by unknown amounts of these effecters. We have chosen to characterize the functional properties of hemoglobins from the earth-living uro- delan S. salamander and to compare the results obtained with those previously reported on the aquatic urodelan, T. cristatus (Condi, et al., 1981). Furthermore, we found that blood from the newt, H. genei, which has no respiratory organs and breathes exclusively through the skin and bucco- faringeal cavity, displays interesting oxygen-carrying properties. Due to the difficulty in capturing the animals only preliminary results on hemoglobins from this species are reported in this paper. From these and previous experimental data it appears that different mechanisms of oxygen affinity 319