Interaction of Hemoglobin with Ions

Abstract

Methods are described for the rapid determination of free ATP and ATP loosely-and firmlybound to oxygenated hemoglobin using anion exchange resin and ultrafiltration. I n hemolyzates of human erythrocytes under simulated in vivo conditions about 200/, of total ATP is firmly-bound t o hemoglobin with an association constant of about 1 x lo8 (M-1).Depending on the total ATP concentration used, equilibrium between free and firmly-bound ATP is reached within 0.2 and 0.5 sec after ATP addition to "stripped" hemolyzates. I n equilibrium the exchange between these two fractions is very slow, not reaching radioactive equilibrium until several days after labelling the free ATP fraction. On the other hand, exchange between free and loosely-bound ATP is very fast.I n addition t o the known two binding sites for ATP in oxy-and deoxyhemoglobin there exists a third binding site which firmly binds ATP and other anions independent of the oxygenation state of the hemoglobin molecule.I n the last few years an increasing number of papers concerning the binding of anions to hemoglobin [I-51 has appeared. By and large the results reported by different authors are in agreement qualitatively. However, quantitatively the results present certain contradictions. This seems to be due mainly to the methods applied, which do not allow direct differentiation between a t least two classes of binding sites having quite different association constants. Indirect differentiation is possible using various graphic methods available. However, under certain conditions the results obtained in this way are unclear [6]. A second difficulty, which may possibly lead to lowered binding values, could be inefficient "stripping" methods to render hemolyzates phosphate free.I n the course of a tracer kinetic analysis of glycolysis in intact human erythrocytes we were especially interested in definite binding data for ATP t o human hemoglobin. No compatibility with the tracer data could be achieved using the binding data available in the literature. ATP binding was therefore reexamined under simulated in vivo conditions, i.e. 5mM oxygenated hemoglobin, p H 7.2, 37" and intracellular ion concentrations. Furthermore, for the evaluation of tracer data the exchange rates between free and loosely-bound and between free and firmly-bound ATP were required but had not been reported in the literature hitherto. The present paper describes the methods elaborated (a) for rendering hemolyzates completely free of total phosphate, (b) for the rapid and direct determination of ATP in the free form and looselyand firmly-bound to hemoglobin, and (c) for measurements of the exchange rates between free ATP and these two classes of bound ATP. The methods involve ultrafiltration, anion exchange resin treatment and the employment of [y-32P]ATP. Some binding and exchange data for simulated in vivo conditions are reported. EXPERIMENTAL PROCEDUREChemicals ATP, ADP and AMP were purchased from Boehringer Mannheim GmbH (Mannheim, Germany), and purified by paper chromatography according to ...