Multiple hemoglobins of the cutthroat trout, Salmo clarki.

Abstract

Nine hemoglobins were purified from blood of Salmo clarki by ion-exchange chromatography and preparative isoelectric focusing. The subunit structures of eight of the purified hemoglobins were studied by electrophoresis of globins in the presence of urea. Six are alpha 2 beta 2 tetramers while two appear to be heterotetramers of the type alpha alpha' beta 2 and alpha alpha' beta beta'. The effects of pH, nucleotides, and temperature on the oxygen equilibria of the purified hemoglobins were studied. Five hemoglobins with isoelectric points from 9.1 to 7.1 and one minor hemoglobin with an isoelectric point of 5.9 appear to have essentially identical oxygen binding properties. All have similar oxygen equilibria which are independent of pH and temperature and not affected by saturating amounts of ATP. Another minor hemoglobin with an isoelectric point below 5.9 has similar oxygen equilibria except for a possible pH dependence. Two hemoglobins, with isoelectric points of 6.5 and 6.4, have oxygen binding properties which are strongly pH and temperature dependent. Addition of ATP or GTP causes a large decrease in the oxygen affinity without affecting the cooperativity of oxygen binding. The effect of GTP is slightly greater than that of ATP. No significant differences were observed in the oxygen equilibria of these two hemoglobins. The red blood cells of S. clarki were found to contain large amounts of both ATP and GTP, with an ATP:GTP ratio of 3:1. Both nucleotides may be important modulators of hemoglobin oxygen affinity in S. clarki, in contrast to the situation in S. gairdneri, in which red blood cell GTP concentrations are considerably lower. The presence of six or possibly seven hemoglobins with identical oxygen binding properties in S. clarki suggests that, to a large extent, the physiological role of multiple hemoglobins in this species involves phenomena not directly related to the oxygen binding properties of the hemoglobins.