Llama Hemoglobin Binding to Heterologous Haptoglobins

Abstract

The plasma protein haptoglobin binds hemoglobin released from lysed erythrocytes. The complex is internalized through the macrophage membrane protein CD163 thus preventing pathological oxidation of biological molecules and tissues. Hemoglobin primary structures can be as little as 50% homologous among species. Haptoglobin also shows structural variation among species and a genetic polymorphism is found in humans and not in other mammals. The goal of this study was to examine the extent to which hemoglobins can bind to haptoglobins from different species. The llama is adapted to high altitude and its hemoglobin might thus differ from those of many other mammals. By using non‐denaturing agarose gel electrophoresis, the charge properties and binding to human haptoglobin was compared for llama, human, dog, pig, horse and goat hemoglobins.Llama hemoglobin was found to be more positively charged than the other mammalian hemoglobins. All hemoglobins tested showed a band of increased anodal mobility when incubated with human plasma which suggested formation of hemoglobin‐haptoglobin complexes. For one plasma sample there was only one new band whereas for a second sample two new anodal hemoglobin bands were formed. This suggested the ability of the different species hemoglobins to bind to different genetic polymorphisms of human haptoglobin. When the different hemoglobins were incubated with purified haptoglobin variants Hp 1:1 and Hp 2:2, one new anodal hemoglobin‐containing band was seen. The new band in the Hp 1:1 samples had higher mobility than those in the Hp 2:2 samples. In all cases the llama hemoglobin‐haptoglobin samples had lesser mobility than those of the other mammals. Because incubation with one of the plasma samples produced two new bands rather than one as with the Hp 1:1 and Hp2:2 it is surmised that this plasma sample had the Hp 2:1 variant and that all animal hemoglobins tested can bind the three genetic variants of human haptoglobin although this variation is regarded to be absent in non‐human species. It is concluded that the binding sites on hemoglobin and haptoglobin for each other have been evolutionarily conserved despite differences in primary structure and marked difference in the charge of llama hemoglobin.Support or Funding InformationThe research was supported by Furman University Research Experience for Undergraduates 2016 session, NSF grant NSF CHE‐1460806 – awarded to K. Buchmueller and T. Hanks at Furman University and by Albany State University, Center for Undergraduate Research