The Microheterogeneity of Human Haptoglobin and Its Complex with Hemoglobin

Abstract

Human haptoglobin, type 1S-1S, isolated from plasma and from ascites fluid was subjected to isoelectric focusing in polyacrylamide gel and its complex with horse cyanomethemoglobin was analyzed by isoelectric focusing in a column using a sucrose density gradient. Both methods revealed microheterogeneity. Similar patterns were obtained consisting of five to eight strong bands with a symmetrical distribution giving the highest yield in the center. An investigation of the nature of these bands was carried out and it was found that the sialic acid content varied in the different components. Experiments were undertaken to show that the multiple bands were not due to the binding of Ampholine to the protein and that this heterogeneity preexisted in haptoglobin prior to its isolation. No observable difference in heterogeneity was observed between samples of haptoglobin isolated by different methods and for complexes obtained by adding hemoglobin to haptoglobin and haptoglobin to hemoglobin.The unfractionated complex and the separated complex components were crystallized.