Isolation and Preliminary Characterization of Immuno-inhibitory Factors from Dog Erythrocytes and Liver

Crude dog liver extract (DIE) inhibits proliferation of dog and human lymphocytes stimulated by phytohemagglutinin and alloantigens. While purifying this activity from dog liver, we observed that dog liver inhibitory factor (DLIF) shared properties with hemoglobin. DLIF migrated with hemoglobin during DEAE cellulose chromatography, and DLIF had oligomeric (61,900) and subunit (17,900 and 15,700) apparent molecular weights (AMW) similar to concurrently analyzed Sigma canine hemoglobin (61,100, 16,700 and 14,900). After separate cross linking, the proteins comigrated on sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) gels. We therefore isolated dog erythrocyte inhibitory factor (DEIF) from red blood cells to determine if DEIF was a hemoglobin derivative. DEIF, like DLIF, separated at an isoelectric point of 5.6. DEIF also had similar subunit AMW (17,600 and 15,300) by SDS PAGE, but DEIF had much lower lymphocyte inhibitory activity (LIA = 2.27) than DLIF (LIA = 100.00). We conclude that DLIF and DEIF are similar to each other and hemoglobin, but further studies are needed to determine the function and exact structure of DLIF and DEIF.