Binding of B Cell - Derived Autocrine Growth Factor to Hemoglobin

Park, Jae Seung; Choi, Il Whan; Shin, Young Mi

Abstract

Normal human B cells produce autocrine growth factor in response to Staphylococcus aureus Cowan I strain (SAC). However, the functional role and molecular nature of the B cell derived-B cell growth factor (B-BCGF) are largely unknown. We have tried to investigate the nature of B-BCGF using mAb for several years. We report here that B- BCGF is capable of binding to hemoglobin (Hb). The concentrated culture supernatant from tonsillar B cells stimulated with SAC for 24 h was loaded into the fast protein liquid chromatography and ion-exchange chromatography. The peak with BCGF activity was shown to have a M.W. of 16-18 Kda in polyacrylamide gel electrophoresis followed by silver stain. Amino acid sequence of the fraction was found to identical to human hemoglobin (Hb) by more than 85%. However, Hb itself had no BCGF activity. The presence of Hb in culture supernatant was due to the contamination of SRBC during B cell purification. SRSC were completely removed from B cells by percoll-gradient centrifugation and B cells were stimulated with SAC and exogenous Hb was added to the cultures. The Hb fraction from FPLC again showed a BCGF activity. These data strongly suggested that BCGF binds to Hb. We confirmed this in dot blot as well as Western blot. The M.W of Hb-binding BCGF was 20 Kda. This information may provide a rapid progress in research of BCGF.