ISOLATION AND CHARACTERIZATION OF A DISULFIDE-LINKED HUMAN STEM-CELL FACTOR DIMER - BIOCHEMICAL, BIOPHYSICAL, AND BIOLOGICAL COMPARISON TO THE NONCOVALENTLY HELD DIMER

Distinct from the noncovalently linked recombinant human stem cell fac tor (rhSCF) dimer, we report here the isolation and identification of an SDS-nondissociable dimer produced during folding/oxidation of rhSCF . Experimental evidence using various cleavage strategies and analyses shows that the isolated dimer is composed of two rhSCF monomers coval ently linked by four disulfide bonds. The cysteines are paired as in t he noncovalently associated dimer except that all pairings are intermo lecular rather than intramolecular. Other structural models, involving intertwining of intramolecular disulfide loops, are ruled out. The mo lecule behaves similarly to the noncovalently associated dimer during ion-exchange or gel permeation chromatography. However, the disulfide- linked dimer exhibits increased hydrophobicity in reverse-phase column s and in the native state does not undergo spontaneous dimer dissociat ion-association as seen for the noncovalent dimer. Spectroscopic analy ses indicate that the disulfide-linked and noncovalently associated rh SCF dimers have grossly similar secondary and tertiary structures. In vitro, the disulfide-linked dimer exhibits approximately 3-fold higher biological activity in supporting growth of a hematopoietic cell line and stimulating hematopoietic cell colony formation from enriched hum an CD34(+) cells. The molecule binds to the rhSCF receptor, Kit, with an efficiency only half that of the noncovalently associated dimer. Fo rmation of intermolecular disulfides in the disulfide-linked dimer wit h retention of biological activity has implications for the three-dime nsional structure of noncovalently held dimer and disulfide-linked dim er.