SPONTANEOUS DISSOCIATION ASSOCIATION OF MONOMERS OF THE HUMAN-STEM-CELL FACTOR DIMER

In its native state, recombinant human-stem-cell-factor (SCF) dimer ca n spontaneously and rapidly undergo hybridization when two different S CF dimer species are incubated together. SCF species differing in mole cular charge, e.g., a wild-type SCF form and a variant with Asp at pos ition 10 instead of Asn, were used in the hybridization studies; the o riginal species and newly formed dimer hybrid can be separated and qua ntified by cationic-exchange h.p.l.c. The hybridization reaches an equ ilibrium where the ratio of hybrid dimer to each of the original speci es is 2. Kinetic studies of the initial rate of hybridization enable a rate constant for monomer dissociation to be determined. This rate co nstant is influenced by pH, temperature and salt concentration. The pH and salt effects suggest that salt bridges between charged amino acid s at the monomer-monomer interface may be present. From the temperatur e effects, the activation energy for monomer dissociation was determin ed to be 85.6 kJ/mol, which is typical for oligomeric proteins. Heavil y glycosylated recombinant SCF from Chinese-hamster ovary cells exchan ged equally well with the bacterially derived non-glycosylated SCF, in dicating that the attached carbohydrate moieties had no effect on mono mer exchange.