SPECTROSCOPIC CHARACTERIZATION OF RECOMBINANT FOLLICLE-STIMULATING-HORMONE

Recombinant follicle stimulating hormone (recFSH, Org. 32489) has been characterized by absorption (UV and IR), (polarized) fluorescence, li near-dichroism (LD) and circular-dichroism (CD) spectroscopy. Absorpti on and fluorescence spectra of the isolated subunits have also been me asured. From the spectra the extinction coefficient, fluorescence quan tum yield and anisotropy have been calculated. Global analysis is used to characterize the bands in the spectra. The adsorption, CD, LD and fluorescence excitation spectra all contain a band around 300 nm that appears to be a sensitive indicator for the intactness of the protein. Evidence is provided for the involvement of tyrosinate in the fluores cence, and for a close contact between the tryptophan (in the beta sub unit) with at least one tyrosine of the alpha subunit. The overall sec ondary structure of recFSH has been determined from its far-UV CD and its IR absorption spectrum. The secondary structure of recFSH is estim ated to contain 15-25% alpha-helix, 15-25%, beta-turn and 30-40% beta- sheet. The beta-sheet in recFSH is almost exclusively antiparallel. Th e results confirm that recFSH contains significantly more alpha-helix than the closed related human glycoproteins, chorionic gonadotropin an d lutropin; however, the alpha-helices may be short and distorted.