Cleavage of recombinant human corticotropin-releasing factor (CRF)-bindingprotein produces a 27-kilodalton fragment capable of binding CRF

CRF is both a peripheral and a central mediator of inflammation, the activi ty of which is modified by the presence of a 37-kDa binding protein (CRF-BP ). The objective of this study was to measure and characterize this protein in the synovial fluid of rheumatoid arthritis patients and to observe the effects of this inflammatory condition on its structure and properties. Mea sured by immunoradiometric assays, the mean CRF-BP concentration in synovia l fluid from 27 arthritic patients was 0.51 nmol/L (SD = 0.24 nmol/L); that for CRF was 6.31 pmol/L. The mean plasma concentration of CRF-BP in 24 con trol subjects was 1.38 nmol/L (SD = 0.35 nmol/L) and that for 10 arthritic patients was 2.89 nmol/L (SD = 0.84 nmol/L). Synovial fluids were found by immunoblotting to contain intact CRF-BP and a 10-kDa C-terminal CRF-BP frag ment; synovial fluid from healthy controls was not examined. We previously reported that after purification of recombinant CRF-BP, spontaneous cleavag e frequently occurs, resulting in a 27-kDa N-terminal and a 10-kDa C-termin al fragment. Because concentrations of native CRF-BP in synovial fluid were insufficient to study the effects of cleavage on ligand binding, they were determined using recombinant human CRF-BP. Tryptophan excitation fluoresce nce spectra of intact and cleaved recombinant CRF-BP revealed that cleavage was accompanied by conformational change in the N-terminal fragment, leadi ng to exposure of the sole tryptophan residue to polar molecules (emission peak shift from 310 to 250 nm). Using gel filtration chromatography to sepa rate the N- and C-terminal fragments, it was found that the N-terminal frag ment of the recombinant protein bound human CRF, although dimerization was somewhat impaired. The C-terminal fragment did not bind CRF. Scatchard anal ysis confirmed that the affinity of both intact and cleaved CRF-BP for CRF was 1 x 10(10) L/mol. We conclude that synovial fluid contains intact CRF-B P in molar excess to CRF and fragmented CRF-BP. The significance of cleavag e and the role of cleavage products have yet to be determined, although the y may represent the generation of a novel bioactivity.