Three-estate unfolding and self-association of maspin, a tumor-suppressingserpin

Maspin is a tumor suppressor protein expressed by normal human mammary epit helium but not by many breast tumor cell lines, Recombinant human maspin (r Maspin) inhibits tumor cell motility, invasion, and metastasis and thus has potential value as an anti-cancer therapeutic, Maspin is a member of the s erpin family and, although the molecular mechanism by which maspin acts is unknown, recent work suggests that tissue plasminogen activator is a potent ial target. A puzzling observation in previous cell culture studies was los s of rMaspin activity at higher protein concentrations, One hypothesis to e xplain these results is self-association of rMaspin at the higher concentra tions, which would be consistent with the tendency of serpins to form nonco valent polymers, This hypothesis is addressed by examining the relationship between rMaspin stability and self-association. Urea denaturation of rMasp in at pH 7 and 25 degrees C and at protein concentrations ranging from 0.01 to 0.2 mg/ml has been monitored by circular dichroism and intrinsic trypto phan fluorescence, Denaturation profiles show a protein concentration depen dence and indicate the presence of at least one unfolding intermediate. The results suggest that destabilization of native monomeric rMaspin leads to partial unfolding and formation of an intermediate which can self-associate .