CHARACTERIZATION OF THE EQUILIBRIUM INTERMEDIATES IN ACID DENATURATION OF HUMAN STEFIN-B

Acid-induced denaturation of recombinant human stefin B was followed u sing circular dichroism (CD) and fluorimetry. By comparing different s pectroscopic probes, a number of equilibrium intermediates were detect ed. In pH denaturation at very low salt concentration (0.03 M NaCl) fo ur states can be distinguished: N - I-N - I-1 - U, where N is the nati ve state, I-N is a native-like intermediate, I-1 is an acid intermedia te state with properties of a molten globule and U is the unfolded sta te. State I-1 exhibits no near-ultraviolet CD but has some residual fa r-ultraviolet CD. It differs from U in its ability to increase fluores cence of 1-anilino-naphthalene 8-sulfonate (ANS). In 0.42 M salt, the pH denaturation is three-state between the dimeric native state N-2 an d intermediates I-N2, and I-2, which are also dimeric according to siz e-exclusion chromatography. The acid intermediate I-2 is more structur ed than I-1: it binds ANS to a lower extent an I-1, its Tyr residues a re protected from the solvent, it shows some near-ultraviolet CD and i ts far-ultraviolet CD is even more intense than that for the native st ate. H-1-NMR spectra confirmed the overall structural features of the acid intermediates. To obtain the enthalpies of unfolding, microcalori metric measurements were performed under conditions where the acid int ermediates are maximally populated (18 degrees C): state I-N from pH 5 .0 to 4.6, 0.03 M salt; state I-2 below pH 3.8, 0.42 M salt; and state I-1 in equilibrium with I-N at pH 4.05, 0.03 M salt. Enthalpies of un folding for states I-N and I-2 were comparable to those of the native state. The enthalpy of unfolding for state I-1 could not be determined .