Resistance of soybean trypsin inhibitor (Kunitz) to denaturation by guanidinium chloride.

Viscosity measurements in 6 M guanidinium chloride (Gdm.Cl) (pH 5.2, 25”) suggest that Kunitz soybean trypsin inhibitor (STI) undergoes a slow unfolding which requires over 2 weeks to reach completion. The reduced viscosity increased during this time from an initial value of 3.5 ml/g to a final value of 16 to 17 ml/g. At pH 7 and 25”, over 4 weeks were required to reach the same final state. Gel chromatography of ST1 in 6 M Gdm. Cl yielded two peaks: one of equivalent hydrodynamic radius (It,) equal to 38 hi and a second of R, = 24 b. Over a time interval of days, the quantity of ST1 of R, = 38 A progressively increased at the expense of material of R, = 24 A. Material from both peaks had the same molecular weight and amino acid composition as native STI, and after renaturing in dilute buffer, both totally inhibited trypsin at a molar ratio of 1:l. Exposure of native STI to 6 M Gdm-Cl also produced a slow disappearance of the CD absorption bands of ST1 in the near-ultraviolet to ultimately yield a featureless spectrum. Lower pH, the presence of reducing agent, or higher temperatures accelerated the rate of unfolding of STI; for example, at 70 ST1 reached its final unfolded state within 15 min. Taken together, the hydrodynamic and spectral data strongly suggest that the final state reached by ST1 in 6 M Gdm.Cl is the disulfide cross-linked randomly coiled polypeptide.