The denaturation of proteins IV. Conalbumin and iron(III)-conalbumin in urea soloution

Abstract

Extensive optical rotation, viscosity, sedimentation, and spectroscopic studies of the urea denaturation of conalbumin and its iron complex are presented and considered in relation to the structure of these molecules. At low pH (< 4.2), conalbumin undergoes reversible changes in optical rotation, sedimentation and viscosity. The extent of these changes is dependent on the ionic strength and is greatly increased in the presence of urea. Over the pH range 5.8–7.8, urea brings about extensive disorganization of both conalbumin and iron-conalbumin. The optical rotation and viscosity changes in urea (> 5 M) solutions of conalbumin are very rapid, those of urea solutions of iron-conalbumin are much slower. In the pH range 8.8–10. iron-conalbumin is also considerably more stable towards urea than conalbumin. Experiments with cysteine present indicate that disulphide bonds are of importance in stabilizing the native configuration of both conalbumin and iron-conalbumin. Both acid and urea bring about similar rapid changes in the region of tyrosine and tryptophan absorption of the ultraviolet spectrum of conalbumin. The analogous spectral changes in urea solutions of iron-conalbumin are much slower. The change of absorbancy of iron-conalbumin at 292 mμ in concentrated urea solutions is first order with respect to time. The apparent order of reaction with respect to urea at pH 5.9 and 30° is 14. The spectroscopic changes accompanying urea denaturation of iron-conalbumin in the pH range 9.6–10.2 are complex and irreversible.Evidence is presented to show that at near-neutral pH, urea denaturation of conalbumin modifies, but does not destroy, the iron complex. Some preliminary experiments on the “renaturation” of conalbumin are reported.