AVIDIN. 4. STABILITY AT EXTREMES OF PH AND DISSOCIATION INTO SUB-UNITS BY GUANIDINE HYDROCHLORIDE

Abstract

Reduction of the disulphide bonds of avidin was dependent on prior denaturation by guanidine hydro-chloride. Molecular weights from sedimentation-diffusion, sedimentation, equilibrium and osmotic-pressure measurements showed that even without reduction avidin dissociated into three sub-units in 6M-guanidine hydrochloride. Dissociation in guanidine hydrochloride solution was accompanied by a denaturation blue shift of the tryptophan absorption bands and by loss of biotin-binding activity. It could be reversed with regain of the normal spectrum and activity by diluting the guanidine hydrochloride tenfold. The reversal showed marked hysteresis with a concentration difference of 2.8 M-guanidine hydrochloride between the forward and reverse branches of the curve. Throughout the whole course of the denaturation the loss of biotin-binding activity was proportional to the denaturation blue shift. This is most readily explicable in terms of a single-step reaction involving simultaneous loss of activity, exposure of tryptophan residues to the solvent and dissociation into sub-units. Avidin was also reversibly denatured below pH 2. Again, the reversal, as measured by the blue shift, was accompanied by hysteresis with a pH difference of 1.5 units between the forward and reverse branches of the curve. No loss of biotin-binding activity was observed on standing for short times at alkaline pH, and spectrophotometric titration with biotin could be performed in 0.5 M-potassium hydroxide. The tyrosine residues, which in avidin ionized reversibly at pH 12.3, regained a proton in 0.5 N-potassium hydroxide when biotin was added. The tyrosine residues of denatured avidin in 3 M-guanidine hydrochloride showed normal ionization (pK 10.1). Measurement of the polarization of fluorescence of avidin labelled with l-dimethylamino-naphthalene-5-sulphonyl groups was consistent with a compact symmetrical conformation of avidin, which changed little when biotin was added. Denaturation approximately halved the rotational relaxation time, which was consistent with the results of molecular weight measurement.