Partial dissociation and renaturation of embryonic chick delta-crystallin. Characterization by ultracentrifugation and circular dichroism

Abstract

1. delta-Crystallin from 15-day-old embryonic chick lenses was characterized by circular dichrosim (CD) spectroscopy. Examination by CD spectroscopy in the far ultraviolet (190-250 nm) demonstrated that the secondary structure of delta-crystallin has at least 75% alpha-helix; the delta-crystallin subunits dissociated in sodium dodecyl sulfate retain their alpha-helical content. This appreciable alpha-helical content of delta-crystallin contrasts with the absence of alpha-helix in other lens crystallins. 2. As judged by CD spectroscopy in the near ultraviolet (250-320 nm) the tertiary structure of embryonic delta-crystallin is not readily disrupted by environmental changes, such as NaCl, KSCN or the non-ionic detergent Emulphogene BC 720, and is stable to temperature fluctuation between 2 and 56 degrees C. 3. Experiments were directed towards deaggregation and renaturation of the four subunits of embryonic delta-crystallin by treatment with urea or guanidine hydrochloride. The native tertiary structure of delta-crystallin was lost above 4 M urea or 2 M guanidine hydrochloride, as judged by CD spectroscopy in the near ultraviolet. Ultracentrifugation at sedimentation equilibrium showed that in 4 M urea delta-crystallin dissociates into dimeric subunits, while in 2 M guanidine hydrochloride delta-crystallin exists as a mixture of dimeric and tetrameric subunits. Dialysis of delta-crystallin from 4 M urea resulted in reaggregation of the subunits into tetramers, about 50% of which showed native tertiary structure. Dialysis from 2 M guanidine hydrochloride also resulted in tetramer formation, and about 35% was recovered with native conformation. Removal of denaturant by dialysis produced no native teritary structure after treatment with 8 M urea, but about 15% native conformation after treatment with 6 M guanidine hydrochloride.