Figure 3.

Gel filtration chromatography of Stv-43. (A) Purified Stv-43 (approximately 3 μg) with excess d-[carbonyl-¹⁴C]biotin was applied to a Superdex 75 HR 10/30 column (1.0 × 30 cm) that had been equilibrated with TTBS. Proteins were eluted at room temperature (22°C) with TTBS, and the absorbance at 280 nm was monitored (solid line). The eluate was collected in 320-μl fractions, and the radioactivity of each fraction was determined (○). The minimum-sized core streptavidin (50.4 kDa) (26) was also analyzed, and the absorbance at 280 nm is shown by a dashed line. (B) Stv-43, which had been stored without bound biotin at 4°C for 7 days, was mixed with excess d-[carbonyl-¹⁴C]biotin. The mixture was analyzed by gel filtration chromatography by the same procedure as in A.