FIGURE 2.

Extent of subunit exchange by nickel affinity chromatography. ¹⁴C-αA R116C and ¹⁴C-WTαA homocomplexes were separately incubated in the presence and absence of nonradioactive 7xHis-αA for 15 h at 37 °C. Following the incubation, samples were exchanged into nickel affinity binding buffer and then loaded onto a charged nickel affinity column. The resin was washed extensively with wash buffer and then eluted with imidazole. The counts per minute of the final eluate was measured to determine the amount of radioactive α-crystallin subunits bound to the resin as a measure of exchange into the nickel binding 7xHis-αA complexes. Data were normalized to the ¹⁴C-WTαA + 7xHis-αA mixture data. The data indicate that the αA R116C subunits show an approximate 4-fold reduction in the total amount of exchange possible.