Figure 9. Subunit exchange studies with αB-crysteine mutants. FRET assay was performed with 50 μg of each donor and acceptor Alexa Fluor labeled protein at 37 ^°C. The rate of subunit exchange was calculated by measuring the increase of acceptor fluorescence intensity. Ft/F0 represents the ratio of acceptor fluorescence intensity at given time intervals to the fluorescence intensity at the baseline. Curve 1 is the NH₂-labeled protein; Curves 2, 3, and 4 are thiol-labeled proteins. The rate of subunit exchange between NH₂ -labeled αBT162C–αBI5C was 65.5x10^-5sec^-1. The interaction rate between thiol-labeled αBT162C subunits was 5.16x10^-5sec^-1. The rate of interaction between thiol-labeled αBI5C subunits was 1.66x10^-6sec^-1. There was no measurable interaction between αBT162C and αBI5C when one of the mutants was labeled with a donor Alexa Fluor and the second mutant was labeled with an acceptor Alexa Flour at thiol residues. The results demonstrate the absence of interaction between NH₂- and COOH-terminal regions of the two subunits and the presence of high degree of interactions between two NH₂-terminal regions or two COOH-terminal regions of  αB-crystallin in an oligomer.