Figure 6. Effect of 50 °C incubation on fluorescence enhancement

1 mM of two Mutant 9 preparations (Mutant 9-1 and Mutant 9-2) and EcR1 were tested in a retinol fluorescence enhancement assay with or without a five minute 50 °C preassay incubation (labeled HT for heat treated). The 50 °C incubation had little effect on EcR1 retinol binding. The heat treatment reduced the ability of Mutant 9 to bind retinol. These results are consistent with the hypothesis that retinol fluorescence enhancement is dependent on a native state of the binding protein. For Mutant 9, incubation at 50 °C was above its transition temperature of 45 °C, the incubation substantially denatured this protein, and according to the hypothesis, rendering it less capable of binding retinol. The 50 °C incubation of EcR1 was below its transition temperature of 60 °C, which did not denature the protein, and according to the hypothesis should have no adverse effect on retinol binding.