High-sensitivity fluorescence anisotropy detection of protein-folding events: Application to alpha-lactalbumin

An experimental procedure has been devised to record simultaneously fluores cence intensity and fluorescence anisotropy, A photoelastic modulator on th e excitation beam enables the anisotropy signal to be recorded in one pass using a single photomultiplier tube and eliminates the need for a polarizer on the emission path, In conjunction with a stopped-flow mixer, providing a time-resolved capability, this procedure was used to study the refolding of apo a-lactalbumin following dilution from guanidinium chloride. Although the fluorescence intensity does not change detectably, the fluorescence an isotropy was found to resolve the conformational changes occurring between the initial unfolded state and the molten globule state formed either kinet ically during refolding at pH 7.0 or at equilibrium at pH 2.0 (A-state), Th is result provides further evidence that fluorescence anisotropy is a valua ble probe of protein structural transitions and that the information it pro vides concerning the rotational mobility of a fluorophore can be complement ary to the information about the local environment provided by fluorescence intensity.