 If the fluorophore is attached to the larger protein in a binding pair, the difference in polarization between bound and unbound states will be smaller (because the unbound protein will already be fairly stable and tumble slowly to begin with) and the measurement will be less accurate.The degree of binding is calculated by using the difference in anisotropy of the partially bound, free and fully bound (large excess of protein) states measured by titrating the two binding partners.If the fluorophore is bound to a relatively large molecule like a protein or an RNA, the change in the mobility accompanying folding can be used to study the dynamics of folding.