Use of intrinsic protein fluorescence to study DNA-protein binding

✍ Scribed by R. Wickett; H.J. Li; I. Isenberg

• Publisher: Elsevier Science
• Year: 1972
• Tongue: English
• Weight: 194 KB
• Volume: 50
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1016/0003-2697(72)90072-3

✦ Synopsis

When a polypeptide binds to DNA, the rotational mobility of a chromophore on the polypeptide is reduced. This simple fact makes it feasible to use polarization of fluorescence methods to study polypeptide-nucleic acid interaction. Previous (l-3) polarization of fluorescence studies of polypeptide-DNA binding have relied upon dye labeling to obtain a fluorescent probe. For polylysine binding, a dye label is needed simply to provide an emitter; for proteins, dye labels are used for their high quantum yield. However a problem exists when dye labels are employed: The dye may itself change the binding parameters, In specific instances (2,3), dye labelling has been shown to affect the binding very little, but the general problem remains. In this paper we demonstrate that the intrinsic emission from the protein may be used to study DNA-protein binding, thereby circumventing the dye labelling problem in a direct manner.

To develop and test the method, we have used the interaction of pancreatic ribonuclease A with DNA ( 46), but we also present results on the salt dissociation of histone IV-DNA complexes.

MATERIALS AND METHODS